Alpha 5 beta 1 integrin binding agents and uses thereof

Abstract

The present disclosure provides α5β1 integrin binding agents (eg, antibodies, including multispecific antibodies such as bispecific antibodies) and uses thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 187,371, filed May 11, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file named "14700-001-228_SEQ_LISTING.txt", created on May 8, 2022, and having a size of 142,533 bytes.

[0003] (Field) The present disclosure relates generally to binding agents, such as antibodies, that bind to alpha5beta1 (α5β1) integrin, including human α5β1 integrin, and methods of use thereof. [Background technology]

[0004] (background) Integrins are transmembrane proteins that bind to extracellular matrix (ECM) components and regulate cell adhesion, migration, and activation. Each integrin is composed of one α and one β transmembrane integrin subunit. There are 18 α and 8 β integrin subunits in the human genome, which combine to generate 23 unique heterodimeric integrins. These heterodimers regulate cell behavior through mechanisms known as "inside-out" and "outside-in" signaling. In the former, intracellular proteins bind to the integrin cytoplasmic domain and stabilize a conformation that binds extracellular ligands with high affinity. Through "outside-in" signaling, the ligand-bound integrin then stimulates intracellular signaling cascades that regulate cell behavior.

[0005] The α5β1 integrin is known as the fibronectin (FN) receptor due to its high affinity for fibronectin (FN) in the extracellular matrix (ECM). This binding is mediated by a ligand-binding site at the interface between the α and β subunits in the headpiece of α5β1 and an arginine-glycine-aspartic acid (RGD) peptide motif in the type III repeats of FN. α5β1 integrin binds additional RGD-containing proteins such as osteopontin and fibrillin, along with proteins lacking the RGD motif, such as CD40L, IL-1b, and the TNF-α converting enzyme ADAM-17. Consistent with the tissue distribution of its ligands, α5β1 is expressed by various cell types, such as endothelial cells, mast cells, and macrophage lineages (e.g., microglia and perivascular macrophages) in peripheral tissues and the central nervous system (CNS).

[0006] The association of α5β1 integrin with tumor angiogenesis has been well established. In addition, it has been demonstrated that α5β1 is present on tumor cells. Antibodies that bind to α5β1 have been shown to not only inhibit angiogenesis but also promote the death of tumor cells expressing α5β1. The association of α5β1 integrin with neuroinflammatory diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) has also been demonstrated. Antibodies that bind to α5β1 have been shown to inhibit angiogenesis in an experimental autoimmune encephalitis (EAE) model of MS and SOD1 in ALS. G93A It has been shown to ameliorate symptoms in transgenic models. Expression of α5β1 may make it a promising target in antiangiogenesis and cancer therapy, as well as in neuroinflammatory disease therapy, but clinical success with antibodies targeting α5 integrin has yet to be achieved.

[0007] Thus, there remains a need in the art for agents that can target α5β1 integrin and treat, prevent, or ameliorate α5-mediated diseases, disorders, or conditions, such as diseases, disorders, or conditions involving cells that express α5β1, such as tumor cells and macrophages. Summary of the Invention

[0008] (overview) The present disclosure provides α5β1 integrin binding agents, such as human α5β1 integrin binding agents. Such agents include antibodies that bind to α5β1 integrin, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to α5β1 integrin. In some embodiments, such antibodies compete for binding to human α5β1 integrin with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Tables 1-6).

[0009] The present disclosure also provides compositions comprising α5β1 integrin binding agents. Such compositions, in some embodiments, comprise an antibody that binds to α5β1 integrin, e.g., a monospecific or multispecific (e.g., bispecific) antibody that binds to α5β1 integrin. Such compositions, in some embodiments, comprise an antibody that competes with an antibody having a heavy chain variable region and a light chain variable region described herein (e.g., Tables 1-6) for binding to human α5β1 integrin.

[0010] The disclosure also provides methods of treating, preventing, or ameliorating an α5β1 integrin-mediated disease, disorder, or condition (including methods of treating, preventing, or ameliorating one or more symptoms of the disease, disorder, or condition) with an α5β1 integrin binding agent, such as an α5β1 integrin binding agent or a composition comprising such an agent, such as a composition comprising such an agent. Such compositions include antibodies that bind to α5β1 integrin, such as monospecific or multispecific (e.g., bispecific) antibodies that bind to α5β1 integrin. [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows exemplary results of a fibronectin (FN) inhibition assay, which is further described in Example 5.

[0012] [Diagram 2] 2A-2B show sequence alignments of the heavy and light chain variable regions of (i) A-15B08, A2-3B06, A2-5D10, C-14D12, and (ii) A2-7A05, A2-7F01, including exemplary consensus sequences for VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. CDR boundaries are indicated according to Kabat, AbM, Chothia, Contact, and IMGT (see also, e.g., Tables 1-6).

[0013] Figures 2C-2D show sequence alignments of the heavy and light chain variable regions of A-15B08-T62A and exemplary human engineered variable regions A-15B08_Low and A-15B08_Low+Mod. CDR boundaries are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0014] Figures 2E-2F show sequence alignments of the heavy and light chain variable regions of A-7A05 and exemplary human engineered variable regions A-7A05_Low and A-7A05_Low+Mod. CDR boundaries are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0015] Figures 2G-2H show sequence alignments of the heavy and light chain variable regions of C-14D12 and exemplary human modified C-14D12_Low and C-14D12_Low+Mod. CDR boundaries are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0016] [Diagram 3] FIG. 3 shows a sequence alignment of exemplary Fc sequences, including variant Fc sequences.

[0017] [Figure 4]4A and 4B show exemplary results of antibodies provided herein discrupting the α5β1-FN integrin-ligand complex, as further described in Example 7.

[0018] [Diagram 5] FIG. 5 shows exemplary results of an α5β1 integrin FN blocking assay, which is further described in Example 8.

[0019] [Figure 6] FIG. 6 shows exemplary results of the efficacy of chimeras compared to hybridomas, which are further described in Example 8.

[0020] [Figure 7] FIG. 7 shows exemplary SDS-PAGE results of various IgG4 chimeric antibodies, which are further described in Example 8.

[0021] [Figure 8] FIG. 8 shows exemplary results of a conformational assay, further described in Example 9.

[0022] [Figure 9] FIG. 9 shows exemplary results of a cell adhesion assay, which is further described in Example 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] (Detailed Description) The present disclosure provides α5β1 integrin binding agents. Such agents include antibodies (e.g., monospecific or multispecific (including bispecific)) that bind to α5β1 integrin, such as antibodies that bind to human α5β1 integrin. Such binding agents are useful in compositions and methods for treating, preventing, or ameliorating α5β1 integrin-mediated diseases, disorders, or conditions, including one or more symptoms of the disease, disorder, or condition. α5β1 integrin-mediated diseases, disorders, and conditions include cancer, angiogenesis-mediated diseases (e.g., diseases involving abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases). In addition, the α5β1 integrin binding agents described herein, such as α5β1 integrin binding antibodies (e.g., monospecific or multispecific antibodies (including bispecific antibodies)), are useful for (i) inhibiting α5β1 integrin binding to fibronectin, (ii) inhibiting angiogenesis, and / or (iii) treating or alleviating one or more symptoms of (i) cancer, (ii) angiogenesis-mediated disease, disorder, or condition, or (iii) inflammatory disease, disorder, or condition. The α5β1 integrin binding agents described herein, such as α5β1 integrin binding antibodies (e.g., monospecific or multispecific antibodies (including bispecific)), are useful in compositions and methods of treating α5β1-mediated diseases, disorders, or conditions.

[0024] The terms "α5 integrin", "CD49e", or "α5 integrin polypeptide" and similar terms refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any naturally occurring α5 integrin from any vertebrate origin, including mammals such as primates (e.g., humans, cynos), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The α5 integrin, also known in the art as integrin α-5, ITGA5 protein, CD49e antigen, glycoprotein Ic (GPIc), VLA5A, FNRA, and fibronectin receptor subunit α, has multiple domains, including a β-propeller (e.g., with seven 60 amino acid FG-GAP repeats), thigh, genu, calf1, calf2, transmembrane, and intracellular domains, and four calcium binding sites. The term α5 integrin encompasses "full length," "unprocessed α5 integrin," and any form of α5 integrin resulting from processing within a cell or any fragment thereof. The term α5 integrin also encompasses naturally occurring variants of α5 integrin, such as SNP variants, splice variants, and allelic variants. α5 integrin associated with β1 as a heterodimer is known in the art to interact with several ligands (e.g., fibronectin), which leads to conformational changes in the protein and signal transduction, resulting in changes in cellular activities such as cell adhesion, proliferation, apoptosis, migration, and phagocytosis. The full length amino acid sequence of human α5 integrin is shown below (exemplary signal sequence = italic letters; exemplary extracellular domain = underlined letters): [ka]

[0025] The full length amino acid sequence of mouse α5 integrin is shown below (exemplary signal sequence = italic letters; exemplary extracellular domain = underlined letters): [ka]

[0026] Other related α5 integrin polypeptides that are also encompassed by the term α5 integrin include fragments, derivatives (e.g., substitution, deletion, truncation, and insertion variants), fusion polypeptides, and interspecies homologs that retain α5 integrin activity and / or are sufficient to generate an anti-α5 integrin immune response. As one skilled in the art will recognize, the α5 integrin binding agents (e.g., antibodies) described herein can bind to α5 integrin polypeptides, α5 integrin polypeptide fragments, α5 integrin antigens, and / or α5 integrin epitopes. The epitopes can be part of a larger α5 integrin antigen, the α5 integrin antigens can be part of a larger α5 integrin polypeptide fragment, and the α5 integrin polypeptide fragments can be part of a larger α5 integrin polypeptide. The α5 integrin can exist in a natural or denatured form. The α5 integrin polypeptides described herein can be isolated from various sources, such as from human tissue types or from another source, or can be prepared by recombinant or synthetic methods. The α5 integrin polypeptide may include a polypeptide having the same amino acid sequence as the corresponding naturally occurring α5 integrin polypeptide. Orthologues to the α5 integrin polypeptide are also known in the art.

[0027] The terms "β1 integrin," "CD29," or "β1 integrin polypeptide," and similar terms, unless otherwise indicated, refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) from any vertebrate origin, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno), dogs, and rodents (e.g., mice and rats), or any naturally occurring β1 integrin. β1 integrin, also known in the art as integrin β-1, ITGB1 protein, CD29 antigen, fibronectin receptor subunit β, and glycoprotein IIa, comprises the β1 headpiece (β1 The term β1 integrin encompasses "full length," unprocessed β1 integrin, and any form of β1 integrin resulting from processing within the cell or any fragment thereof. The term β1 integrin also encompasses SNP-based integrins. The present invention also encompasses naturally occurring variants of β1 integrin, such as variants, splice variants, and allelic variants. It is known in the art that β1 integrin associated with α5 as a heterodimer interacts with several ligands (e.g., fibronectin), which leads to changes in protein conformation and signal transduction, resulting in changes in cellular activities such as cell adhesion, proliferation, apoptosis, migration, and phagocytosis. The full-length amino acid sequence of human β1 integrin is shown below (exemplary signal sequence = italicized letters; exemplary extracellular domain = underlined letters): [ka]

[0028] The full length amino acid sequence of mouse β1 integrin is shown below (exemplary signal sequence = italic letters; exemplary extracellular domain = underlined letters): [ka]

[0029] The terms "α4 integrin", "CD49d", or "α4 integrin polypeptide" and similar terms refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any naturally occurring α4 integrin from any vertebrate origin, including mammals such as primates (e.g., human, cyno), dogs, and rodents (e.g., mouse and rat), unless otherwise indicated. The α4 integrin, also known in the art as integrin α-4, ITGA4 protein, CD49d, VLA-4 subunit α, has multiple domains including a β-propeller (e.g., with seven 60 amino acid FG-GAP repeats), thigh, genu, calf1, calf2, transmembrane, and intracellular domains, and also has three calcium binding sites. The term α4 integrin encompasses "full length", unprocessed α4 integrin, and any form of α4 integrin resulting from processing within the cell, or any fragment thereof. The term α4 integrin also encompasses naturally occurring variants of α4 integrin, such as SNP variants, splice variants, and allelic variants. It is known in the art that α4 integrin associated with β1 as a heterodimer interacts with several ligands (e.g., VCAM1, fibronectin), which leads to protein conformational changes and signal transduction, resulting in altered cellular activities such as cell adhesion, proliferation, migration, and phagocytosis. The full-length amino acid sequence of human α4 integrin is shown below (exemplary signal sequence = italicized letters; exemplary extracellular domain = underlined letters): [ka]

[0030] The term "fibronectin" or "FN" and similar terms refer to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) or any naturally occurring fibronectin from any vertebrate origin, including mammals such as primates (e.g., humans, cynos), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. Fibronectin exists as a dimer or multimer linked through disulfide bonds and has a multimodular structure composed primarily of three distinct repeats named FN-I, FN-II, and FN-III. In the dimeric form, each of the two fibronectin subunits consists of 12 FN-I, 2 FN-II, and 15-17 FN-III modules, respectively. The term fibronectin also encompasses naturally occurring variants of fibronectin, such as SNP variants, splice variants, and allelic variants. Fibronectin is an essential component of the extracellular matrix and has multiple protein binding domains, including domains for fibrin binding, collagen binding, fibulin-1-binding, heparin binding, and syndecan binding. Fibronectin is known in the art to interact with integrins (e.g., via RGD) and is a ligand for α5β1 integrin, α8β1 integrin, and αvβ3 integrin. The full-length amino acid sequence of human fibronectin is shown below (exemplary signal sequence = italics): [ka] TIFF2024517953000008.tif177170

[0031] As used herein, the term "binding agent" or its grammatical equivalents refers to a molecule (e.g., an antibody) that has one or more antigen-binding sites that bind to an antigen. In some embodiments, the α5β1 integrin binding agent described herein is an antibody, antibody fragment, or other peptide-based molecule that binds to α5β1 integrin, such as human α5β1 integrin.

[0032] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and in the broadest sense, specifically covering, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies with full-length heavy and / or light chains. The present disclosure also includes antibody fragments (and / or polypeptides comprising antibody fragments) that retain α5 integrin binding properties. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of an antibody, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single chain antibody molecules, dual variable region antibodies, single variable region antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, diabodies, didiabodies, disulfide-linked Fvs (dsFv), single domain antibodies (e.g., VHH, nanobodies), or other fragments (e.g., fragments consisting of non-covalently coupled heavy and light chain variable regions). In general, the variable region domains can be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable regions. For example, the present disclosure also includes tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the variable region domain may be a dimer and may include a VH-VH, VH-VL, or VL-VL dimer that binds to α5 integrin. If desired, the VH and VL chains may be covalently coupled, either directly or via a linker, to form a single chain Fv (scFv). For ease of reference, scFv proteins are included in the category of "antibody fragments" herein. Another form of antibody fragment is a peptide that includes one or more complementarity determining regions (CDRs) of an antibody.CDRs (also termed "minimal recognition units" or "hypervariable regions") can be obtained by constructing polynucleotides that encode the CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, Genetic Manipulation of Monoclonal Antibodies, Ritter et al., eds., Monoclonal Antibodies Production, Engineering and Clinical Application, Cambridge University Press (1995), p. 166; and Ward et al., Genetic Manipulation and Expression of Monoclonal Antibodies, Birch et al., eds., Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. (1995), p. 137). Antibodies). Antibody fragments may be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of new antigen receptors (v-NAR), and bis single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005).The binding agent, in some embodiments, comprises a light chain and / or a heavy chain constant region, such as one or more constant regions comprising one or more of IgG1, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, the antibody can comprise an epitope-binding fragment of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibody can be an α5β1-binding antibody, including an antagonistic or agonistic antibody.

[0033] As used herein, the term "monospecific" when used in reference to a binding agent (e.g., an antibody) means a binding agent that has one or more binding sites that each bind to the same epitope of the same antigen.

[0034] The term "bispecific" when used in reference to a binding agent (e.g., an antibody) refers to a binding agent that can specifically bind to at least two distinct antigenic determinants, e.g., two binding sites, formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), each of which binds to a different antigen or to a different epitope on the same antigen. Such bispecific binding agents (e.g., antibodies) may have a 1+1 format. Other bispecific binding agent (e.g., antibody) formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific binding agent (e.g., an antibody) comprises two antigen-binding sites, each may bind to a different antigenic determinant. Such bispecific binding agents (e.g., antibodies) may bind to two different epitopes on the same antigen (e.g., an epitope on α5β1 integrin) or different antigens (e.g., an epitope on α5β1 integrin and an epitope on αvβ1 integrin).

[0035] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that, when compared and aligned for best correspondence (with gaps introduced, if necessary), are the same or have a specified percentage of the same nucleotides or amino acid residues, without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for best correspondence as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween, in length. In some embodiments, the identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, such as the coding regions of target proteins or antibodies. In some embodiments, the identity exists over a region of the nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween in length.In some embodiments, the identity exists over a region longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, such as a nucleotide sequence encoding a protein of interest.

[0036] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue that has a side chain with similar chemical characteristics. Families of amino acid residues with similar side chains are generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In general, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody that contains that amino acid sequence to a target binding site. Methods for identifying conservative substitutions of amino acids that do not abolish binding are well known in the art.

[0037] The term "polypeptide" refers to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may contain non-amino acids (e.g., it may be interrupted by non-amino acids). The term also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, or any other manipulation or modification, such as, for example, linkage to or conjugation with a moiety (direct or indirect), such as a labeling moiety. Also included within the definition are polypeptides that contain, for example, one or more analogs of an amino acid (such as, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in some embodiments, the polypeptides may exist as a single chain.

[0038] As used herein, an "antigen" is a site or molecule that contains an epitope to which a binding agent (e.g., an antibody) can bind. Thus, an antigen can be bound by an antibody. In some embodiments, the antigen that is bound by the binding agent (e.g., an antibody) described herein is α5β1 integrin (e.g., human α5β1 integrin) or a fragment thereof.

[0039] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a "linear" epitope), or an epitope can include amino acids of two or more discontinuous regions of the polypeptide (a "conformational," "non-linear," or "discontinuous" epitope), such as human α5β1 integrin. Those skilled in the art will generally recognize that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether the amino acids are folded into the native three-dimensional protein structure. In another embodiment, an antibody requires that the amino acid residues that make up an epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to that epitope.

[0040] If two antibodies (antibody and reference antibody) recognize the same, overlapping or adjacent epitopes in three-dimensional space, the antibody binds to the "epitope" or "essentially the same epitope" or "same epitope" as the reference antibody. The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping or adjacent epitopes in three-dimensional space is a competitive assay, which can be configured in several different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of unlabeled antibody to block the binding of labeled antibody is measured using radioactive, fluorescent or enzyme labeling.

[0041] "Epitope binning" is the process of classifying antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for distinguishing the epitope recognition characteristics of different antibodies, for example, using competitive assays. Such assays can be combined with computational processes to cluster antibodies based on their epitope recognition characteristics and identify antibodies with distinct binding specificities.

[0042] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," "selectively bind," "immunospecifically recognize," and "immunospecific" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope), as such binding is understood by those of skill in the art. In some embodiments, "specifically bind" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with higher affinity, or any combination of the above, than it does with another substance, such as a related or unrelated protein. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, Biacore™, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, an antibody or antigen-binding domain binds or specifically binds to an antigen if it binds to the antigen with higher affinity than to any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the background signal or noise, and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Paul, ed., Fundamental Immunology, 332-36 (2nd ed., 1989). In some embodiments, the extent of binding of an antibody or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the antibody or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds the antigen with a Ka that is at least 2 logs, 2.5 logs, 3 logs, 4 logs, or more than the Ka that the molecule binds to another antigen.In some embodiments, the molecule that specifically binds to an antigen does not cross-react with other proteins. In some embodiments, the molecule that specifically binds to an antigen does not cross-react with other non-α5β1 integrin proteins. In some embodiments, "specifically binds" refers to, for example, a polypeptide or molecule that binds to a protein or target with a K of about 0.1 mM or less, but more typically less than about 1 μM. D In some embodiments, "specifically binds" means that a polypeptide or molecule binds with a K that is at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. D Specific binding means binding to a target at a specific site. Due to sequence identity between homologous proteins in different species, specific binding may include a polypeptide or molecule that recognizes a protein or target in more than one species. Similarly, due to homology within a region of the polypeptide sequence of different proteins, specific binding may include a polypeptide or molecule that recognizes more than one protein or target. It is understood that in some embodiments, a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, a polypeptide or molecule can, in some embodiments, specifically bind to more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on a polypeptide or molecule. For example, an antibody can, in some instances, include two identical antigen-binding sites that each specifically bind to the same epitope on two or more proteins. In some alternative embodiments, an antibody can be bispecific and include at least two antigen-binding sites with different specificities. Generally, but not necessarily, reference to "binding" means "specific binding".

[0043] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by conventional methods known in the art, such as those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. In one embodiment, "K D " or "Kd value" can be measured, for example, by biolayer interferometry (BLI) using an OctetQK384 system (ForteBio, Menlo Park, Calif.). Alternatively, K D can also be measured using, for example, a radiolabeled antigen binding assay (RIA) performed using a Fab version of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881), or a surface plasmon resonance (SPR) assay by Biacore, for example using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). "ON rate" or "rate of binding" or "on-rate" or "k on " and "OFF rate" or "rate of dissociation" or "dissociation rate" or "k off" can also be determined by the same SPR or BLI techniques described above, for example, using an OctetQK384 system (ForteBio, Menlo Park, Calif.) or a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), respectively.

[0044] The term "compete" when used in the context of an α5β1 integrin binding agent (e.g., an antibody) refers to binding agents that compete for the same epitope or binding site on a target, including competition between such binding agents as determined by an assay in which the binding agent being tested prevents or inhibits specific binding of a reference molecule (e.g., a reference ligand, or a reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., α5β1 integrin). Many types of competitive binding assays can be used to determine whether a test binding agent competes with a reference molecule for binding to α5β1 integrin (e.g., human α5β1 integrin). Examples of assays which can be employed include solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., (1990) Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigen (e.g., α5β1 integrin, such as human α5β1 integrin) bound to a solid surface or cells bearing either an unlabeled test antigen binding protein (e.g., a test α5β1 integrin antibody) or a labeled reference antigen binding protein (e.g., a reference α5β1 integrin antibody).Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cell in the presence of a test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and / or antibodies that bind to adjacent epitopes (e.g., similar or overlapping epitopes) close enough to the epitope bound by the reference that steric hindrance of the antibodies occurs. Additional details regarding methods for determining competitive binding are described herein, as shown in Example 6. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 20%, e.g., at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%, 99% or more.

[0045] The term "constant region" or "constant domain" as used herein is a well-known antibody terminology in the art and refers to the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exert various effector functions, such as interaction with Fc receptors. The term includes the portion of an immunoglobulin molecule that has a generally more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0046] Antibody "effector function" means a biological activity attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an amino acid sequence variant Fc region) and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0047] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, such as, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the Fc region boundaries of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as extending from an amino acid residue at position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Exemplary Fc region sequences are shown below (CH2 domain = bold letters; CH3 domain = underlined letters): [ka]

[0048] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain), and can be assessed using a variety of assays, such as those disclosed.

[0049] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature and which has not been engineered, modified, and / or altered (e.g., isolated, purified, selected, inclusion of other sequences such as variable region sequences, or combination with other such sequences) by man. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Exemplary IgG1 and IgG4 Fc sequences are shown in FIG. 3.

[0050] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region described herein can have at least about 80% homology with the native sequence Fc region and / or with the Fc region of the parent polypeptide, or at least about 90% homology thereto, e.g., at least about 95% homology thereto. The variant Fc region herein described herein may have lost effector function (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are shown below (CH2 domain = bold letters (amino acid changes underlined); CH3 domain = underlined letters): [ka] Exemplary variant Fc sequences are shown in FIG. 3, including the following variants (according to the EU numbering system): N297A / Q (N297A or N297Q), LALA (L234A, L235A), LALAPS (L234A, L235A, P331S), LALAPG (L234A, L235A, P329G), and TM (L234F, L235E, P331S).

[0051] As used herein, the term "heavy chain" when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, the amino terminal portion of which contains a variable region of about 120-130 or more amino acids and the carboxy terminal portion of which contains one or more constant regions. "Heavy chain" can also refer to any of the distinct types based on the amino acid sequence of the constant domains that give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, respectively, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ).

[0052] As used herein, the term "light chain" when used in reference to an antibody can refer to a polypeptide chain of about 25 kDa, the amino terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, e.g., lambda (λ) and kappa (κ). Light chain amino acid sequences are well known in the art.

[0053] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to a portion of an antibody that contains the amino acid residues that interact with an antigen and confer on the binding fragment, domain, or region its specificity and affinity for the antigen (e.g., CDRs). As used herein, "antigen-binding fragment" includes "antibody fragments" that contain a portion of an antibody such as one or more CDRs (such as the antigen-binding or variable regions of an antibody).

[0054] Antibodies as described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies, etc.), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single chain Fvs (scFvs) (e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0055] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain one or more antigen-binding sites that bind to the α5β1 integrin antigen.

[0056] An antibody can be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b). In some embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass thereof.

[0057] In some embodiments, the antibody is a four-chain antibody unit comprising two heavy (H) / light (L) chain pairs, in which the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In some embodiments, the H and L chains comprise a constant region, e.g., a human constant region. In some embodiments, the L chain constant region of such an antibody is a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such an antibody comprises a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In some embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., an IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0058] The antibody or fragment thereof may preferentially bind to α5β1 integrin, such as human α5β1 integrin. This means that the antibody or fragment thereof binds to α5β1 integrin with a higher affinity than it binds to an unrelated control protein and / or binds to human α5β1 integrin with a higher affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof may specifically recognize and bind to α5β1 integrin or a portion thereof. "Specific binding" indicates that the antibody or fragment thereof binds to α5β1 integrin with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than its affinity to an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof may bind substantially exclusively to α5β1 integrin (e.g., be able to distinguish α5β1 integrin from other known polypeptides (e.g., due to a measurable difference in binding affinity)). In some embodiments, the α5β1 integrin binding agent (eg, an antibody) may react with an α5β1 integrin sequence other than the human α5β1 integrin sequence (eg, a cynomolgous α5β1 integrin sequence).

[0059] The term "variable region" or "variable domain" generally refers to a portion of an antibody's light or heavy chain located at the amino terminus of the light or heavy chain, having a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, and used for the binding and specificity of each particular antibody to its particular antigen. The variable region of the heavy chain may also be referred to as "VH". The variable region of the light chain may also be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region differ significantly in sequence between antibodies. The V regions mediate antigen binding and define the specificity of a particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the approximately 110 amino acid span of the variable region. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches of about 15-30 amino acids called framework regions (FR) separated by shorter regions of higher variability (e.g., very high variability) called "hypervariable regions" or "complementarity determining regions". Each of the heavy and light chain variable regions contains four FRs (FR1, FR2, FR3, and FR4), which are largely in a β-sheet structure and connected by three hypervariable regions that form loops connecting and, in some cases, forming part of the β-sheet structure. The hypervariable regions in each chain are held in close proximity together with the hypervariable regions of the other chains by the FRs and contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant regions are not directly involved in binding the antibody to the antigen, but exert various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies.The variability of the sequence is concentrated in CDR, and the less variable part of the variable region is called framework region (FR). The CDR of the light chain and the heavy chain are mainly responsible for the interaction of the antibody with the antigen. In a specific embodiment, the variable region is a human variable region.

[0060] The terms "hypervariable region", "HVR", "HV", "complementarity determining region" or "CDR" as used herein refer to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several hypervariable region descriptions are in use and are encompassed herein. The Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme allows for insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The "Contact" hypervariable regions are based on an analysis of the available complex crystal structures. The residues of each of these hypervariable regions or CDRs are described below.

[0061] A universal numbering system, the ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)), has been developed and is widely used. IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC) for humans and other vertebrates. Herein, CDRs are referred to in terms of both amino acid sequence and location within the light or heavy chain. The "location" of the CDRs within the structure of an immunoglobulin variable domain is easily identified by using a numbering system that aligns the variable domain sequence according to structural features that are conserved across species and reside within structures called loops, allowing the CDR and framework residues to be readily identified. This information can be used to graft and replace CDR residues from one immunoglobulin into an acceptor framework, usually derived from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun (Honegger and Pluckthun, J. Mol. Biol. 309: 657-670 (2001). Correspondences between numbering systems, for example between Kabat numbering and the IMGT unique numbering system, are well known to those of skill in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra), and are also exemplified below. The Exemplary system presented herein combines Kabat and Chothia. [Table 1]

[0062] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region", or "CDR" are used interchangeably.

[0063] The term "vector" refers to a material used to carry or contain a nucleic acid sequence, for example, to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell chromosome. Furthermore, the vector can include one or more selection marker genes and appropriate expression control sequences. Selection marker genes that can be included provide, for example, resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply important nutrients not included in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single vector expression, the encoding nucleic acid can be operatively linked to one common expression control sequence, or can be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of the nucleic acid molecule into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule will be expressed in sufficient amounts to produce the desired product (e.g., the α5β1 integrin binding agent described herein), and it will be further understood that the expression level can be optimized to obtain sufficient expression, using methods well known in the art.

[0064] "α5β1 integrin-mediated disease" and "α5β1 integrin-mediated disorder" and "α5β1 integrin-mediated condition" are used interchangeably and refer to any disease, disorder, or condition that is caused or results from α5β1 integrin or the interaction of α5β1 integrin with fibronectin in whole or in part, and / or alternatively, any disease, disorder, or condition in which it is desirable to inhibit the in vivo action of the interaction of α5β1 integrin with fibronectin. α5β1 integrin-mediated diseases, disorders, or conditions include cancer, angiogenesis-mediated diseases (e.g., diseases involving abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases, such as MS and ALS). In some embodiments, α5β1 integrin-mediated diseases include diseases, disorders, or conditions that are cancers characterized by or associated with tumor cells that express or overexpress α5β1 integrin. In some embodiments, α5β1 integrin mediated diseases include diseases, disorders, or conditions that are characterized by or associated with abnormally increased angiogenic activity of cells (e.g., tumor cells). In some embodiments, α5β1 integrin mediated diseases are diseases, disorders, or conditions that are specifically associated with abnormal angiogenesis (e.g., ocular diseases such as diabetic retinopathy or age-induced macular degeneration). In some embodiments, α5β1 integrin mediated diseases include diseases, disorders, or conditions that are inflammatory diseases characterized by or associated with inflammatory immune responses (e.g., inflammatory autoimmune diseases such as multiple sclerosis). In some embodiments, α5β1 integrin mediated diseases include diseases, disorders, or conditions that are neuroinflammatory diseases characterized by or associated with neurodegeneration (e.g., MS or ALS).

[0065] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of one or more symptoms, eliminate one or more symptoms and / or their underlying causes, prevent the occurrence of one or more symptoms and / or their underlying causes, and / or ameliorate or treat damage caused by or associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0066] The term "therapeutically effective amount" as used herein refers to an amount of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition and / or symptoms associated therewith. A therapeutically effective amount of an agent, such as a therapeutic agent, can be the amount necessary to (i) reduce or ameliorate the progression or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of an α5β1 integrin binding agent, such as an antibody described herein). The "therapeutically effective amount" of a substance / molecule / agent (e.g., an α5β1 integrin antibody) of the present disclosure can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In some embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (e.g., or drug) effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0067] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, exerts the intended prophylactic effect, e.g., prevents or delays the onset (or reoccurrence) of a disease, disorder, or condition, or reduces the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptoms. A complete therapeutic or prophylactic effect may not necessarily occur by administration of a single dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0068] As used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency for use in animals, especially humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias.

[0069] As used herein, "carrier" includes carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. In many cases, the carrier is a pH-buffered aqueous solution. Examples of carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which a therapeutic is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, and the like.Oral compositions such as formulations can contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences", (1990) Mack Publishing Co., Easton, PA. Compositions such as pharmaceutical compounds may contain, for example, a prophylactically or therapeutically effective amount of an α5β1 integrin binding agent (e.g., an antibody) in isolated or purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject (e.g., a patient). The formulation should be appropriate for the mode of administration.

[0070] In some embodiments, the present disclosure provides α5β1 integrin binding agents that can be used as therapeutic agents herein. Such agents include antibodies that bind to α5β1 integrin (e.g., monospecific or multispecific (including bispecific)). Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof with increased or decreased affinity or other properties.

[0071] In some embodiments, described herein are α5β1 integrin binding agents (e.g., antibodies) that bind to α5β1 integrin, such as α5β1 integrin polypeptides, α5β1 integrin polypeptide fragments, α5β1 integrin peptides, or α5β1 integrin epitopes. In some embodiments, the α5β1 integrin binding agent is a human, humanized, or chimeric antibody (e.g., comprising a human constant region) that binds to α5β1 integrin, such as α5 integrin polypeptides, α5 integrin polypeptide fragments, α5 integrin peptides, or α5 integrin epitopes. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, can bind to α5β1 integrin expressed on the surface of a mammalian (e.g., human) cell, such as an α5β1 integrin-expressing tumor cell. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) binds to an α5β1 integrin extracellular epitope (e.g., an α5β1 integrin epitope) exposed on a cell, such as a tumor cell. In some embodiments, described herein are α5β1 integrin binding agents (e.g., antibodies) that bind to α5β1 integrin or portions thereof, such as human α5β1 integrin. In some embodiments, the α5β1 integrin is human α5β1 integrin. In some embodiments, the α5β1 integrin binding agent is a human α5β1 integrin binding agent (e.g., an antibody that binds to human α5β1 integrin). Exemplary amino acid sequences of human α5 integrin and human β1 integrin are described herein.

[0072] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) described herein competes for binding to an α5β1 integrin, such as human α5β1 integrin, with an α5β1 integrin binding agent (e.g., an antibody) comprising the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 listed in Tables 1 to 6. Thus, in some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein compete for binding to α5β1 integrin, such as human α5β1 integrin, with an α5β1 integrin binding agent (e.g., an antibody) that comprises one, two, and / or three VH CDRs and / or one, two, and / or three VL CDRs from an antibody designated as follows: (a) an antibody designated A-15B08; (b) an antibody designated A2-3B06; (c) an antibody designated A2-5D10; (d) an antibody designated A2-7A05; (e) an antibody designated A2-7F01; or (f) an antibody designated C-14D12, as shown in Tables 1-6. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) described herein competes for binding to α5β1 integrin, such as human α5β1 integrin, with an α5β1 integrin binding agent (e.g., an antibody) that comprises one, two, and / or three VH CDRs and one, two, and / or three VL CDRs from an antibody designated as: (a) an antibody designated A-15B08; (b) an antibody designated A2-3B06; (c) an antibody designated A2-5D10; (d) an antibody designated A2-7A05; (e) an antibody designated A2-7F01; or (f) an antibody designated C-14D12, as shown in Tables 1-6.In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) described herein competes for binding to α5β1 integrin, such as human α5β1 integrin, with an α5β1 integrin binding agent (e.g., an antibody) comprising a VH region and a VL region from an antibody designated as: (a) an antibody designated A-15B08; (b) an antibody designated A2-3B06; (c) an antibody designated A2-5D10, (d) an antibody designated A2-7A05, (e) an antibody designated A2-7F01, or (f) an antibody designated C-14D12, as shown in Tables 1 to 6. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein, with respect to binding to α5β1 integrin, such as human α5β1 integrin, include: (a) a VH region comprising the amino acid sequence of SEQ ID NO: 25 or a humanized variant thereof and a VL region comprising the amino acid sequence of SEQ ID NO: 26 or a humanized variant thereof; (b) a VH region comprising the amino acid sequence of SEQ ID NO: 42 or a humanized variant thereof and a VL region comprising the amino acid sequence of SEQ ID NO: 43 or a humanized variant thereof; (c) a VH region comprising the amino acid sequence of SEQ ID NO: 51 or a humanized variant thereof and a VL region comprising the amino acid sequence of SEQ ID NO: 52 or a humanized variant thereof. (d) a VH region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 77 and a VL region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 78; (e) a VH region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 91 and a VL region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 92; or (f) a VH region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 109 and a VL region or humanized variant thereof comprising the amino acid sequence of SEQ ID NO: 110.

[0073] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein comprise the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 set forth in Tables 1-6. Thus, in some embodiments, an α5β1 integrin binding agent (e.g., an antibody) described herein comprises one, two, and / or three heavy chain CDRs and / or one, two, and / or three light chain CDRs as set forth in Tables 1-6 from: (a) an antibody designated A-15B08; (b) an antibody designated A2-3B06; (c) an antibody designated A2-5D10; (d) an antibody designated A2-7A05; (e) an antibody designated A2-7F01; or (f) an antibody designated C-14D12. In some embodiments, an α5β1 integrin binding agent (e.g., an antibody) described herein comprises one, two, and / or three heavy chain CDRs and one, two, and / or three light chain CDRs as set forth in Tables 1-6 from: (a) an antibody designated A-15B08; (b) an antibody designated A2-3B06; (c) an antibody designated A2-5D10; (d) an antibody designated A2-7A05; (e) an antibody designated A2-7F01; or (f) an antibody designated C-14D12.

[0074] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) comprises a VH region comprising a VH CDR1, a VH CDR2, and / or a VH CDR3 of any one of the binding agents described herein (see, e.g., Table 1, Table 2, Table 3, Table 4, Table 5, Table 6) and a VL region comprising a VL CDR1, a VL CDR2, and / or a VL CDR3. Thus, in some embodiments, the α5β1 integrin binding agent (e.g., an antibody) described herein comprises one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 1. In some embodiments, the α5β1 integrin binding agents described herein comprise one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 2. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein comprise one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 3. In some embodiments, the α5β1 integrin binding agents described herein comprise one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 4.In some embodiments, the α5β1 integrin binding agents described herein comprise one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 5. In some embodiments, the α5β1 integrin binding agents described herein comprise one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 6. In some embodiments, an α5β1 integrin binding agent (e.g., an antibody) described herein is multispecific (e.g., bispecific) and comprises one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6. and a second binding domain that comprises one, two, and / or three heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or one, two, and / or three light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from a binding agent that binds to a second target antigen that is not α5β1 integrin (e.g., αvβ3 integrin, α4β1 integrin, α4β7 integrin, TREM2, TNFα, IL-6, IL-1β, CSF1, CSF-1R, C1Q, CD40L, FGFR, IL-12, and type I interferon).

[0075] The antibody designated A-15B08 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 1, and in some embodiments may comprise a VH sequence which is SEQ ID NO:25 or a humanized variant thereof and a VL sequence which is SEQ ID NO:26 or a humanized variant thereof.

[0076] The antibody designated A2-3B06 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 2, and in some embodiments may comprise a VH sequence which is SEQ ID NO: 42 or a humanized variant thereof and a VL sequence which is SEQ ID NO: 43 or a humanized variant thereof.

[0077] The antibody designated A2-5D10 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 3, and in some embodiments may comprise a VH sequence which is SEQ ID NO: 51 or a humanized variant thereof and a VL sequence which is SEQ ID NO: 52 or a humanized variant thereof.

[0078] The antibody designated A2-7A05 comprises the CDR sequences by Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 4, and in some embodiments may comprise a VH sequence that is SEQ ID NO: 77 or a humanized variant thereof and a VL sequence that is SEQ ID NO: 78 or a humanized variant thereof.

[0079] The antibody designated A2-7F01 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 5, and in some embodiments may comprise a VH sequence which is SEQ ID NO:91 or a humanized variant thereof and a VL sequence which is SEQ ID NO:92 or a humanized variant thereof.

[0080] The antibody designated C-14D12 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact, or IMGT as shown in Table 6, and in some embodiments may comprise a VH sequence which is SEQ ID NO: 109 or a humanized variant thereof and a VL sequence which is SEQ ID NO: 110 or a humanized variant thereof. (Table 1: Antibody A-15B08) [Table 2] The NST in VH CDR2 is alternatively NSA or X1SX2, where X1 is N, Q, S, or A and / or X2 is A or T. (Table 2: Antibody A2-3B06) [Table 3] (Table 3: Antibody A2-5D10) [Table 4] NST in VH CDR2 is alternatively NSA or X1SX2, where X1 is N, Q, S, or A and / or X2 is A or T. C in VL CDR1 is alternatively S, A, or V. (Table 4: Antibody A2-7A05) [Table 5] (Table 5: Antibody A2-7F01) [Table 6] (Table 6: Antibody C-14D12) [Table 7]

[0081] In some embodiments, the α5β1 integrin binding agent, such as the human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises a VH region or a VH domain. In another embodiment, the α5β1 integrin binding agent, such as the human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises a VL region or a VL domain. In some embodiments, the α5β1 integrin binding agent, such as the human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), has a combination of (i) a VH domain or a VH region; and / or (ii) a VL domain or a VL region.

[0082] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises a heavy chain having a combination of (i) a VH domain that includes the CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT as set forth in any one of Tables 1-6; and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any of the VH domains described herein, as well as the following: [ka] Another exemplary IgG heavy chain comprises any VH domain having the CDRs described herein, as well as the following: [ka] An exemplary Fc sequence includes the CH1, hinge, CH2, and CH3 amino acid sequences of:

[0083] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises a light chain having a combination of: (i) a VL domain comprising the CDRs set forth in any one of Tables 1-6; and (ii) a light chain constant domain (CL). Exemplary light chains (e.g., for pairing with an IgG heavy chain) include any VL domain having the CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT described herein, and the following: [ka] The CL amino acid sequence of

[0084] In some embodiments, an alpha5beta1 integrin binding agent (e.g., an antibody, such as a bispecific antibody), such as a human alpha5beta1 integrin binding agent described herein, comprises: (a) a heavy chain having a combination of (i) a VH domain having CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT as set forth in any one of Tables 1-6, and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL domain having CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT as set forth in any one of Tables 1-6, and (ii) a light chain constant domain (CL).

[0085] In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more CDRs (such as six CDRs) identified in Table 1, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more (including six) CDRs identified in Table 2, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more (including six) CDRs identified in Table 3, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more (including six) CDRs identified in Table 4, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more (including six) CDRs identified in Table 5, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody), comprises one or more (including six) CDRs identified in Table 6, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3.In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody such as a bispecific antibody), comprises one or more (including six) CDRs identified in Tables 1, 2, 3, 4, 5, and / or 6, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3.

[0086] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 1. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 1. In yet another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 1, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 1, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0087] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 2. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 2. In yet another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 2, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 2, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0088] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 3. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 3. In yet another embodiment, the α5β1 integrin binding agent, such as the human α5β1 integrin binding agent described herein (e.g., an antibody such as a bispecific antibody), comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 3, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 3, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0089] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 4. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 4. In yet another embodiment, the α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody such as a bispecific antibody), comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 4, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 4, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0090] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 5. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 5. In yet another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 5, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 5, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0091] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three VH CDRs), e.g., VH CDR1, VH CDR2, and / or VH CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 6. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody), such as a human α5β1 integrin binding agent described herein, comprises one or more CDRs (including three CDRs), e.g., VL CDR1, VL CDR2, and / or VL CDR3, according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 6. In yet another embodiment, the α5β1 integrin binding agent, such as a human α5β1 integrin binding agent described herein (e.g., an antibody such as a bispecific antibody), comprises one or more CDRs (including three VH CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 6, e.g., VH CDR1, VH CDR2, and / or VH CDR3, and one or more CDRs (including three VL CDRs) according to Kabat and / or Chothia, AbM, Contact, or IMGT listed in Table 6, e.g., VL CDR1, VL CDR2, and / or VL CDR3.

[0092] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise two or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise three or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise four or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise five or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise six or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108.

[0093] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-6. In another embodiment, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., one, two, or three) VL CDRs listed in Tables 1-6. In yet another embodiment, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., one, two, or three) VH CDRs listed in Tables 1-6, and one or more VL CDRs listed in Tables 1-6. Thus, in some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a VH CDR1 having the amino acid sequence of any one of SEQ ID NOs: 1, 7, 12, 13, 18, 27, 31, 34, 35, 38, 53, 59, 64, 65, 70, 93, 97, 100, 101, and 105. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a VH CDR2 having the amino acid sequence of any one of SEQ ID NOs: 2, 8, 14, 19, 24, 28, 54, 60, 66, 71, 76, 79, 82, 84, 87, and 90. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH CDR3 having an amino acid sequence of any one of SEQ ID NOs: 3, 9, 15, 20, 29, 32, 36, 39, 55, 61, 67, 72, 80, 83, 85, 88, 94, 98, 102, and 106. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH CDR1 and / or a VH CDR2 and / or a VH CDR3 independently selected from the VH CDR1, VH CDR2, VH CDR3 described in any one of the amino acid sequences described in Tables 1-6.In some embodiments, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a VL CDR1 having the amino acid sequence of any one of SEQ ID NOs: 4, 10, 16, 21, 30, 33, 37, 40, 44, 46, 47, 49, 56, 62, 68, 73, 95, 99, 103, and 107. In another embodiment, the α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise a VL CDR2 having the amino acid sequence of any one of SEQ ID NOs: 5, 11, 22, 41, 57, 63, and 74. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a VL CDR3 having an amino acid sequence of any one of SEQ ID NOs: 6, 17, 23, 45, 48, 50, 58, 69, 75, 81, 86, 89, 96, 104, and 108. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody such as a bispecific antibody) described herein comprises a VL CDR1 and / or a VL CDR2 and / or a VL CDR3 independently selected from the VL CDR1, VL CDR2, and VL CDR3 set forth in any one of the amino acid sequences set forth in Tables 1 to 6.

[0094] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27, 53, or 93; (ii) SEQ ID NO: 7, 31, 59, or 97; (iii) SEQ ID NO: 12, 34, 64, or 100; (iv) SEQ ID NO: 13, 35, 65, or 101; and (v) SEQ ID NO: 18, 38, 70, or 105; (2) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28, 54, or 79; (ii) SEQ ID NO: 8, 60, or 82; (iii) SEQ ID NO: 14, 66, or 84; (iv) SEQ ID NO: 19, 71, or 87; and (v) SEQ ID NO: 24, 76; or 90. and / or: (1) a heavy chain variable (VH) region comprising a VH CDR2 and a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29, 55, 80, or 94; (ii) SEQ ID NO: 9, 32, 61, 83, or 98; (iii) SEQ ID NO: 15, 36, 67, 85, or 102; and / or: (1) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, 30, 44, 56, or 95; (ii) SEQ ID NO: 10, 33, 46, 62, or 99; (iii) SEQ ID NO: 16, 37, 47, 68, or 103; and (iv) SEQ ID NO: 21, 40, 49, 73, or 107. CDR1; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:5 or 57, (ii) SEQ ID NO:11 or 63, and (iii) SEQ ID NO:22, 41, or 74; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:6, 45, 58, 81, or 96, (ii) SEQ ID NO:17, 48, 69, 86, or 104, and (iii) SEQ ID NO:23, 50, 75, 89, or 108.

[0095] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27, 53, or 93; (ii) SEQ ID NO: 7, 31, 59, or 97; (iii) SEQ ID NO: 12, 34, 64, or 100; (iv) SEQ ID NO: 13, 35, 65, or 101; and (v) SEQ ID NO: 18, 38, 70, or 105; (2) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28, 54, or 79; (ii) SEQ ID NO: 8, 60, or 82; (iii) SEQ ID NO: 14, 66, or 84; (iv) SEQ ID NO: 19, 71, or 87; and (v) SEQ ID NO: 24, 76; or 90. and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29, 55, 80, or 94; (ii) SEQ ID NO: 9, 32, 61, 83, or 98; (iii) SEQ ID NO: 15, 36, 67, 85, or 102; and (iv) SEQ ID NO: 20, 39, 72, 88, or 106.

[0096] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, 30, 44, 56, or 95, (ii) SEQ ID NO: 10, 33, 46, 62, or 99, (iii) SEQ ID NO: 16, 37, 47, 68, or 103, and (iv) SEQ ID NO: 21, 40, 49, 73, or 107; (2) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5 or 57, (ii) SEQ ID NO: 11 or 63, and (iii) SEQ ID NO: 22, 41, or 74. and (3) a light chain variable (VL) region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 6, 45, 58, 81, or 96, (ii) SEQ ID NO: 17, 48, 69, 86, or 104, and (iii) SEQ ID NO: 23, 50, 75, 89, or 108.

[0097] In some embodiments, described herein is an antibody or fragment thereof that binds to α5β1 integrin, comprising: (a): (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27, 53, or 93; (ii) SEQ ID NO: 7, 31, 59, or 97; (iii) SEQ ID NO: 12, 34, 64, or 100; (iv) SEQ ID NO: 13, 35, 65, or 101; and (v) SEQ ID NO: 18, 38, 70, or 105; (2) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28, 54, or 79; (ii) SEQ ID NO: 8, 60, or 82; (iii) SEQ ID NO: 14, 66, or 84; (iv) SEQ ID NO: 19, 71, or 87; and (v) SEQ ID NO: 24, 76; or 90. and / or: (1) a heavy chain variable (VH) region comprising a VH CDR2 and a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29, 55, 80, or 94, (ii) SEQ ID NO: 9, 32, 61, 83, or 98, (iii) SEQ ID NO: 15, 36, 67, 85, or 102, and (iv) SEQ ID NO: 20, 39, 72, 88, or 106; and / or: (1) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, 30, 44, 56, or 95, (ii) SEQ ID NO: 10, 33, 46, 62, or 99, (iii) SEQ ID NO: 16, 37, 47, 68, or 103, and (iv) SEQ ID NO: 21, 40, 49, 73, or 107. (2) a VL CDR2 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:5 or 57, (ii) SEQ ID NO:11 or 63, and (iii) SEQ ID NO:22, 41, or 74; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:6, 45, 58, 81, or 96, (ii) SEQ ID NO:17, 48, 69, 86, or 104, and (iii) SEQ ID NO:23, 50, 75, 89, or 108.

[0098] In some embodiments, described herein is an antibody or fragment thereof that binds to α5β1 integrin, comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 1, 27, 53, or 93; (ii) SEQ ID NO: 7, 31, 59, or 97; (iii) SEQ ID NO: 12, 34, 64, or 100; (iv) SEQ ID NO: 13, 35, 65, or 101; and (v) SEQ ID NO: 18, 38, 70, or 105; (2) a VH CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, 28, 54, or 79; (ii) SEQ ID NO: 8, 60, or 82; (iii) SEQ ID NO: 14, 66, or 84; (iv) SEQ ID NO: 19, 71, or 87; and (v) SEQ ID NO: 24, 76; or 90. and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 3, 29, 55, 80, or 94; (ii) SEQ ID NO: 9, 32, 61, 83, or 98; (iii) SEQ ID NO: 15, 36, 67, 85, or 102; and (iv) SEQ ID NO: 20, 39, 72, 88, or 106.

[0099] In some embodiments, described herein is an antibody or fragment thereof that binds to α5β1 integrin, comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 4, 30, 44, 56, or 95, (ii) SEQ ID NO: 10, 33, 46, 62, or 99, (iii) SEQ ID NO: 16, 37, 47, 68, or 103, and (iv) SEQ ID NO: 21, 40, 49, 73, or 107; (2) a VL CDR1 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 5, or 57, (ii) SEQ ID NO: 11, or 63, and (iii) SEQ ID NO: 22, 41, or 74. and (3) a light chain variable (VL) region comprising a VL CDR3 having an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 6, 45, 58, 81, or 96, (ii) SEQ ID NO: 17, 48, 69, 86, or 104, and (iii) SEQ ID NO: 23, 50, 75, 89, or 108.

[0100] In some embodiments, described herein are: (i) an antibody designated A-15B08 comprising a VH sequence which is SEQ ID NO:25 or a humanized variant thereof and a VL sequence which is SEQ ID NO:26 or a humanized variant thereof; (ii) an antibody designated A2-3B06 comprising a VH sequence which is SEQ ID NO:42 or a humanized variant thereof and a VL sequence which is SEQ ID NO:43 or a humanized variant thereof; (iii) an antibody designated A2-5D10 comprising a VH sequence which is SEQ ID NO:51 or a humanized variant thereof and a VL sequence which is SEQ ID NO:52 or a humanized variant thereof; (iv) an antibody designated A2-5D10 comprising a VH sequence which is SEQ ID NO:77 or a humanized variant thereof. and a humanized variant of SEQ ID NO: 78 or a humanized variant thereof; (v) an antibody designated A2-7F01 comprising a VH sequence of SEQ ID NO: 91 or a humanized variant thereof and a VL sequence of SEQ ID NO: 92 or a humanized variant thereof; or (vi) an antibody or fragment thereof that binds to α5β1 integrin comprising all three heavy chain complementarity determining regions (CDRs) and / or all three light chain CDRs from an antibody designated C-14D12 comprising a VH sequence of SEQ ID NO: 109 or a humanized variant thereof and a VL sequence of SEQ ID NO: 110 or a humanized variant thereof. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A-15B08 (by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-3B06 (by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-5D10 (by Kabat and / or Chothia, AbM, Contact, or IMGT).In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody named A2-7A05 (by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody named A2-7F01 (by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody named C-14D12 (by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the antibody or fragment thereof competes for said binding with an antibody or fragment thereof comprising: (i) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A-15B08 (see, e.g., Table 1); (ii) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A2-3B06 (see, e.g., Table 2); (iii) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A2-5D10 (see, e.g., Table 3); or (iv) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated C-14D12 (see, e.g., Table 6). In some embodiments, the antibody or fragment thereof competes for said binding with an antibody or fragment thereof that comprises: (i) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A2-7A05 (see, e.g., Table 4), or (ii) all three heavy chain CDRs and / or all three light chain CDRs from an antibody designated A2-7F01 (see, e.g., Table 5).

[0101] In some embodiments, described herein is an antibody or fragment thereof that binds to α5β1 integrin, the antibody comprising: (a) a heavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VH CDR3 amino acid sequence set forth in Tables 1-6; and / or (b) a light chain variable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 amino acid sequence set forth in Tables 1-6. In some embodiments, the antibody comprises a heavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VH CDR3 amino acid sequence set forth in Tables 1-6. In some embodiments, the antibody comprises a light chain variable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 amino acid sequence set forth in Tables 1-6.

[0102] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0103] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:1; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:2; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:4; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0104] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:7; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:8; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:9; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:10; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0105] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:12; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:2; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:4; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0106] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 16; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0107] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:18; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:19; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:20; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:21; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:22; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:23.

[0108] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:1; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:24; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:4; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0109] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35, and 38; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 19, 24, and 28; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 32, 36, and 39; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 33, 37, and 40; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 41, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23.

[0110] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:27; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:28; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:29; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:30; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0111] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:31; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:8; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:32; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:33; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0112] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:34; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:28; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:29; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:30; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0113] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:35, (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:14; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:36; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:37; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:17.

[0114] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:38; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:19; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:39; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:40; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:41; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:23.

[0115] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:27; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:24; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:29; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:30; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:6.

[0116] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 12, 13, and 18; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 46, 47, and 49; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22; and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 48, and 50.

[0117] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:1; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:2; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:44; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:45.

[0118] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:7; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:8; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:9; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:46; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:45.

[0119] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:12; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:2; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:44; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:45.

[0120] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 13; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 47; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 48.

[0121] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:18; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:19; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:20; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:49; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:22; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:50.

[0122] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:1; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:24; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:3; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:44; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:45.

[0123] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 59, 64, 65, and 70; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 60, 66, 71, and 76; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 61, 67, and 72; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 62, 68, and 73; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 63, and 74, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 69, and 75.

[0124] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:53; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:54; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:55; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:58.

[0125] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:59; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:60; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:61; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:62; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:63; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:58.

[0126] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:64; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:54; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:55; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:58.

[0127] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:65; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:66; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:67; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:68; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:63; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:69.

[0128] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:70; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:71; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:72; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:73; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:74; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:75.

[0129] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:53; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:76; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:55; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:58.

[0130] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 59, 64, 65, and 70; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 82, 84, 87, and 90; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 83, 85, and 88; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 62, 68, and 73; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 63, and 74, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 86, and 89.

[0131] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:53; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:79; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:80; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:81.

[0132] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:59; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:82; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:83; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:62; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:63; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:81.

[0133] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:64; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:79; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:80; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:81.

[0134] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:65; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:84; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:85; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:68; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:63; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:86.

[0135] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:70; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:87; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:88; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:73; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:74; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:89.

[0136] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:53; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:90; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:80; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:56; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:57; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:81.

[0137] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 93, 97, 100, 101, and 105; (2) a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 19, 24, and 28; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 94, 98, 102, and 106; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 95, 99, 103, and 107; (2) a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22, and (3) a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 96, 104, and 108.

[0138] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:93; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:28; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:94; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:95; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:96.

[0139] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:97; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:8; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:98; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:99; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:96.

[0140] In some embodiments, described herein are antibodies comprising: (a) a heavy chain. In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 100; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 94; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 95; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 96.

[0141] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 101; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 102; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 103; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 104.

[0142] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 105; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 19; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 106; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 107; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 108.

[0143] In some embodiments, described herein are antibodies comprising: (a): a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO:93; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO:24; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO:94; and (b): a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO:95; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO:96.

[0144] In some embodiments, described herein are antibodies comprising the VH and / or VL regions described herein, further comprising human framework sequences. In some embodiments, the VH and / or VL regions further comprise framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

[0145] In some embodiments, the antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized, human, or chimeric antibodies. In some embodiments, the antibodies described herein are Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single chain antibody molecules, dual variable region antibodies, single variable region antibodies, linear antibodies, V regions, or multispecific antibodies formed from antibody fragments.

[0146] In some embodiments, the CDRs disclosed herein comprise consensus sequences derived from a group of related antibodies (see, e.g., Tables 1-6). As described herein, "consensus sequence" refers to an amino acid sequence that has conserved amino acids common among several sequences and variable amino acids that differ within a given amino acid sequence. Exemplary CDR consensus sequences provided include CDRs corresponding to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and / or CDRL3. Exemplary consensus sequences of CDRs of α5β1 integrin binding agents (e.g., antibodies such as bispecific antibodies) are shown in Figures 2A and 2B. In some embodiments, the CDRs disclosed herein comprise exemplary consensus sequences derived from a group of related antibodies (see, e.g., Tables 1-6, e.g., the first exemplary group of Tables 1, 2, 3, and 6 and the second exemplary group of Tables 4 and 5). Exemplary consensus sequences for the CDRs of an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) are shown in Figures 2A and 2B. Thus, in some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein has the following CDR: (a): (1) amino acid sequence [ka] (wherein X1 is a naturally occurring amino acid (e.g., S, T, or D); (2) [ka] (wherein each (or either) of X1 and / or X2 is independently a naturally occurring amino acid (e.g., X1 is N, Q, S, or A and / or X2 is A or T); and (3) [ka] (wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and / or X 10 Each (or any) of is independently a naturally occurring amino acid (e.g., X1 is Y, G, or A, X2 is D, G, or P, X3 is Y, L, or S, X4 is D, L, or F, X5 is G, R, or I, X6 is D or R, X7 is W, D, or Y, X8 is F, A, or G, X9 is absent, M, or S, and / or X 10 (b): (1) a heavy chain variable (VH) region comprising a VH CDR3 having the amino acid sequence [ka] (wherein each (or any) of X1, X2, X3, X4, and / or X5 is independently a naturally occurring amino acid (e.g., X1 is R or S, X2 is S or T, X3 is N, R, or S, X4 is S, F, C, A, or V, and / or X5 is L or F); (2) a VL CDR1 having the amino acid sequence [ka] and (3) a VL CDR2 having the amino acid sequence [ka] (wherein X1 is a naturally occurring amino acid (e.g., L, Y, or H). Thus, in some embodiments, an α5β1 integrin binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a light chain variable (VL) region comprising a VL CDR3 having the amino acid sequence: [ka] (2) VH CDR1 having the following structure: [ka] (wherein each (or any) of X1, X2, X3, and / or X4 is a naturally occurring amino acid (e.g., X1 is K or N, X2 is I or S, X3 is D or N, and / or X4 is S or T); and (3) a VH CDR2 having an amino acid sequence of [ka] (wherein X1 is a naturally occurring amino acid (e.g., X1 is absent, L, or F); and / or (b): (1) a heavy chain variable (VH) region comprising a VH CDR3 having the amino acid sequence [ka] (2) a VL CDR1 having the amino acid sequence [ka] and (3) a VL CDR2 having the amino acid sequence [ka] (wherein each (or either) of X1 and / or X2 is a naturally occurring amino acid (e.g., X1 is F or L and / or X2 is T or G)).

[0147] In some embodiments, described herein is a binding agent (e.g., an antibody) that binds to essentially the same epitope as any one of the antibodies described herein or a fragment thereof. In some embodiments, described herein is a binding agent (e.g., an antibody) that competes with any one of the antibodies described herein or a fragment thereof for binding to human α5β1 integrin. In some embodiments, the binding agent is an antibody or a fragment thereof.

[0148] In some embodiments, the CDRs of an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, can be determined by the Kabat system (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al., (1991) Sequences of proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242.).

[0149] In certain aspects, the CDRs of an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, can be determined by the Chothia system, herein referred to as "Chothia CDRs" (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1):175-82; and U.S. Patent No. 7,709,226).

[0150] In one aspect, the CDRs of an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, can be determined by the ImMunoGeneTics (IMGT) system, e.g., as described in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212, and are referred to herein as "IMGT CDRs."

[0151] In certain embodiments, the CDRs of an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, can be determined by the AbM system, e.g., as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745, herein referred to as "AbM CDRs." See also, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," Chapter 31, pp. 422-439, in Antibody Engineering, edited by Kontermann and Dubel, Springer-Verlag, Berlin (2001).

[0152] In some embodiments, the CDRs of an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, can be determined by the Contact system, and are referred to herein as "Contact CDRs" (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5: 732-745). Contact CDRs are based on the analysis of available complex crystal structures.

[0153] In some embodiments, one or more CDR positions along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein (see, e.g., the CDRs by Kabat and / or Chothia, AbM, Contact, or IMGT in Tables 1-6) may be altered (e.g., by one or more amino acid modifications) by only 1, 2, 3, 4, 5, or 6 amino acid positions, so long as binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the positions defining any of the CDRs in Tables 1, 2, 3, 4, 5, or 6 (by Kabat and / or Chothia, AbM, Contact, or IMGT) may be altered by shifting the N-terminal and / or C-terminal boundaries of that CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the current CDR positions, so long as binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In another embodiment, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein (see, e.g., the CDRs by Kabat and / or Chothia, AbM, Contact, or IMGT in Tables 1-6) may be altered (e.g., shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be 1, 2, 3, 4, 5 or more amino acids shorter than one or more of the CDRs set forth in SEQ ID NOs:1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs set forth in SEQ ID NOs:1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to alpha5beta1 integrin (e.g., human alpha5beta1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In another embodiment, the amino terminus of CDR1, CDR2, and / or CDR3 of a VH and / or VL described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to alpha5beta1 integrin (e.g., human alpha5beta1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the carboxy terminus of CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to alpha5beta1 integrin (e.g., human alpha5beta1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the amino terminus of CDR1, CDR2, and / or CDR3 of the VH and / or VL described herein may be shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs set forth in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to alpha5beta1 integrin (e.g., human alpha5beta1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In some embodiments, the carboxy terminus of CDR1, CDR2, and / or CDR3 of VH and / or VL described herein may be shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (by Kabat and / or Chothia, AbM, Contact, or IMGT), so long as binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, such as the binding assays and conditions described in the "Examples" section herein, can be used to confirm whether binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained. For example, Example 2 described herein describes an assay for measuring binding to α5β1 integrin (eg, human α5β1 integrin).

[0154] In another embodiment, the α5β1 integrin binding agent (e.g., antibody) presented herein, such as the human α5β1 integrin binding agent that binds to α5β1 integrin, comprises conservative sequence modifications (e.g., one or more amino acid modifications in one or more CDRs as described above).For a polypeptide that is an α5β1 integrin binding agent (e.g., antibody), such as the human α5β1 integrin binding agent, conservative sequence modifications include conservative amino acid substitutions, including those in which an amino acid residue is replaced with an amino acid residue that has a similar side chain.Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, a predicted non-critical amino acid residue in an α5β1 integrin binding agent is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the nucleotide and amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to the CDRs set forth in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6.Thus, for example, each such CDR may contain up to five conservative amino acid substitutions, such as up to four (or less) conservative amino acid substitutions, such as up to three (or less) conservative amino acid substitutions, such as up to two (or less) conservative amino acid substitutions, or as few as one conservative amino acid substitution.

[0155] The present disclosure provides variants of the antibodies described herein (see, e.g., Table 1, Table 3). The antibody designated A-15B08-T62A described in Example 8 below is such an exemplary antibody variant. A-15B08-T62A was generated by replacing the threonine residue at position 62 in CDRH2 of antibody A-15B08 with an alanine to remove a potential N-glycosylation site. The VH, VL, and CDR sequences according to various numbering schemes of A-15B08-T62A are shown in Figures 2C and 2D. More specifically, the antibody designated A-15B08-T62A comprises a VH comprising the amino acid sequence of SEQ ID NO: 135 and a VL comprising the amino acid sequence of SEQ ID NO: 26. The six CDR sequences of A-15B08-T62A according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2C and 2D.

[0156] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 135. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 135; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0157] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 135, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0158] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:135 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0159] If desired, the α5β1 integrin binding agent (e.g., an antibody), including the human α5β1 integrin binding agent, is linked or conjugated (directly or indirectly) to a moiety having an effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or immune mobilizing activity, to form an antibody-drug conjugate (ADC). The moiety that is linked or conjugated (directly or indirectly) includes a cytotoxic or non-cytotoxic drug. Alternatively, or in addition, the α5β1 integrin binding agent (e.g., an antibody), including the human α5β1 integrin binding agent, is optionally linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety that has reporter activity (e.g., a detectable label or reporter). It will be appreciated that the characteristics of the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent described herein, also apply to polypeptides that include α5β1 integrin binding agent fragments.

[0160] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent described herein, is conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or to a diagnostic or detectable agent (e.g., a labeled agent, such as a labeled antibody). The conjugated or recombinantly linked antibody may be useful for diagnosing, treating, and / or preventing α5β1 integrin-mediated diseases, disorders, and conditions, such as, for example, cancer (e.g., cancer associated with or characterized by tumor cells that express or overexpress α5β1 integrin), angiogenesis-mediated diseases (e.g., diseases associated with or characterized by abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases such as MS and ALS).

[0161] Such diagnosis and / or detection (e.g., by diagnostic and / or detectable agents) can, for example, involve the interaction of an α5β1 integrin binding agent (e.g., an antibody) with, for example: an enzyme (such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase); a prosthetic group (such as, but not limited to, streptavidin / biotin or avidin / biotin); a fluorescent material (such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin); a luminescent material (such as, but not limited to, luminol); a bioluminescent material (such as, but not limited to, luciferase, luciferin, or aequorin); a chemiluminescent material (such as, but not limited to, acridinium-based compounds or HALOTAG); a radioactive material (such as, but not limited to, Examples of ions include, but are not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, and 140La. , 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn, etc.); positron emitting metals using various positron emission tomography; and non-radioactive paramagnetic metal ions (e.g., a labeling agent such as a labeled antibody).

[0162] A labeling agent (e.g., a labeled antibody) that specifically binds to α5β1 integrin can be used for diagnostic purposes to detect, diagnose, or monitor α5β1 integrin-mediated diseases, disorders, or conditions. Described herein is a method for detecting α5β1 integrin-mediated diseases, disorders, or conditions, comprising: (a) assaying the expression of α5β1 integrin in a cell or tissue sample of a subject using one or more α5β1 integrin binding agents (e.g., antibodies) that specifically bind to α5β1 integrin as described herein; and (b) comparing the level of α5β1 integrin with a control level (e.g., a level in a normal tissue sample, such as from a patient without an α5β1 integrin-mediated disease, disorder, or condition, or from the same patient before the onset of the disease), whereby an increase in the assayed level of α5β1 integrin compared to the control level of α5β1 integrin indicates an α5β1 integrin-mediated disease, disorder, or condition. Also described herein is a diagnostic assay for diagnosing an α5β1 integrin-mediated disease, disorder, or condition, comprising: (a) assaying for the level of α5β1 integrin in an individual's cell or tissue sample using one or more α5β1 integrin binding agents (e.g., antibodies) that specifically bind to α5β1 integrin as described herein; and (b) comparing the level of α5β1 integrin to a control level (e.g., the level in a normal tissue sample), whereby an increase in the assayed α5β1 integrin level compared to the control level of α5β1 integrin indicates an α5β1 integrin-mediated disease, disorder, or condition.In some embodiments, described herein is a method for treating α5β1 integrin-mediated disease, disorder, or condition in a subject, comprising: (a) using one or more α5β1 integrin binding agents (e.g., antibodies) that specifically bind to α5β1 integrin as described herein, assaying the level of α5β1 integrin in a cell or tissue sample of the subject; and (b) comparing the level of α5β1 integrin with a control level (e.g., the level in a normal tissue sample), whereby an increase in the assayed α5β1 integrin level compared to the control level of α5β1 integrin indicates an α5β1 integrin-mediated disease, disorder, or condition.In some embodiments, the method further comprises (c) administering an effective amount of the α5β1 integrin binding agent (e.g., antibodies) described herein to a subject identified as having the α5β1 integrin-mediated disease, disorder, or condition. A more definitive diagnosis of an α5β1 integrin-mediated disease, disorder, or condition may enable medical professionals to employ preventative or proactive treatments earlier, thereby preventing the onset or further progression of an α5β1 integrin-mediated disease, disorder, or condition.

[0163] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein, is a component in a kit. In some embodiments, the kit includes an α5β1 integrin binding agent (e.g., an antibody) or a composition (e.g., a pharmaceutical composition) including the α5β1 integrin binding agent (e.g., the antibody) packaged in a suitable packaging material. The kit optionally includes a label or insert that includes a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components contained therein.

[0164] Also described herein are α5β1 integrin binding agents (e.g., antibodies) recombinantly linked or conjugated (covalently or non-covalently conjugated, directly or indirectly) to a heterologous protein or polypeptide (or fragment thereof), e.g., a polypeptide (e.g., of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate a fusion protein, and uses thereof. In particular, described herein are fusion proteins comprising an antigen-binding fragment (e.g., comprising CDR1, CDR2, and / or CDR3 of VH and / or VL) of an α5β1 integrin binding agent (e.g., antibody), such as a human α5β1 integrin binding agent, described herein, and a heterologous protein, polypeptide, or peptide. In some embodiments, the heterologous protein, polypeptide, or peptide to which the α5β1 integrin binding agent (e.g., an antibody) is linked is useful for targeting the α5β1 integrin binding agent to a particular cell (e.g., an α5β1 integrin-expressing cell, such as a tumor cell).

[0165] Additionally, α5β1 integrin binding agents (e.g., antibodies), such as the human α5β1 integrin binding agents described herein, can be linked (directly or indirectly) to a marker or "tag" sequence, such as a peptide, to facilitate purification. In some embodiments, the amino acid sequence of the marker or tag is a hexa-histidine peptide, such as the tag provided in the pQE vector (see, e.g., QIAGEN), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine allows for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the "FLAG" tag.

[0166] Methods for linking or conjugating (directly or indirectly) moieties (polypeptides) to antibodies are well known in the art, any one of which can be used to generate the antibody-drug conjugates or fusion proteins described herein.

[0167] In some embodiments, the α5β1 integrin binding agent (e.g., antibody) described herein is a fusion protein. The term "fusion protein" as used herein refers to a polypeptide that includes the amino acid sequence of a binding agent (e.g., antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not normally part of the antibody (e.g., a non-α5β1 integrin binding antibody)). In some embodiments, the fusion protein maintains the biological activity of the α5β1 integrin binding agent. In some embodiments, the fusion protein includes the VH region, VL region, VH CDR (one, two, or three VH CDRs), and / or VL CDR (one, two, or three VL CDRs) of an α5β1 integrin antibody, where the fusion protein is linked to an α5β1 integrin epitope, an α5β1 integrin fragment, and / or an α5β1 integrin polypeptide.

[0168] Fusion proteins can be generated, for example, by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be employed to alter the activity of an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein, including, for example, an α5β1 integrin binding agent with higher affinity and lower dissociation rate. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent, can be modified by subjecting it to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an α5β1 integrin binding agent described herein can be recombined with one or more components, motifs, sections, portions, domains, fragments, etc., of one or more heterologous molecules.

[0169] The α5β1 integrin binding agents (e.g., antibodies), such as the human α5β1 integrin binding agents described herein, may also be attached to solid supports, which are useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0170] An α5β1 integrin binding agent (e.g., an antibody) described herein, such as a human α5β1 integrin binding agent, can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0171] The linker may be a "cleavable site" that facilitates release of the linked or conjugated agent within the cell, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates (e.g., antibody-drug conjugates) of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0172] Conjugates of antibodies and drugs, including those in which the drug is a drug for the preparation of ADCs, can be made using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). Additionally, the present disclosure contemplates that conjugates of antibodies and drugs, including those in which the drug is a drug for the preparation of ADCs, can be prepared using any suitable method disclosed in the art (see, for example, Bioconjugate Techniques, eds. Hermanson et al., 2nd ed., 2008).

[0173] Conventional conjugation strategies for antibodies and drugs, including those where the drug is a drug for the preparation of ADCs, are based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, resulting in heterogeneous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. These include engineering "thiomabs" that contain cysteine ​​substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not interfere with immunoglobulin folding and assembly or alter the antigen. In another method, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from a stop to a selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids.

[0174] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent described herein, is conjugated to a cytotoxic agent. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent disclosed herein, can be optionally conjugated to one or more cytotoxic agents disclosed herein or known in the art to generate an ADC. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, such as, but not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. In some embodiments, the cytotoxic agent is a radioisotope, resulting in a radioconjugate or radioconjugated drug. A variety of radionuclides are available for the production of radioconjugated drugs, including, but not limited to, 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re, and 212Bi.Conjugates of a polypeptide or molecule with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothenes, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used. Conjugates of polypeptides or molecules with cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), as well as bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0175] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent described herein, is conjugated to a drug, such as a signal transduction modulator, a proapoptotic agent, a mitotic inhibitor, an antitumor antibiotic, an immunomodulatory agent, a nucleic acid for gene therapy, an alkylating agent, an antiangiogenic agent, an antimetabolite, a boron-containing agent, a chemoprotective agent, a hormonal agent, an antihormonal agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor. In some embodiments, the mitotic inhibitor is a dolastatin, an auristatin, a maytansinoid, and a plant alkaloid. In some embodiments, the drug is a dolastatin, an auristatin, a maytansinoid, and a plant alkaloid. An example of an auristatin is monomethylaurisatin F (MMAF) or monomethylauristatin E (MMAE). Examples of maytansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In some embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracycline, calicheamicin, and duocarmycin. In some embodiments, the actinomycin is a pyrrolobenzodiazepine (PBD).

[0176] An α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein, can be monospecific, bispecific, trispecific, or of higher multispecificity. Such an agent can include an antibody. A multispecific antibody, such as a bispecific antibody, is a monoclonal antibody that has binding specificity for at least two different targets (e.g., α5β1 integrin and αv integrin) or two different epitopes on the same target (e.g., a bispecific antibody directed to α5β1 integrin having a first binding domain for a first epitope of α5β1 integrin and a second binding domain for a second epitope of α5β1 integrin). In some embodiments, a multispecific (e.g., bispecific) antibody can be constructed based on the sequence of an antibody described herein, for example, the CDR sequence of Table 1, Table 2, Table 3, Table 4, Table 5, and / or Table 6. In some embodiments, a multispecific antibody described herein is a bispecific antibody. In some embodiments, the bispecific antibody is a murine, chimeric, human, or humanized antibody. In some embodiments, one of the binding specificities of the multispecific antibody is for α5β1 integrin and the other is for any other target (e.g., αvβ3 integrin). In some embodiments, the multispecific (e.g., bispecific) antibody can comprise two or more target (e.g., antigen) binding domains, where the different binding domains are specific for separate targets (e.g., a first binding domain that binds to α5β1 integrin and a second binding domain that binds to another target (e.g., αvβ3 integrin). In some embodiments, the multispecific (e.g., bispecific) antibody molecule can bind to more than one (e.g., two or more) epitopes on the same target (e.g., α5β1 integrin).

[0177] Methods for producing multispecific antibodies, such as by co-expression of two immunoglobulin heavy-chain light-chain pairs, in which the two heavy chains have different specificities, are known in the art (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies, edited by Kontermann (2011).

[0178] Exemplary structures of multispecific antibodies are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10: 1-18; Brinkman et al., 2017, MABS, 9:2, 182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28:3, 251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0179] For example, bispecific antibody molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen binding sites added; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. As non-limiting examples, BsIgG formats can include crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab.

[0180] In some embodiments, BslgG comprises a heavy chain engineered to heterodimerize. For example, the heavy chain can be engineered to heterodimerize using the "knobs-into-holes", SEED platform, common heavy chain (e.g., in κλ-body) strategy, and by using a heterodimeric Fc region. Strategies to avoid homodimeric heavy chain pairing in BsIgG, such as knobs-into-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity, are known in the art.

[0181] Another bispecific antibody format is an IgG with additional antigen-binding sites added. For example, a monospecific IgG can be engineered to have bispecificity by adding additional antigen-binding units onto the monospecific IgG, for example at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Non-limiting examples of added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al., Mol. Immunol. 67(2015):95-106. In some embodiments, an exemplary antibody format is a B-body format for monospecific or multispecific (e.g., bispecific antibodies), e.g., as described in International Patent Application Publication No. WO2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0182] Bispecific antibody fragments (BsAbs) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In some embodiments, bispecific antibody fragments contain heavy and light chain regions connected by a peptide linker that allows efficient expression of the BsAb in a single host cell. Non-limiting examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, sc diabodies, sc diabody-CH3, diabody-CH3, triple bodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies.

[0183] A bispecific fusion protein comprises an antibody fragment linked to another protein. For example, a bispecific fusion protein can be linked to another protein to add additional specificity and / or functionality. In some embodiments, a dock-and-lock (DNL) method can be used to create a bispecific antibody molecule with higher valency. For example, bispecific antibody fusions to albumin binding proteins or human serum albumin can extend the serum half-life of the antibody fragment. In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. Exemplary bispecific antibody conjugates include the CovX body format, in which a low molecular weight drug is site-specifically conjugated to a single reactive lysine on each Fab arm or antibody or fragment thereof. In some embodiments, the conjugation improves serum half-life.

[0184] Methods for producing multispecific antibodies (including bispecific antibodies) are known in the art. For example, multispecific antibodies (including bispecific antibodies) can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly, or by expression of the component antibodies in a single host cell. Purification of multispecific (e.g., bispecific) antibody molecules can be performed by a variety of methods known in the art, such as affinity chromatography.

[0185] In some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent disclosed herein, can be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, in some embodiments, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent, can be provided in any antibody format disclosed herein or known in the art. As a non-limiting example, the α5β1 integrin binding agent (e.g., an antibody), such as the human α5β1 integrin binding agent, can be provided in any antibody format disclosed herein or known in the art. The antibody derivatives can be selected from (CreativeBiolabs); Duobody platform (Genmab); Dock-and-Lock platform; Knobs-into-Hole (KIH) platform; humanized bispecific IgG antibody (REGN1979) (Regeneron); Mab2 bispecific antibody (F-Star); DVD-lg = dual variable domain immunoglobulin (AbbVie); kappa-lambda body; TBTI = tetravalent bispecific tandem Ig; and CrossMab (Roche).

[0186] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain and one or more additional binding domains that bind to one or more targets that are not α5β1 integrin (e.g., αv integrins). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain that comprises the CDRs of the VH and / or VL amino acid sequences of Table 1 (according to Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain that comprises the CDRs of the VH and / or VL amino acid sequences of Table 2 (according to Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain that comprises the CDRs of the VH and / or VL amino acid sequences of Table 3 (according to Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain that comprises the CDRs of the VH and / or VL amino acid sequences of Table 4 (according to Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain that comprises the CDRs of the VH and / or VL amino acid sequences of Table 5 (according to Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, the multispecific (e.g., bispecific) antibody disclosed herein comprises an α5β1 integrin binding domain comprising the CDRs of the VH and / or VL amino acid sequences of Table 6 (according to Kabat and / or Chothia, AbM, Contact, or IMGT).

[0187] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs listed in Table 1 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs listed in Table 2 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs listed in Table 3 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs listed in Table 4 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 by Kabat and / or Chothia, AbM, Contact, or IMGT). In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs set forth in Table 5 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 according to Kabat and / or Chothia, AbM, Contact, or IMGT).In some embodiments, described herein are multispecific (e.g., bispecific) antibodies that comprise a binding domain that binds to α5β1 integrin that comprises the VH and VL CDRs set forth in Table 6 (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 according to Kabat and / or Chothia, AbM, Contact, or IMGT).

[0188] Antibodies that bind to α5β1 integrin may be obtained by any suitable method, including, but not limited to, immunization with whole tumor cells containing α5β1 integrin and recovery of antibodies, recombinant techniques, or screening a library of antibodies or antibody fragments using an α5β1 integrin extracellular domain epitope. Monoclonal antibodies can be made using a variety of known techniques (see, e.g., Coligan et al., eds., Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991); Kennett, McKearn, and Bechtol, eds., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, (1980); Harlow and Lane, eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," p. 93 in Glover et al., 2nd ed., DNA Cloning 2: Expression Systems, Oxford University Press 1995). See, for example, "Antibody Production by Immunization of Human α5β1 Integrin Antigens," in ...

[0189] In some embodiments, monoclonal antibodies or antibody fragments can be isolated from an antibody phage library, such as those described herein. In some embodiments, an antibody phage library can be generated using techniques described, for example, in "Antibody Phage Display: Methods and Protocols," edited by PM O'Brien and R. Aitken, Humana Press, Totawa NJ, 2002. In some embodiments, antibody clones can be selected by screening a phage library. A phage library can contain phages displaying various fragments (e.g., Fab, scFv) of an antibody variable region (Fv) fused to a phage coat protein. Such a phage library is screened for antibodies against a desired antigen. Clones expressing Fv fragments (e.g., Fab, scFv) capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles.

[0190] For example, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994), variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked via a short, flexible peptide, or as Fab fragments in which they are fused to constant domains, respectively, and interact non-covalently.

[0191] For example, as described in Winter et al., supra, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding clones. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies to a wide range of non-self antigens as well as self antigens without any immunization, as described, for example, by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding highly variable CDR3 regions and achieving in vitro rearrangement, e.g., as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0192] Screening of the library can be accomplished by various techniques known in the art. For example, α5β1 integrin (e.g., α5β1 integrin polypeptide, fragment, or epitope) can be used to coat the wells of an adsorption plate, expressed on host cells immobilized on the adsorption plate or used for cell sorting, or conjugated to biotin for capture on streptavidin-coated beads, or any other method for panning the display library. The use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and WO92 / 09690, and low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992) can facilitate the selection of antibodies with slow dissociation kinetics (e.g., good binding affinity).

[0193] An α5β1 integrin binding agent (e.g., an antibody) can be obtained by designing an appropriate antigen screening procedure to select a phage clone of interest and then constructing a full-length α5β1 integrin binding agent (e.g., an antibody) clone using the VH and / or VL sequences (e.g., Fv sequences) from the phage clone of interest, or various CDR sequences from the VH and VL sequences, and appropriate constant region (e.g., Fc) sequences as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0194] Similarly, human antibodies that bind to α5β1 integrin can be made by any of a number of techniques, including, but not limited to, Epstein-Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from a human immunoglobulin V-region phage library, or other procedures known in the art and based on the disclosure herein. Methods for obtaining human antibodies from transgenic animals are further described, for example, in Bruggemann et al., Curr. Opin. Biotechnol., 8: 455-58, 1997; Jakobovits et al., Ann. NY Acad. Sci., 764: 525-35. 1995; Green et al., Nature Genet., 7: 13-21, 1994; Lonberg et al., Nature, 368: 856-859, 1994; Taylor et al., Int. Immun. 6: 579-591, 1994; and U.S. Patent No. 5,877,397.

[0195] For example, human antibodies that bind to α5β1 integrin can be obtained from transgenic animals that have been engineered to produce specific human antibodies in response to antigenic challenge. For example, International Patent Publication No. WO 98 / 24893 discloses transgenic animals with human Ig loci that do not produce functional endogenous immunoglobulins due to inactivation of endogenous heavy and light chain loci. Transgenic non-primate mammalian hosts capable of mounting an immune response to immunogens have also been described, where the antibodies have primate constant and / or variable regions, and the endogenous immunoglobulin encoding loci have been replaced or inactivated. For example, International Patent Publication No. WO 96 / 30498 discloses the use of the Cre / Lox system to modify the immunoglobulin loci of a mammal, e.g., to replace all or part of the constant or variable regions, resulting in the formation of modified antibody molecules. For example, International Patent Publication No. WO 94 / 02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus. For example, U.S. Patent No. 5,939,598 discloses a method for producing a transgenic mouse lacking endogenous heavy chains and expressing a foreign immunoglobulin locus containing one or more heterologous constant regions. Using transgenic animals such as those described herein, an immune response can be generated against a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human-derived monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art and are used, for example, in immunizing transgenic mice, as described, for example, in International Patent Publication No. WO 96 / 33735. Monoclonal antibodies can be tested for their ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.

[0196] The present disclosure provides a humanized antibody that binds to α5β1 integrin, such as human α5β1 integrin. A variety of methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from an "import" variable domain. Humanized antibodies that bind α5β1 integrin can be produced using techniques known to those of skill in the art (e.g., Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Pat. Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0197] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six complementarity determining regions (CDRs) of a parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9:133-139, 1995) determined that only about one-third of the residues in the CDRs actually contact the antigen, and named these "specificity determining residues" or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, for example, Kashmiri et al., Methods 36: 25-34, 2005).

[0198] The choice of human variable domains, both light and heavy, used to make a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent can be selected as the human framework for the humanized antibody (see, for example, Sims et al., (1993) J. Immunol. 151:2296; Chothia et al., (1987) J. Mol. Biol. 196:901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used in several different humanized antibodies (see, e.g., Carter et al., (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al., (1993) J. Immunol., 151:2623). In some cases, the framework is selected from the V subclass, which is the most abundant human subclass. L 6 subgroup I(V L 6I) and V H Subgroup III(V H III) In another method, human germline genes are used as the source of the framework regions.

[0199] In another framework based on CDR comparison, called Superhumanization, FR homology is irrelevant. This method consists of comparing non-human sequences with functional human germline gene repertoires. Then, genes that code for the same or closely related canonical structure as the mouse sequence are selected. Then, among the genes that share the canonical structure with the non-human antibody, those with the highest homology in the CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169: 1119-1125, 2002).

[0200] Furthermore, it is generally desirable that antibodies be humanized while retaining their affinity for the antigen and other favorable biological properties. To this end, according to one method, humanized antibodies are prepared by a process of analysis using three-dimensional models of the parent and humanized sequences of the parent and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that depict and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0201] Another method for antibody humanization is based on a measure of antibody humanness called Human String Content (HSC). This method compares mouse sequences with a repertoire of human germline genes, and scores differences as HSC. Target sequences are then humanized by maximizing their HSC rather than using a global identity measure to generate multiple diverse humanized variants. (See, for example, Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0202] In addition to the above methods, empirical methods may be used to generate and select humanized antibodies. These methods include those based on generating and enriching large libraries of humanized variants or selecting the best clones using high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as by bacterial colony screening (see, for example, Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

[0203] In the FR library approach, a collection of residue variants is introduced into a FR at a particular position, followed by library selection to select the FR that best supports the grafted CDR. The substituted residues include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from a more restricted collection of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0204] In FR shuffling, instead of generating a combinatorial library of selected residue variants, entire FRs are combined with non-human CDRs (see, e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The library can be screened for binding in a two-step selection process in which the VL is first humanized, followed by the VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies exhibit improved biochemical and physicochemical properties, such as improved expression, increased affinity and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

[0205] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and the evaluation of sequential replacement and binding of non-human fragments into a human FR library. It starts with the CDR3 region of the non-human VH and VL chains, and successively replaces other regions of the non-human antibody, including CDR1 and CDR2 of both VH and VL, into human FRs. This methodology usually results in the retention of epitopes and the identification of antibodies from multiple subclasses with distinct human V-segment CDRs. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies. (See, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0206] "Human engineering" methods involve altering non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by making specific changes to the amino acid sequence of the antibody to produce modified antibodies that have reduced immunogenicity in humans, but still maintain the desired binding properties of the original non-human antibody. In general, this technique involves classifying amino acid residues of a non-human (e.g., murine) antibody as "low risk", "medium risk", or "high risk" residues. The classification is performed using an overall risk / reward calculation that assesses the predicted benefit of making a particular substitution (e.g., for immunogenicity in humans) compared to the risk that the substitution will affect folding of the resulting antibody and / or be replaced with a human residue. A specific human amino acid residue to be replaced at a given position (e.g., low or medium risk) of a non-human (e.g., murine) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or consensus human antibody sequence. Amino acid residues at low ("Low") and / or moderate ("Mod") risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence by alignment. Techniques for making human modified proteins are described in more detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication No. WO 93 / 11794.

[0207] Exemplary humanized antibodies made based on the above-mentioned human engineering methods are provided herein, including humanized antibodies designated A-15B08_Low, A-15B08_Low+Mod, A2-7A05_Low, A2-7A05_Low+Mod, C-14D12_Low, and C-14D12_Low+Mod. The VH, VL, and CDR sequences of these humanized antibodies according to various numbering schemes (e.g., Kabat, AbM, Chothia, Contact, and IMGT) are shown in Figures 2C, 2D, 2E, 2F, 2G, and 2H.

[0208] More specifically, the antibody designated A-15B08_Low comprises a VH comprising the amino acid sequence of SEQ ID NO: 136 and a VL comprising the amino acid sequence of SEQ ID NO: 137. The six CDR sequences of A-15B08_Low according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2C and 2D.

[0209] The antibody designated A-15B08_Low+Mod comprises a VH comprising the amino acid sequence of SEQ ID NO: 138 and a VL comprising the amino acid sequence of SEQ ID NO: 139. The six CDR sequences of A-15B08_Low+Mod according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2C and 2D.

[0210] The antibody designated A2-7A05_Low comprises a VH comprising the amino acid sequence of SEQ ID NO: 140 and a VL comprising the amino acid sequence of SEQ ID NO: 141. The six CDR sequences of A2-7A05_Low according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2E and 2F.

[0211] The antibody designated A2-7A05_Low+Mod comprises a VH comprising the amino acid sequence of SEQ ID NO: 142 and a VL comprising the amino acid sequence of SEQ ID NO: 143. The six CDR sequences of A2-7A05_Low+Mod according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2E and 2F.

[0212] The antibody designated C-14D12_Low comprises a VH comprising the amino acid sequence of SEQ ID NO: 144 and a VL comprising the amino acid sequence of SEQ ID NO: 145. The six CDR sequences of C-14D12_Low according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2G and 2H.

[0213] The antibody designated C-14D12_Low+Mod comprises a VH comprising the amino acid sequence of SEQ ID NO: 146 and a VL comprising the amino acid sequence of SEQ ID NO: 147. The six CDR sequences of C-14D12_Low+Mod according to Kabat, AbM, Chothia, Contact, and IMGT are shown in Figures 2G and 2H.

[0214] In some embodiments, provided herein is an α5β1 integrin binding agent (e.g., an antibody or fragment thereof that binds to α5β1 integrin, e.g., human α5β1 integrin) comprising one or more CDR sequences from A-15B08_Low, A-15B08_Low+Mod, A2-7A05_Low, A2-7A05_Low+Mod, C-14D12_Low and C-14D12_Low+Mod as shown in Figures 2C, 2D, 2E, 2F, 2G, and 2H.

[0215] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 136. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 137. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 136; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 137. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0216] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 139. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 138; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 139. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In some embodiments, the CDRs are according to Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0217] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 141. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 140; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 141. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0218] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 143. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 142; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 143. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0219] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 144. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 144; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 145. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0220] In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 147. In some embodiments, the α5β1 integrin binding agent provided herein comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 set forth in the VH comprising the amino acid sequence of SEQ ID NO: 146; and a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 set forth in the VL comprising the amino acid sequence of SEQ ID NO: 147. In some embodiments, the CDRs are according to the Kabat numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the CDRs are according to the Chothia numbering. In some embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT. In some embodiments, the CDRs are according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0221] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 136, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 137, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0222] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 138, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 139, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0223] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 140, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 141, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0224] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 143, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0225] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 144, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 145, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0226] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 146, and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 147, and the binding of the antibody or fragment thereof to α5β1 integrin (e.g., human α5β1 integrin) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0227] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:136 and a VL comprising the amino acid sequence of SEQ ID NO:137.

[0228] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:138 and a VL comprising the amino acid sequence of SEQ ID NO:139.

[0229] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:140 and a VL comprising the amino acid sequence of SEQ ID NO:141.

[0230] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:142 and a VL comprising the amino acid sequence of SEQ ID NO:143.

[0231] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:144 and a VL comprising the amino acid sequence of SEQ ID NO:145.

[0232] In some embodiments, the antibody or fragment thereof provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:146 and a VL comprising the amino acid sequence of SEQ ID NO:147.

[0233] In some embodiments, the α5β1 integrin binding agent described herein comprises a non-antibody protein scaffold. Non-limiting examples of such non-antibody protein scaffolds include fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, fynomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz inhibitors. Methods for producing such non-antibody protein scaffolds are well known in the art, and any one of them can be used to produce the α5β1 integrin binding agent comprising a non-antibody protein scaffold (see, for example, Simeon and Chen, Protein Cell, 9(1):3-14 (2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1):293-320 (2017)).

[0234] Further provided are materials for producing α5β1 integrin binding agents, such as human α5β1 integrin binding agents, and fragments thereof. For example, isolated cells (e.g., hybridomas, transformed or transfected cells) can produce the α5β1 integrin binding agent (e.g., an antibody or antibody fragment). In this regard, the cells (e.g., isolated cells) may produce an antibody or fragment thereof comprising a VH and VL as shown in Tables 1, 2, 3, 4, 5, or 6 for A-15B08, A2-3B06, A2-5D10, A2-7A05, A2-7F01, or C-14D12AB1, respectively, or as shown in Figures 2C-H for A-15B08-T62A, A-15B08_Low, A-15B08_Low+Mod, A2-7A05_Low, A2-7A05_Low+Mod, C-14D12_Low, and C-14D12_Low+Mod. In some embodiments, the polynucleotides described herein may include one or more nucleic acid sequences encoding an α5β1 integrin binding agent (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotide is an isolated and / or recombinant polynucleotide. In various aspects, the isolated polynucleotide is an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VH). L ), wherein the V H and V L contains the same CDRs as shown in Table 1, the CDRs as shown in Table 2, the CDRs as shown in Table 3, the CDRs as shown in Table 4, the CDRs as shown in Table 5, the CDRs as shown in Table 6, or the CDRs as shown in Figures 2C-2H (according to Kabat and / or Chothia, AbM, Contact, or IMGT).

[0235] In some embodiments, one or more vectors (e.g., expression vectors) can contain one or more polynucleotides for expression of the one or more polynucleotides in a suitable host cell. Such vectors are useful, for example, for amplifying the polynucleotides in a host cell to produce useful amounts thereof, and for expressing a binding agent, such as an antibody or an antibody fragment, using recombinant techniques.

[0236] In some embodiments, the one or more vectors are expression vectors in which one or more polynucleotides encoding antibody sequences are operably linked to one or more polynucleotides comprising expression control sequences. Self-replicating recombinant expression constructs, such as plasmids and viral DNA vectors incorporating one or more polynucleotides encoding antibody sequences that bind to α5β1 integrin, are specifically envisioned. Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct (e.g., expression vector) will be utilized. Promoter and enhancer sequences are generally selected for their ability to increase gene expression, and operator sequences are generally selected for their ability to regulate gene expression. Expression constructs (e.g., expression vectors) also include sequences encoding one or more selectable markers that allow for the identification of host cells that carry the construct. Expression constructs (e.g., expression vectors) also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. In some embodiments, expression constructs (e.g., expression vectors) can also include sequences necessary for replication in a host cell.

[0237] Exemplary expression control sequences include, for example, the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29: 2494-2502, 1991; Boshart et al., Cell, 41: 521-530, 1985); the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); the Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); the simian virus 40 promoter; DRA (downregulated in adenomas; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293: G923-G934, 2007); MCT1 (monocarboxylate transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liver Physiol., G977-G979. 2005); and Math1 (mouse atonal homolog 1; Shroyer et al., Gastroenterology, 132: 2477-2478, 2007), where for expression in mammalian cells the promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In another variation, the promoter is an epithelial-specific promoter or an endothelial-specific promoter. The polynucleotide also optionally includes an appropriate polyadenylation sequence (e.g., SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0238] If desired, the one or more polynucleotides also optionally contain a nucleotide sequence encoding a secretory signal peptide fused in-frame with the polypeptide sequence. The secretory signal peptide directs the secretion of the antibody polypeptide by cells expressing the one or more polynucleotides and is cleaved from the polypeptide secreted by the cells. In addition, the one or more polynucleotides optionally contain a sequence whose only intended function is to facilitate large-scale production of the vector. Polynucleotides for gene therapy can be produced and administered using procedures described in the literature for various transgenes. See, for example, Isner et al., Circulation, 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy, 7: 989-1011, 1996.

[0239] In some embodiments, the polynucleotide may further comprise additional sequences that facilitate uptake by a host cell and expression of the antibody or fragment thereof (and / or any other peptides). In some embodiments, a "naked" transgene (e.g., a transgene without a viral, liposomal, or other vector that facilitates transfection) encoding an antibody or fragment thereof described herein is employed.

[0240] Any suitable vector may be used to introduce one or more polynucleotides encoding the antibody, or fragment thereof, into the host. Exemplary vectors that have been described include, but are not limited to, replication-deficient retroviral vectors, such as lentiviral vectors (see, e.g., Kim et al., J. Virol., 72(1): 811-816, 1998; Kingsman and Johnson, Scrip Magazine, October 1998, pp. 43-46); parvoviral vectors, such as adeno-associated viral (AAV) vectors (see, e.g., U.S. Pat. Nos. 5,474,9351; 5,139,941; 5,622,856; 5,658,776; 5,773,289; 5,789,390; 5,834,441; 5,863,541; 5,851,521; 5,252,479; Gnatenko et al., J. Invest. Med., 2003, 14: 111-112, 1998; and U.S. Pat. Nos. 5,474,9351, 5,139,941, 5,622,856, 5,658,776, 5,773,289, 5,789,390, 5,834,441, 5,863,541, 5,851,521, 5,252,479; Gnatenko et al., J. Invest. Med., 2003, 14: 111-112, 1998). 45: 87-98, 1997); adenovirus (AV) vectors (see, e.g., U.S. Pat. Nos. 5,792,453; 5,824,544; 5,707,618; 5,693,509; 5,670,488; and 5,585,362; Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584, 1992; Stratford Perricaudet et al., J. Clin. Invest., 90: 626-630, 1992; and Rosenfeld et al., Cell, 68: 143-155, 1992); adenovirus-adeno-associated virus chimeras (U.S. Pat. No. 5,856,152) or vaccinia virus or herpes virus vectors (U.S. Pat. Nos. 5,879,934; 5,849,571; 5,830,727; 5,661,033; 5,328,688); lipofectin mediated gene transfer (BRL); liposomal vectors (U.S. Pat. No. 5,631,237); and combinations thereof.Any of these expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994). Optionally, the viral vectors can be made replication-deficient, for example, by deleting or disrupting a selection gene required for viral replication.

[0241] Other postulated non-viral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695, 1990), DEAE dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81: 7161-7165, 1987), and ELISA (Graham and Van Der Eb, Virology, 52: 456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7: 2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10: 689-695, 1990). 1984), direct microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985, DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979; Felgner, Sci Am., 276(6): 102-6, 1997; Felgner, Hum Gene Ther., 7(15): 1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 1791-3, 1996), and cellular endothelial cell culture (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 1791-3, 1996). 8463-8467, 1987), gene bombardment using high-velocity microprojectiles (Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990), and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262: 4429-4432, 1987; Wu and Wu, Biochemistry, 27: 887-892, 1988; Wu and Wu, Adv.Drug Delivery Rev., 12: 159-167, 1993).

[0242] The expression vector (or the antibody or fragment thereof described herein) can be entrapped in liposomes. See, for example, Ghosh and Bachhawat, p. 87-104, Wu G, Wu C, eds., Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, New York: Marcel Dekker, (1991); Radler et al., Science, 275(5301): 810-814, 1997). Also contemplated are various commercial approaches involving "lipofection" technology. In some embodiments, the liposome can be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with cell membranes and promote intracellular internalization of DNA encapsulated within the liposome (see, e.g., Kaneda et al., Science, 243: 375-378, 1989). In some embodiments, the liposome is complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (see, e.g., Kato et al., J. Biol. Chem., 266: 3361-3364, 1991). In some embodiments, the liposome is complexed or employed in conjunction with both HVJ and HMG-1. Such expression constructs have been successfully employed for the transfer and expression of nucleic acids in vitro and in vivo. In some embodiments, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), is placed into a liposome and the liposome is targeted to cells (e.g., tumor cells, etc.) that express α5β1 integrin on their surface.

[0243] The cells may comprise one or more polynucleotides or one or more vectors, for example, the cells are transformed or transfected with one or more polynucleotides encoding an α5β1 integrin binding agent (e.g., an antibody) including a human α5β1 integrin binding agent, or one or more vectors comprising the one or more polynucleotides. In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody) including a human α5β1 integrin binding agent comprising one or more (including six) CDRs having at least 75% identity (e.g., 75%, 80%, 85%, 90%, 95%, 100%) to the CDRs of A-15B08 (see, e.g., Table 1). In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody) including a human α5β1 integrin binding agent comprising one or more (including six) CDRs having at least 75% identity (e.g., 75%, 80%, 85%, 90%, 95%, 100%) to the CDRs of A2-3B06 (see, e.g., Table 2). H and V L In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent comprising: H and V L In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent comprising: H and V L In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent comprising: H and V L In some embodiments, the cells express and produce an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent comprising: H and V LThe present invention expresses and produces α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, including human α5β1 integrin binding agents, including human α5β1 integrin binding agents (e.g., antibodies), ... Similarly, a polypeptide (e.g., an α5β1 integrin binding agent such as an antibody (including a human α5β1 integrin binding agent)) may be glycosylated or non-glycosylated and / or may be covalently modified to include one or more water soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0244] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are useful for amplifying polynucleotides and for expressing polypeptides encoded by the polynucleotides. In this regard, a process for the manufacture of an α5β1 integrin binding agent (e.g., an antibody) can include culturing the host cells and isolating the α5β1 integrin binding agent. The transfer of naked DNA expression constructs into cells can be achieved using particle bombardment methods due to the ability to accelerate DNA-coated microprojectiles to high velocities, allowing the microprojectiles to break through cell membranes and enter cells without killing the cells (see, e.g., Klein et al., Nature, 327: 70-73, 1987). Several devices have been developed for accelerating small particles. One such device relies on high voltage ejection to generate an electric current and thereby provide the transport force (see, for example, Yang et al., Proc. Natl. Acad. Sci USA, 87: 9568-9572, 1990). The microprojectiles used consist of biologically inert materials such as tungsten or gold beads. The host cell may be isolated and / or purified. The host cell may also be a cell transformed in vitro to cause transient or permanent expression of a polypeptide in vivo. The host cell may also be an isolated cell transformed ex vivo or may be introduced after transformation to produce a polypeptide in vivo, for example for therapeutic purposes. The definition of a host cell explicitly excludes transgenic humans.

[0245] Various methods for producing antibodies from polynucleotides are generally well known. For example, basic molecular biology procedures are described by Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). In addition, many publications describe techniques suitable for preparing antibodies by DNA manipulation, constructing expression vectors, and transforming and culturing suitable cells (see, for example, Mountain and Adair, Chapter 1, in Biotechnology and Genetic Engineering Reviews, edited by Tombs, Intercept, Andover, UK, 1992); and Current Protocols in Molecular Biology, edited by Ausubel, Wiley Interscience, New York, 1999).

[0246] An α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, can be produced using any suitable method, including those described above, e.g., isolated from an immunized animal, recombinantly or synthetically produced, or genetically engineered. Antibody fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. For example, papain or pepsin digestion of a whole antibody yields a 5S fragment designated F(ab')2 or two monovalent Fab fragments and an Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to yield a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described, for example, in Edelman et al., Methods in Enzymology, 1: 422 Academic Press (1967); Nisonoff et al., Arch. Biochem. Biophys., 89: 230-244, 1960; Porter, Biochem. J., 73: 119-127, 1959; in U.S. Pat. No. 4,331,647; and by Andrews, SM and Titus, JA in Current Protocols in Immunology, edited by Coligan et al., John Wiley & Sons, New York (2003), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5.

[0247] The α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, can be genetically engineered. For example, the α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, comprises a variable region or variable domain, for example, produced by recombinant DNA engineering techniques. In this regard, the variable region is optionally modified by insertion, deletion, or change in the amino acid sequence of the antibody to produce the antibody of interest, including those described above. Polynucleotides encoding the complementarity determining regions (CDRs) of interest, such as those listed in Tables 1-6, are prepared, for example, by using the polymerase chain reaction to synthesize the variable regions using mRNA from antibody-producing cells as a template (see, for example, Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," p. 166 in Monoclonal Antibodies: Production, Engineering and Clinical Application, eds., Ritter et al., (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," p. 137 in Monoclonal Antibodies: Principles and Applications, eds., Birch et al., (Wiley Liss, Inc. 1995); and Larrick et al., "Methods: A Handbook of Methods in Enzymology." (See, A Companion to Methods in Enzymology, 2: 106-110, 1991). Current antibody engineering techniques allow the construction of modified variable region domains that contain at least one CDR and, optionally, one or more framework amino acids from a first antibody, as well as the remainder of the variable region domain from a second antibody. Such techniques are used, for example, to humanize an antibody or to improve its affinity for a binding target.

[0248] A "humanized antibody" is an antibody in which the CDRs of the heavy and light variable chains of a non-human immunoglobulin have been transferred into a human variable domain. The constant region may be absent, but if present, it is optionally substantially identical to a human immunoglobulin constant region, e.g., in some embodiments, at least about 85-90%, about 95%, 96%, 97%, 98%, 99% or more identical. Thus, in some instances, all parts of a humanized immunoglobulin, possibly excepting the CDRs, are substantially identical to the corresponding parts of a native human immunoglobulin sequence. For example, a humanized antibody is a human immunoglobulin (e.g., a host antibody) in which hypervariable region residues of the host antibody have been replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.

[0249] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein are useful in compositions and methods for treating, preventing, or alleviating α5β1 integrin-mediated diseases, disorders, or conditions, including one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject is diagnosed with α5β1 integrin-mediated diseases, disorders, or conditions. α5β1 integrin-mediated diseases, disorders, and conditions include, but are not limited to, cancer (e.g., cancer associated with or characterized by tumor cells that express or overexpress α5β1 integrin), angiogenesis-mediated diseases (e.g., diseases associated with or characterized by abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases such as MS and ALS).

[0250] In some embodiments, described herein is a method for treating cancer or tumor in a subject, comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the subject is diagnosed with cancer.

[0251] In some embodiments, described herein are methods for alleviating one or more symptoms associated with cancer or a tumor in a subject, comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0252] In some embodiments, described herein is a method for (i) treating an angiogenesis-mediated disease, disorder, or condition, or (ii) inhibiting angiogenesis in a subject (e.g., having a tumor), comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the subject is diagnosed with an angiogenesis-mediated disease, disorder, or condition.

[0253] In some embodiments, described herein is a method for alleviating one or more symptoms associated with an angiogenesis-mediated disease, disorder, or condition in a subject, comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0254] In some embodiments, described herein is a method for treating an inflammatory disease, disorder, or condition, such as a neuroinflammatory disease, disorder, or condition (e.g., MS, ALS), in a subject, comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein. In some embodiments, the subject is diagnosed with an inflammatory disease, disorder, or condition, such as a neuroinflammatory disease, disorder, or condition (e.g., MS, ALS).

[0255] In some embodiments, described herein are methods for alleviating one or more symptoms associated with an inflammatory disease, such as a neuroinflammatory disease, disorder, or condition (e.g., MS, ALS) in a subject, comprising administering to the subject an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0256] The subject of the above-described methods can be administered one or more therapeutic agents described herein in combination with an α5β1 integrin binding agent (e.g., an antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (e.g., an antibody) described herein.

[0257] In some embodiments, the antibody is a human antibody, such as, but not limited to, an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, for example, as described in Kabat et al., (1991) "Sequences of Proteins of Immunological Interest", 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242. If the antibody contains a constant region, preferably the constant region is also derived from human germline immunoglobulin sequences. A human antibody may contain amino acid residues not encoded by human germline immunoglobulin sequences, e.g., to improve the activity of the antibody, but does not contain CDRs derived from other species (e.g., a murine CDR placed within a human variable framework region).

[0258] In some embodiments, the α5β1 integrin binding agent (e.g., antibody) binds to and kills tumor cells in cell culture. Such cell culture can include tumor cells that express or overexpress α5β1 integrin. Tumor cells include, but are not limited to, breast cancer cells, bladder cancer cells, melanoma cells, prostate cancer cells, mesothelioma cells, lung cancer cells, testicular cancer cells, thyroid cancer cells, squamous cell carcinoma cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, gastric cancer cells, bladder cancer cells, and pancreatic cancer cells.

[0259] In some embodiments, described herein is a method of inhibiting abnormal angiogenesis in a subject (e.g., having a tumor). For example, the method includes administering an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent described herein, in an amount effective to inhibit abnormal angiogenesis. In some embodiments, the method includes administering an α5β1 integrin binding agent (e.g., an antibody) that competes with antibody A-15B08, antibody A2-3B06, antibody A2-5D10, antibody A2-7A05, antibody A2-7F01, and / or antibody C-14D12AB1 (see, e.g., CDRs and VH / VL of Tables 1, 2, 3, 4, 5, and / or 6) for binding to human α5β1 integrin, and / or that competes with antibody A-15B08, antibody A2-3B06, antibody A2-5D10, antibody A2-7A05, antibody A2-7F01, and / or antibody C-14D12AB1 (see, e.g., CDRs and VH / VL of Tables 1, 2, 3, 4, 5, and / or 6) for binding to human α5β1 integrin. , antibody A2-5D10, antibody A2-7A05, antibody A2-7F01, and / or antibody C-14D12 (see, e.g., CDRs and VH / VL in Tables 1, 2, 3, 4, 5, and / or 6), resulting in inhibition of abnormal angiogenesis. In some embodiments, one or more binding agents (e.g., antibodies), polynucleotides, vectors, and / or cells as described above can be used in methods of inhibiting abnormal angiogenesis in vivo (e.g., in methods of treating cancer in a subject).

[0260] Also provided is a method of modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject. For example, the method includes administering to the subject a composition comprising an α5β1 integrin binding agent (e.g., an antibody) in an amount effective to modulate tumor growth in the subject. "Tumor" refers to any neoplastic cell growth or proliferation, whether malignant or benign, and includes all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals typically characterized by unregulated or abnormal cell proliferation, including, but not limited to, all malignant neoplasms, such as: carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Examples of cancer include, but are not limited to, breast cancer (including metastatic breast cancer), cervical cancer, colon cancer, colorectal cancer (including metastatic colorectal cancer), lung cancer (including non-small cell lung cancer), fibrosarcoma, non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia, bladder cancer, pancreatic cancer, renal cell carcinoma, splenic cancer, prostate cancer such as hormone-refractory prostate cancer, liver cancer, head and neck cancer, gastric cancer, bladder cancer, melanoma, ovarian cancer, mesothelioma, soft tissue cancer, gastrointestinal stromal tumor, glioblastoma multiforme, and multiple myeloma. Also provided are methods of treating, preventing, or ameliorating cancer by administering an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, alone or in combination with another agent, to a subject in need thereof. Also provided are methods for treating, preventing, or ameliorating one or more symptoms of cancer by administering an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, to a subject in need thereof, alone or in combination with another agent.

[0261] "Inhibiting" abnormal angiogenesis does not require 100% inhibition. Any inhibition that reduces tumor growth and / or metastasis is envisioned. Similarly, "modulating" tumor growth means reducing the size of the tumor, slowing the rate of tumor growth, or inhibiting an increase in the size of an existing tumor. Complete disappearance of the tumor is not required; any reduction in tumor size or any slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, tumor cell elimination can be enhanced, for example, by at least about 5%, at least about 10%, or at least about 20%, compared to the level of elimination observed in the absence of the method (e.g., in a biologically matched control subject or a specimen not exposed to the agent of the method). The effect is detected, for example, by a reduction in tumor size or tumor metastasis, a reduction or maintenance of the level of a tumor marker, or a reduction or maintenance of a tumor cell population. In some embodiments, tumor cell removal is improved, e.g., by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%), compared to tumor cell removal in the absence of the α5β1 integrin binding agent (e.g., an antibody) of the method.

[0262] The particular dosing regimen of an α5β1 integrin binding agent (e.g., an antibody) for a particular subject will depend, in part, on the agent used, the amount of agent administered, the route of administration, and the cause and extent of any side effects. The amount of agent administered to a subject (e.g., a mammal such as a human) should be sufficient to produce a desired response over a reasonable time frame. Thus, in some embodiments, the amount of an α5β1 integrin binding agent (e.g., an antibody) or pharmaceutical composition described herein administered to a subject is an effective amount. In some embodiments, the amount of an α5β1 integrin binding agent (e.g., an antibody) or pharmaceutical composition described herein administered to a subject is a therapeutically effective amount. In some embodiments, the method includes, for example, administering about 0.1 μg / kg up to about 100 mg / kg or more. Some conditions or pathologies require long-term treatment, which may or may not require administering multiple doses of multiple α5β1 integrin binding agents (e.g., antibodies), such as multiple human α5β1 integrin binding agents (e.g., antibodies), over multiple administrations.

[0263] Suitable routes of administration of compositions comprising an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent (e.g., an antibody), are well known in the art. More than one route can be used to administer an agent (e.g., an antibody), but one particular route may provide a more rapid and effective response than another route. Depending on the situation, a composition comprising an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, is applied or instilled into a body cavity, absorbed through the skin or mucous membrane, ingested, inhaled, and / or introduced into the circulation. For example, it may be desirable to deliver a composition comprising an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), via injection by intravenous, subcutaneous, intraperitoneal, intracerebral (intracembidium), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, ​​vaginal, or rectal means, by a sustained release system, or by an implantable device. If desired, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), is administered locally via administration into an artery or vein that supplies nutrients to the area of ​​interest, for example, via a hepatic artery for delivery to the liver. Alternatively, an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), is administered locally by implantation of a membrane, sponge, or another suitable material onto which the binding agent has been absorbed or encapsulated. When an implantable device is used, the device, in one embodiment, is implanted into any suitable tissue or organ and delivery of the α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), is by, for example, diffusion, timed-release bolus, or continuous administration. In another embodiment, the α5β1 integrin binding agent (e.g., an antibody) is administered directly to tissue exposed during tumor resection or other surgical procedure.

[0264] The present disclosure provides compositions, such as pharmaceutical compositions, that include an α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody) and a carrier (e.g., a pharma- ceutical acceptable carrier). The particular carrier employed may depend on chemico-physical considerations, such as solubility and non-reactivity with the binding agent or co-therapy, as well as on the route of administration. Pharmaceutically acceptable carriers are well known in the art, examples of which are described herein. Exemplary pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Injectable formulations are further described, for example, in Pharmaceutics and Pharmacy Practice, edited by Banker and Chalmers, JB Lippincott Co., Philadelphia. Pa., pp. 238-250 (1982), and in ASHP Handbook on Injectable Drugs, 4th ed., pp. 622-630 (1986) by Toissel. Pharmaceutical compositions including α5β1 integrin binding agents (e.g., antibodies), such as human α5β1 integrin binding agents, are in one embodiment packaged in a container with packaging material that provides instructions for use of such pharmaceutical compositions. Typically, such instructions include substantial labeling describing the reagent concentrations and, in some embodiments, the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be required to reconstitute the pharmaceutical composition.

[0265] In some embodiments, the methods described herein further include administering one or more additional agents, such as therapeutic agents, which may be present in the composition, may be administered together with the α5β1 integrin binding agent, such as a human α5β1 integrin binding agent (e.g., an antibody), or may be provided in a separate composition using the same or different administration route. The one or more additional agents, such as therapeutic agents, may be administered together with the α5β1 integrin binding agent (e.g., an antibody) or separately (e.g., simultaneously, alternating, sequentially) (e.g., for combination therapy). Such additional therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapies and immunotherapeutic agents, cytotoxic agents, chemotherapeutic agents, and inhibitors.

[0266] Therapeutic antibodies that can be used (e.g., for combination therapy) with the α5β1 integrin binding agents (e.g., antibodies) described herein include αvβ3 binding antibodies (e.g., etaracizumab), α4β1 binding antibodies (e.g., natalizumab), α4β7 binding antibodies (e.g., vedolizumab), TREM2 binding antibodies (e.g., AL002), TNFα binding antibodies (e.g., adalimumab), CSF1 binding antibodies (e.g., MCS110), CSF-1R binding antibodies (e.g., AL002), TNFα binding antibodies (e.g., adalimumab), CSF-2 binding antibodies (e.g., AL002), TNFα binding antibodies (e.g., adalimumab), CSF-3 binding antibodies (e.g., AL002), TNFα binding antibodies (e.g., adalimumab), CSF-4 binding antibodies (e.g., AL002), TNFα binding antibodies (e.g., adalimumab), CSF-5 binding antibodies (e.g., adalimumab), CSF-6 binding antibodies (e.g., adalimumab), CSF-7 binding antibodies (e.g., adalimumab), CSF-8 binding antibodies (e.g., adalimumab), CSF-9 binding antibodies (e.g., adalimumab), CSF-1R ... These include, but are not limited to, antibodies that bind to type I interferon (IFN) (e.g., sifalimumab), antibodies that bind to type I interferon (IFN) (e.g., sifalimumab), antibodies that bind to type I interferon (IFN) (e.g., sifalimumab), antibodies that bind to type I interferon (IFN) (e.g., sifalimumab), IL-1β binding antibodies (e.g., canakinumab, gevokizumab), IL-6 binding antibodies (e.g., tocilizumab), IL-12 binding antibodies (e.g., ustekinumab), and antibodies that bind to type I interferon (IFN) (e.g., sifalimumab).

[0267] Immunotherapies and immunotherapeutic agents that can be used (e.g., for combination therapy) with the α5β1 integrin binding agents (e.g., antibodies) described herein include, but are not limited to, cytokines, interleukins, tumor necrosis factors, and combinations thereof. In some embodiments, the immunotherapies include immunotherapeutic agents that modulate immune responses, such as checkpoint inhibitors or checkpoint agonists. In some embodiments, the immunotherapeutic agent is an antibody modulator targeting PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3 and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFβ, OX40, 41BB, LIGHT, CD40, GITR, TGF-β, TIM-3, SIRP-α, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, among others known in the art. In some embodiments, the immunotherapeutic agent is an agent that increases natural killer (NK) cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the suppression of an immune response. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or suppressor cell activity. In some embodiments, the immunotherapeutic agent is an agent or therapy that inhibits Treg activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of an inhibitory immune checkpoint receptor.

[0268] In some embodiments, the immunotherapeutic agent comprises a T cell modulator selected from an agonist or activator of a costimulatory molecule. In one embodiment, the agonist of a costimulatory molecule is selected from GITR, OX40, ICOS, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3, or an agonist of a CD83 ligand (e.g., an agonist antibody or antigen-binding fragment thereof, or a soluble fusion). In another embodiment, the combination of effector cells comprises a bispecific T cell engager (e.g., a bispecific antibody molecule that binds CD3 and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2, among others).

[0269] Cytotoxic agents that can be used (e.g., for combination therapy) with the α5β1 integrin binding agents (e.g., antibodies) described herein include substances that inhibit or prevent cell function and / or cause cell death or destruction. Exemplary cytotoxic agents include radioisotopes (e.g., I 131 , I 125 , Y 90 , and Re 186 ); chemotherapeutic agents; and toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof).

[0270] Chemotherapeutic agents that can be used in conjunction with (e.g., for combination therapy) the α5β1 integrin binding agents (e.g., antibodies) described herein include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; and charismatic drugs. statins; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and biceresin); cryptophycins (particularly crytophycin 1 and crytophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatins; chlorambucil, chlor Nitrogen mustards such as nafazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Enediyne antibiotics (e.g., antibiotics such as calicheamicin, particularly calicheamicin γ1I and calicheamicin ω11 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin ), caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline o-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin mitomycins such as cyclosporine, marcelomycin, and mitomycin C; mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folate analogues such as terin, methotrexate, pteropterin, trimetrexate;purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane; fluororic acid folic acid supplements such as aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elformithine, elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa;Taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France;Chlorambucil;GEMZAR® gemcitabine;6-thioguanine;Mercaptopurine;Methotrexate;Platinum analogues such as cisplatin and carboplatin;Vinblastine;Platinum;Etoposide (VP-16);Ifosfamide;Mitoxantrone;Vincristine;Navelbine® vinorelbine;Novantrone;Teniposide;Edatrexate;Daunomycin;Aminopterin;Xeloda;Ibandronate;Irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin);Topoisomerase inhibitors RFS 2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;combretastatin;leucovorin (LV);oxaliplatin including oxaliplatin treatment regimens (FOLFOX);These include, but are not limited to, inhibitors of PKC-α, Raf, H-Ras, and EGFR that reduce cell proliferation (e.g., erlotinib (Tarceva™)), as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the above. Also, anti-hormonal agents that act to regulate or inhibit the action of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and FARESTON toremifene; agents that inhibit the production of estrogen by the adrenal gland, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. aromatase inhibitors, which inhibit the enzyme aromatase, which regulates cell growth; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways implicated in abnormal cell proliferation, such as, for example, PKC-α, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; gene therapy vaccines, such as, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH;Also included are vinorelbine and esperamicin, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above.

[0271] Inhibitors that can be used (e.g., for combination therapy) with the α5β1 integrin binding agents (e.g., antibodies) described herein include, but are not limited to, kinase inhibitors such as FAK inhibitors (e.g., GSK2256098), MEK inhibitors (e.g., cobimetinib, rametinib, binimetinib, selumetinib), tyrosine kinase inhibitors (e.g., cabozantinib); EGFR inhibitors (e.g., erlotinib); Janus kinase (JAK) 1 selective inhibitors (e.g., baricitinib, tofacitinib, upadacitinib), CSF-1R inhibitors (e.g., BLZ945); C-kit inhibitors (e.g., masitinib); and FGFR inhibitors (e.g., erdafitinib).

[0272] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) disclosed herein can be used in combination with an inhibitor of PD-1 or an inhibitor of PD-L1, such as an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody, such as nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, or avelumab.

[0273] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) disclosed herein can be used in combination with CTLA-4 inhibitors, e.g., anti-CTLA-4 antibodies, e.g., ipilimumab (Yervoy), or with antibodies against cytokines, or with bispecific antibodies that bind PD-L1 and CTLA-4 or PD-1 and CTLA-4, or with other anti-cancer agents.

[0274] The additional agent may be a pharma- ceutically acceptable salt, ester, amide, hydrate, and / or prodrug of any of the therapeutic agents listed above or other agents.

[0275] The additional therapeutic agent may be a pharma- ceutically acceptable salt, ester, amide, hydrate, and / or prodrug of any of the therapeutic agents listed above or other agents.

[0276] It is understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Accordingly, the following examples are intended to illustrate, but not limit, the disclosure. EXAMPLES

[0277] (Example) Example 1: Antibody generation and initial screening for α5 integrin binding Four NZBW and four CD-1 mice were injected with 100 μg of purified recombinant human α5β1 integrin heterodimer (rh-α5β1; Acro Biosystems, Newark, DE; Catalog No. IT1-H52W5). Four weeks later, cells from the spleen and draining lymph nodes were fused to generate hybridomas. Approximately 4000 hybridoma supernatants (2,500 from the NZBW lineage and 1,500 from the CD-1 lineage) were screened for positivity by flow cytometry on K562 cells (ATCC® CCL-243™; Manassas, VA 20110) that had been activated with 10 ng / mL PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich, St. Louis, MO, Catalog No. 5.00582) for 24 hours, cryopreserved, and thawed immediately before use. Briefly, hybridoma supernatants were incubated with activated K562 cells for 20 min, washed, then incubated with fluorescently conjugated detection antibodies for 20 min, washed, resuspended in 7-aminoactinomycin D, and mean fluorescence intensity (MFI) was measured with a Guava cytometer (Luminex Corporation, Austin, TX 78727). As shown in Table 7, 249 positive clones were selected for further screening based on hybridoma supernatants that had significantly higher (>1.5x) MFI than hybridoma medium alone (MFI of 125 for K562 cells).

[0278] (Table 7) [Table 8] TIFF2024517953000034.tif245170TIFF2024517953000035.tif59170

[0279] Example 2: Screening for α5 binding specificity 249 positive hybridoma supernatants selected as described in Example 1 were screened for reactivity to rh-α5β1 in a plate-based ELISA. Immulon4 HBX ELISA 96-well plates (Thermo Fisher Scientific, Waltham, MA, Catalog No. 3855) were coated with 1 μg / mL rh-α5β1 (R&D Systems, Minneapolis, MN 55413, Catalog No. 3230-α5) in PBS supplemented with 0.5 mM MgCl2, MnCl2, and CaCl2 and incubated overnight at 4°C. Plates were washed three times with Wash Buffer (1x Tris-buffered saline containing 0.05% Tween 20), blocked with 2% BSA in 1x TBS (prepared from 10x Thermo Scientific Blocker BSA (10x) in TBS; Thermo Fisher Scientific, Waltham, MA, Catalog No. 37520) for 2 hours at room temperature (RT), and then incubated with hybridoma supernatants diluted 1:10 in standard diluent (2% BSA, 1x TBS, 0.05% Tween 20). After 1 hour incubation at room temperature, the wells were washed 3 times, incubated with biotinylated goat anti-mouse secondary antibody at a dilution of 1:8000 for 1 hour (Invitrogen, Carlsbad, CA, Catalog No. 62-6540), washed 3 times, then incubated with poly-HRP streptavidin (Thermo Fisher Scientific, Waltham, MA, Catalog No. N200) for 30 minutes, washed 4 times, and incubated with TMB (Thermo Fisher Scientific, Waltham, MA, Catalog No. N301) for 5-10 minutes, followed by the addition of ELISA Stop Solution (Invitrogen, Carlsbad, CA, Catalog No. SS04). Absorbance was measured at 450 nm. The results are shown in Tables 8A and 8B.

[0280] (Table 8A) [Table 9] TIFF2024517953000037.tif248170TIFF2024517953000038.tif248170TIFF2024517953000039.tif202170

[0281] (Table 8B) [Table 10] TIFF2024517953000041.tif248170TIFF2024517953000042.tif248170TIFF2024517953000043.tif49170

[0282] In parallel, 249 hybridomas were screened for specificity for the human α5 subunit by testing for reactivity to rh-α4β1 (R&D Systems, Minneapolis, MN 55413; Catalog Nos. 3230-α5 and 5668-α4). Results are shown in Tables 8A and 8B. Any hybridomas that reacted with both rh-α4β1 and rh-α5β1 appear to be β1 subunit specific and are not considered α5 specific. Using the protocol described above, 29 hybridoma supernatants reacted strongly (>1.0 absorbance 450 nm) with rh-α4β1 and were not considered α5 specific.

[0283] In addition, the hybridomas were tested for cross-reactivity to recombinant mouse α5β1 (rm-α5β1; R&D Systems, Minneapolis, MN 55413; Catalog No. 7728-A5) using the same protocol as above, except that the plates were coated with rm-α5β1. The results are shown in Tables 8A and 8B. Three hybridomas showed strong binding to rm-α5β1 (>1.0 absorbance 450 nm), but also had strong reactivity to rh-α4β1.

[0284] Of the 220 hybridomas that showed specific binding to the α5 subunit, 28 were selected for further characterization and were various anti-α5 binding antibodies with no cross-reactivity to rh-α4β1 and absorbance 450 nm readings ranging from 0.32 to 2.3. None had detectable cross-reactivity to rm-α5β1 in this 1:10 dilution single point assay.

[0285] Example 3: Selection of antibodies with high α5 binding affinity Kinetic analysis was performed on 28 hybridoma supernatants selected as described in Example 2 using a ForteBio Octet BMIA instrument (ForteBio, Fremont, CA) to determine the equilibrium dissociation constants (K D =k off / k on ) was calculated. Each clone supernatant was loaded using an anti-mouse IgG Fc biosensor. The assay steps were as follows: Sensor Check (30 sec) → Load Ab (700 sec) → Quench (480 sec) → Baseline (480 sec) → Ab Assoc. (600 sec) → Dissociation (600 sec). Kinetic data are shown in Table 9. Twenty of the hybridomas had K values ​​below 10 nanomolar (nM) ranging from 0.4 to 7.8 nM. D These were selected for further characterization.

[0286] (Table 9) [Table 11]

[0287] Example 4: Variable region sequencing to identify unique antibody sequences DNA sequencing of the heavy and light chain variable regions of 20 clones selected as described in Example 3 was performed. DNA was isolated from hybridoma cell pellets and sequenced using the Sanger method. Sequence alignment revealed nine unique sequence heavy and light chain pairs that were assigned group numbers 1 to 9. One antibody clone was selected to represent each unique sequence group, as shown in Table 10.

[0288] (Table 10) [Table 12]

[0289] Example 5: Selection of antibodies that inhibit the binding of fibronectin to α5β1 To determine whether the nine unique hybridoma clones selected as described in Example 4 were able to inhibit the binding of α5β1 integrin to fibronectin (FN), antibodies were first purified from hybridoma supernatants by protein A chromatography, protein concentrations were measured by BCA assay (Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA, catalog no. 23225), and then tested in a quantitative FN inhibition assay in an ELISA format.

[0290] Immulon4 HBX ELISA 96-well plates were coated with FN by overnight incubation with 2.5 μg / mL human FN (R&D Systems, Minneapolis, MN 55413, Catalog No. 1918-FN) in 1× PBS (0.01 M phosphate buffer and 0.154 M NaCl, pH 7.4) at 4° C. Plates were then washed three times with Wash Buffer (1× Tris-buffered saline containing 0.05% Tween 20) and blocked with 2% BSA in 1× TBS for 2 hours at room temperature (RT). Antibodies were diluted in standard diluent (2% BSA, 1x TBS, 0.05% Tween20) containing 0.1 μg / mL rh-α5β1-6xHis tagged protein (Acro Biosystems, Newark, DE, Cat. No. IT1-H52W5) to generate an 11-point 1:3 antibody dilution series ranging from 10,000 ng / mL to 0.17 ng / mL. Data were normalized across different assay runs using an isotype control antibody (Control Ab; Ms IgG2a EMD Millipore Corp, Billerica, MA, Cat. No. PP102). For the assay, 100 uL of antibody dilution series / His tagged α5β1 mixture was added to the wells after removing the blocking solution and washing the wells three times. After 1 hour at room temperature, wells were washed 3 times, incubated with biotinylated anti-6xHis-tag Ab (Invitrogen, Carlsbad, CA, Catalog No. MAI-21315-BTIN) at 1:1000 in standard diluent for 1 hour, washed 3 times, incubated with poly-HRP streptavidin (Thermo Fisher Scientific, Waltham, MA, Catalog No. N200) for 30 minutes, washed 4 times, and incubated with TMB substrate (Thermo Fisher Scientific, Waltham, MA, Catalog No. N301) for 2-5 minutes, followed by addition of ELISA Stop Solution (Invitrogen, Carlsbad, CA, Catalog No. SS04). Absorbance was measured at 450 nm.Data points were normalized to isotype control Ab values ​​at each concentration and reported as % absorbance 450 nm normalized to control Ab. A nonlinear regression analysis was used to fit a curve (4 parameters) to the data using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA). The results are shown in Figure 1 and Table 11.

[0291] (Table 11) [Table 13]

[0292] Three classes of antibodies were identified, defined by their ability to strongly inhibit (>=93% maximal inhibition), partially inhibit (<=60% maximal inhibition), or not inhibit FN binding (Table 11). Six antibodies that strongly or partially inhibited α5β1 binding to FN were selected for further characterization.

[0293] Example 6: Competitive binding assay to identify epitope-binding groups Epitope analysis by competitive binding studies was performed using Biolayer Interferometry (BLI) for six antibodies selected as described in Example 5. To determine whether this antibody-antigen binding prevents or allows binding to the antibody on the sensor, anti-α5β1 FN blocking antibodies A2-7A05, C-14D12, and A-15B08 were immobilized on the sensor and incubated with each of the other antibodies preincubated with rh-α5β1. The magnitude of binding (response) observed was compared to the binding of the antigen alone under the same conditions. If the overall response was greater than 120% of the antigen binding alone, the antibodies can pair with each other. If the response was less than 80% of the antigen binding alone, the antibodies block each other. The protocol was as follows: preincubate the panel of antibodies with the antigen (1 hour). Equilibrate the sensor (30 seconds (s)) → Load lead Ab onto the sensor (700 seconds) → Quench (480 seconds) → Read baseline (480 seconds) → Measure pre-incubated Ab+Ag binding with the loaded sensor (600 seconds). The results are shown in Table 12A and Table 12B.

[0294] For example, rh-α5β1 complexed with A2-7A05 or A2-7F01 was able to bind to C-14D12 and A-15B08 on the sensor. rh-α5β1 complexed with A-15B08, A2-3B06, A2-5D10, or C-14D12 was able to bind to A2-7A05 on the sensor. A2-3B06 and C-14D12 were unable to bind to A-15B08 on the sensor when complexed with α5β1, and A-15B08 and A2-3B06 complexed with rh-α5β1 were unable to bind to C-14D12 on the sensor. The results in Tables 12A and 12B show that two epitope groups are presented by these six antibodies. A2-7A05 and A2-7F01 represent one group, and A-15B08, A2-3B06, A2-5D10, and C-14D12 represent the second group.

[0295] (Table 12A) [Table 14] *Premixed with 20nM rh-α5β1

[0296] (Table 12B) [Table 15]

[0297] Example 7: Antibody Binding Disrupts the α5β1-FN Integrin-Ligand Complex Surface plasmon resonance (SPR) and cell-based assays were used to test the effect of antibodies on the dissociation of rh-α5β1 protein bound to human FN protein. The antibodies tested are representative of two groups of antibodies identified in Example 6 that define two different epitope binding groups and have distinct ligand-blocking properties. These are A-15B08, a potent blocker of FN binding, and A2-7A05, a partial blocker of FN binding. In addition, the small molecule antagonist cyclic RGD (cRGD; Creative-Peptides, Shirley, New York, 11967, catalog number CP22175) was tested as a control drug that inhibits the binding of α5β1 integrin to FN by competing in the ligand-binding pocket.

[0298] Description of the SPR method used: FN protein dissolved in water was manually printed on bare gold coated (47 nm thick) PlexArray Nanocapture Sensor Chip (Plexera Bioscience, Seattle, WA) at 40% humidity. The chip was incubated at 4°C overnight at 80% humidity and rinsed for 10 min with 10x PBST (0.1 M phosphate buffer, 1.54 M NaCl, pH 7.4, 0.5% Tween 20), 10 min with 1x PBST (0.01 M phosphate buffer, pH 7.4, 0.154 M NaCl, 0.05% Tween 20), and twice with deionized water for 10 min. The chip was then blocked overnight with 5% (w / v) skim milk in water, washed for 10 min with 10x PBST, 10 min with 1x PBST, and twice for 10 min with deionized water, and then dried under a stream of nitrogen before use. SPR measurements were performed using a high-throughput surface plasmon resonance imaging (SPRi) platform, PlexArray HT (Plexera Bioscience, Seattle, WA). Collimated light (660 nm) passes through a coupling prism, is reflected off an SPR-active gold surface, and is received by a CCD camera. Buffer and sample were pumped by a non-pulsating piston pump into a 30 μL flow cell mounted on a coupling prim. Each SPR measurement cycle included four steps: washing with 1x PBS running buffer at a constant rate of 2μL / s to obtain a stable baseline, injection of 400nM rh-α5β1 at 5uL / s for 300s (to equilibrate) for binding to FN, followed by injection of running buffer alone at 2μL / s for 50s to allow dissociation of rh-α5β1, and a final injection of 1μM antibody at 2μL / s for 250s. All measurements were performed at 25°C. SPR binding responses (au) were recorded and plotted over time.

[0299] Both antibodies positively affected the dissociation of rh-α5β1 protein bound to FN, but to different extents, and cRGD had no effect. Figure 4A shows the overlay of SPR responses with and without the addition of antibody or cRGD. Injection of either antibody resulted in a transient increase in SPR response indicative of the formation of a ternary complex of FN, rh-α5β1 protein, and antibody. Remarkably, the A-15B08 antibody, previously shown by ELISA to be a potent blocker of FN binding, caused rapid dissociation of the FN-α5β1 complex, as detected by SPR response, decreasing to nearly baseline within the 250 seconds between the time the antibody was injected and the SPR response was measured. This propensity of A-15B08 to rapidly dissociate the complex contrasts with published results showing significantly slower dissociation rates with other anti-α5β1 antibodies under similar conditions (Mould et al., J Bio Chem., 30; 291 (40): 20993-21007 (2016)). The A2-7A05 antibody, shown by ELISA to only partially block α5β1 binding to FN, did not cause such a rapid reduction in the SPR response, but did result in a slower partial dissociation comparable to the results previously published by Mould et al. (2016) for several other antibodies. In contrast, injection of the small molecule antagonist cRGD had no discernible effect on the dissociation rate of the complex. Both antibodies have similar binding affinities for the rh-α5β1 protein (K D 1.31E-09 and 0.8E-09, see also Table 9, suggesting that the difference in dissociation rates induced by these antibodies is not a specific function of binding affinity, but rather an intrinsic property of the binding interaction. As previously shown by Mould et al. (2016), ligand site competitive antagonists such as cRGD are not as effective at dissociating the ligand-integrin complex as are allosteric antagonist antibodies, suggesting that an allosteric mechanism is responsible for the dissociation activity seen with the antibodies tested here.

[0300] The A-15B08 and A2-7A05 antibodies and cRGD were also tested for their ability to induce dissociation of cellular α5β1 integrin from FN. U87MG cells (HTB-14™, ATCC, Manassas, VA), originally derived from a glioblastoma tumor, express α5β1 integrin and are known to adhere to FN-coated plates. After overnight incubation on FN-coated plates, U87MG cells form a loosely packed monolayer with an elongated spindle shape. When the cells are induced to detach from the plate, for example with trypsin, the attachment points are released and the cells become rounded. This type of morphological change was used to assess whether the antibodies and the small molecule inhibitor cRGD can induce U87MG cells to detach from FN-coated plates.

[0301] Cell detachment activity was assessed using the following method: 96-well cell culture plates (Thermo Fisher Scientific, Waltham, MA, catalog number 165306) were coated with FN (human fibronectin, R&D Systems, Minneapolis, MN 55413, catalog number 1918-FN) by incubation of 32 μg / mL FN in 1× PBS (50 uL per well) for 1 hour at 37°C, then washed twice with EMEM medium (ATCC, Manassas, VA, catalog number 30-2003) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, catalog number 30-2021). U87MG cells were plated overnight at 20,000 cells per well in EMEM medium + 10% FBS to allow maximum attachment. The next day, the medium was replaced with fresh medium containing a 1:5 dilution series of antibodies A-15B08, A2-7A05, or isotype control IgG4 at concentrations of 2.0, 0.4, 0.08, and 0.016 μg / mL or cRGD at 20, 4, 0.8, and 0.16 μM. All experiments were performed in duplicate. Cell morphology was assessed and images were captured using a 10x objective on an ECHO Rebel light microscope (ECHO, San Diego, CA).

[0302] The results show that antibody A-15B08, a representative of potent FN blocking antibodies, caused significant cell rounding due to detachment of cell contacts at an antibody concentration of only 0.4 μg / mL, which was observed at both 1 and 3 hours, the latter time when images were captured (FIG. 4B). The partial antagonist A2-7A05 also showed cell detachment at an antibody concentration of 0.4 μg / mL, but at a much lower level and only evident at the 3 hour time point. The ability of this weaker antibody to detach adherent cells is consistent with it being only a partial inhibitor. The IgG4 isotype control and cRGD treatment did not show significant cell detachment at any of the concentrations or time points assayed (FIG. 4B). The extent to which the two antibodies dissociated cells from FN-coated wells is consistent with the SPR dissociation data obtained with purified protein (Figure 4A), in which the strong antagonist antibody caused rapid dissociation of the integrin-FN complex and the partial antagonist only weakly induced dissociation.

[0303] Example 8: Generation of human IgG4 chimeras and removal of N-glycosylation sites in CDRH2 does not significantly affect the FN blocking activity of the antibody. Antibody expression plasmids were constructed as human IgG4 chimeras with variable domains from antibodies A-15B08, C-14D12, and A2-7A05. In addition, the threonine residue at position 62 of CDRH2 of antibody A-15B08 was changed to alanine to remove a potential N-glycosylation site, and the IgG4 clone was named A-15B08-T62A. The VH and VL sequences and the six CDR sequences (with various numbering schemes) of A-15B08-T62A are shown in Figures 2C and 2D. The accuracy of the sequences was verified using Sanger sequencing, and the plasmid concentration was determined by measuring the absorbance at a wavelength of 260 nm. The expression clones were transfected into suspension-adapted CHO K1 cells and grown in animal component-free serum-free medium. The supernatant was collected by centrifugation followed by filtration (0.2 μm filter). Antibodies were purified using MabSelect™ SuRe™ (Cytiva, Marlborough, MA). Purity was determined by analytical size-exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7um 7.8×300mm; Agilent Technologies, Santa Clara, CA) with PBS as elution buffer at 0.8 mL / min. Yields were determined by absorbance at 280 nm. All four chimeric antibodies A-15B08, A-15B08-T62A, C-14D12, and A2-7A05 were expressed at high levels with yields of 55, 53, 43, and 24 mg, respectively, from 250 mL culture.

[0304] Freeze-thaw stability was tested on small aliquots of each chimeric antibody by three repeated overnight storage at -80°C followed by thawing on ice. All four chimeric antibodies were stable to three freeze-thaw cycles as judged by their ability to inhibit α5β1 integrin binding to fibronectin (FN) compared to antibodies stored only at 4°C. ELISA methods were used as described in Example 2. IC50s were calculated by nonlinear regression analysis curve fitting (four parameters) of the ELISA data using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA) (Figure 5). The resulting IC50s indicate that three freeze-thaws (F / T) achieved only marginal antibody potency in the FN blocking assay (Table 13). In addition, the potency of the IgG4 chimeras was compared to that of the mouse hybridomas from which they were derived using the same FN blocking ELISA and IC50 determination methods. The results show that the chimera was slightly more potent than the hybridoma, approximately 2-fold (see Figure 6 and Table 14), in all cases except for A2-7A05, where the difference was less (IC50 0.115 vs. 0.130). (Table 13) [Table 16] (Table 14) [Table 17]

[0305] The glycosylation status of A-15B08 was determined by comparing the mobility of its heavy chain on SDS-PAGE with that of A-15B08-T62A, which contains a T to A mutation at position 62 of the heavy chain variable domain in the putative N-glycosylation site NST. 2 μg of each antibody was separated by SDS-PAGE using Bolt™ 4-12.5% ​​Tris-Bis Plus (Invitrogen, Carlsbad, CA, Catalog No. NW04120BOX) mini protein gels run with MOPS buffer (Invitrogen, Carlsbad, CA, Catalog No. B0001). The mobility of the antibody heavy chain A-15B08-T62A with the mutated N-glycosylation site migrated similarly to the other two antibody heavy chains from C-14D12 and A2-7A05, which do not contain the putative N-glycosylation site. Notably, the heavy chain of A-15B08 migrated slower than the other three antibodies, providing evidence that it was, in fact, glycosylated at this site (Figure 7). Based on this data, it is expected that antibody A2-5D10 (not mutant and not tested by SDS-PAGE) will also be glycosylated at a similar NST sequence in its CDRH2.

[0306] Example 9: Antibody binding to α5β1 integrin shifts its conformation to an inactive form The ability of anti-α5 antibodies to modulate the conformation of integrins from an active to an inactive conformation was assayed using 12G10 (mouse anti-human integrin β1 / CD29 antibody, Novus Biologicals, Littleton, CO, catalog number NB100-63255), an antibody that preferentially binds to the β1-chain when the integrin is in the active or open conformation. Nunc MaxiSorp flat-bottom 96-well plates (Invitrogen, Waltham, MA, catalog number 44-2404-21) were coated with 2 μg / mL of 12G10 antibody in 0.2 M carbonate-bicarbonate buffer, pH 9.4 (Thermo Scientific, Rockford, IL, catalog number 28382) by overnight incubation at 4° C. Plates were then washed three times with wash buffer (1x Tris-buffered saline with 0.05% Tween 20) and then blocked with 2% BSA in 1x TBS for 2 hours at room temperature (RT). Human IgG4 chimeric versions of antibodies were diluted in standard diluent (2% BSA, 1x TBS, 0.05% Tween 20) containing 0.05 μg / mL rh-α5β1-6xHis tagged protein (Acro Biosystems, Newark, DE, Catalog No. IT1-H52W5) and 0.5 mM MnCl2 (TEKnova, Hollister, CA, Catalog No. M0350) to generate a seven-point 1:5 antibody dilution series ranging from 10,000 ng / mL to 0.64 ng / mL. In addition to the human IgG4 chimeric antibodies A-15B08, C-14D12, and A2-7A05, three other antibodies tested included an IgG4 isotype control (human IgG4, kappa, anti-fluorescein Ab00102-13.0, Absolute Antibody, Wilton, UK), the anti-integrin α-5 clone SNAKA51 (MilliporeSigma, St. Louis, MO, catalog no. MABT201), which is known to drive integrin α-5 into its active conformation, and finally the 12G10 antibody itself, which can directly compete with 12G10 bound to MaxiSorp plates.For the assay, the blocking solution was removed and the wells were washed three times before adding 100 μL of the antibody dilution series, His-tagged α5β1 mixture to the wells. After 1 h at room temperature, the wells were washed three times, incubated with biotinylated anti-6xHis-tag Ab (Invitrogen, Carlsbad, CA, Catalog No. MAI-21315-BTIN) at 1:1000 in standard diluent for 1 h, washed three times, incubated with poly-HRP streptavidin (Thermo Fisher Scientific, Waltham, MA, Catalog No. N200) for 30 min, washed three times, and incubated with TMB substrate (Thermo Fisher Scientific, Waltham, MA, Catalog No. N301) for 2–5 min, followed by the addition of ELISA Stop Solution (Invitrogen, Carlsbad, CA, Catalog No. SS04). Absorbance was measured at 450 nm. Data points were normalized to the absorbance of wells without antibody for each dilution series and reported as percent binding. A curve (3 parameters) was fitted to the data using nonlinear regression analysis with GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA). The results are shown in Figure 8.

[0307] The results show that two antibodies, A-15B08 and C-14D12, previously shown to strongly inhibit α5β1 binding to FN, reduced α5β1 integrin binding to 12G10 by approximately 50%, while antibody A2-7A05, which partially inhibits binding to FN, did not reduce α5β1 integrin binding to 12G10. Similarly, the IgG4 isotype control did not reduce α5β1 integrin binding to 12G10. The SNAKA51 antibody, known to shift α5β1 integrin to an active conformation, increased α5β1 integrin binding to 12G10. Without wishing to be bound by any theory, antibodies that strongly inhibit α5β1 integrin binding to its primary ligand, FN, may do so in part by shifting the conformation of α5β1 integrin to an inactive conformational state.

[0308] Example 10: Antibody inhibition of cell adhesion to fibronectin The cell surface α5β1 integrin receptor interacts with FN to promote cell adhesion. To test whether a human IgG4 chimeric anti-α5β1 antibody inhibits adhesion, a plate-based cell adhesion assay was developed for use with the U87MG cell line (HTB-14™, ATCC, Manassas, VA), which is derived from a glioblastoma tumor and known to express α5β1 integrin. U87MG cells were grown to 80% confluence in EMEM medium (ATCC, Manassas, VA, catalog number 30-2003) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, catalog number 30-2021) in a 5% CO2, 37°C incubator. Cells were dissociated from flasks using 0.05% trypsin, 0.02% EDTA (Lifeline Cell Technology, Frederick, MD, catalog number CM0017), washed once with Dulbecco's modified 1x PBS (DPBS) containing calcium and magnesium (HyClone, Logan, UT, catalog number SH30028-02), and resuspended at a concentration of 2 million cells per ml in unsupplemented EMEM medium. Cells were rested for 30 minutes in an incubator before being mixed with a six-point 1:3 dilution series of antibodies ranging from 3 to 0.01 μg / mL final concentrations in a 96-well polypropylene round-bottom plate. 100,000 rested cells were premixed with each dilution of antibody to a final volume of 100 μL and then transferred to a human FN-coated 96-well plate (R&D Systems, Minneapolis, MN, catalog number CWP001). The cell-antibody mixture was incubated for 1 hour at 37° C., 5% CO2. Non-adherent cells were removed from the wells by inverting the plates onto an absorbent pad, then washed twice with 200 uL per well of 1×DPBS without calcium and magnesium (EMD Millipore Corp, Billerica, MA, Catalog No. TMS-012-A).To facilitate cell counting, 100 μL of 1×DPBS (containing Hoechst 33342 dye (Thermo Fisher Scientific, Waltham, MA, Cat. No. 62249) at the recommended dilution (1:2000) was added to each well to fluorescently stain the nuclei, and cells that remained attached to the wells were counted using image analysis software provided by the ImageXpress Pico Automated Cell Imaging System (Molecular Devices, San Jose, CA), which in this case is based on counting fluorescently stained nuclei using a DAPI filter to detect Hoechst nuclear staining. A curve (4 parameters) was fitted to the data using nonlinear regression analysis with GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA).

[0309] The results are shown in FIG. 9. The average number of U87MG cells that adhered to FN-coated wells in the absence of antibody was 37,022 with a standard deviation (stdev) of 2,364 (n=16). The average number of cells from wells without FN coating was 628 with a standard deviation of 143 (n=12). The highest concentration of antibody tested at 3 μg / mL inhibited adhesion by 78%-86% for antibodies A-15B08, A-15B08-T62A, and C-14D12, which are potent blockers of FN binding, and by 25% for antibody A2-7A05, which was shown to be a partial blocker of FN binding (Table 15). The cell adhesion blocking assay data demonstrated the inhibitory effect of the antibodies on α5β1 binding to Fn and on cell adhesion. (Table 15) [Table 18] (Implementation) 1. Regarding binding to α5β1 integrin: (A) (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 42 and a light chain variable region having the amino acid sequence of SEQ ID NO: 43; (iii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; (iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 109 and a light chain variable region having the amino acid sequence of SEQ ID NO: 110; (v) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 135 and a light chain variable region having the amino acid sequence of SEQ ID NO: 136; (vi) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 136 and a light chain variable region having the amino acid sequence of SEQ ID NO: 137; (vii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 138 and a light chain variable region having the amino acid sequence of SEQ ID NO: 139; (viii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 144 and a light chain variable region having the amino acid sequence of SEQ ID NO: 145; (ix) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 146 and a light chain variable region having the amino acid sequence of SEQ ID NO: 147; or (B) (i) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 77 and a light chain variable region having the amino acid sequence of SEQ ID NO: 78; (ii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 91 and a light chain variable region having the amino acid sequence of SEQ ID NO: 92; (iii) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 140 and a light chain variable region having the amino acid sequence of SEQ ID NO: 141; and / or (iv) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 142 and a light chain variable region having the amino acid sequence of SEQ ID NO: 143. An antibody or fragment thereof that competes with an antibody comprising the compound. 2. An antibody or fragment thereof that binds to α5β1 integrin, comprising: (a) Below: (1): (i) SEQ ID NO: 1, 27, 53, or 93, (ii) SEQ ID NO: 7, 31, 59, or 97, (iii) SEQ ID NO: 12, 34, 64, or 100, (iv) SEQ ID NO: 13, 35, 65, or 101, and (v) SEQ ID NO: 18, 38, 70, or 105 V having an amino acid sequence selected from the group consisting of H CDR1; (2): (i) SEQ ID NO: 2, 28, 54, or 79, (ii) SEQ ID NO: 8, 60, or 82, (iii) SEQ ID NO: 14, 66, or 84, (iv) SEQ ID NO: 19, 71, or 87, and (v) SEQ ID NO: 24, 76, or 90 V having an amino acid sequence selected from the group consisting of H CDR2; and (3): (i) SEQ ID NO: 3, 29, 55, 80, or 94, (ii) SEQ ID NO: 9, 32, 61, 83, or 98, (iii) SEQ ID NO: 15, 36, 67, 85, or 102, and (iv) SEQ ID NO: 20, 39, 72, 88, or 106 V having an amino acid sequence selected from the group consisting of H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1): (i) SEQ ID NO: 4, 30, 44, 56, or 95, (ii) SEQ ID NO: 10, 33, 46, 62, or 99, (iii) SEQ ID NO: 16, 37, 47, 68, or 103, and (iv) SEQ ID NO: 21, 40, 49, 73, or 107 V having an amino acid sequence selected from the group consisting of L CDR1; (2): (i) SEQ ID NO: 5 or 57, (ii) SEQ ID NO: 11 or 63, and (iii) SEQ ID NO: 22, 41, or 74 V having an amino acid sequence selected from the group consisting of L CDR2; and (3): (i) SEQ ID NO: 6, 45, 58, 81, or 96, (ii) SEQ ID NO: 17, 48, 69, 86, or 104, and (iii) SEQ ID NO: 23, 50, 75, 89, or 108 V having an amino acid sequence selected from the group consisting of L CDR3 The light chain variable (V L )region The antibody or fragment thereof comprising: 3. An antibody or fragment thereof that binds to α5β1 integrin, comprising: (1): The following (i) SEQ ID NO: 1, 27, 53, or 93, (ii) SEQ ID NO: 7, 31, 59, or 97, (iii) SEQ ID NO: 12, 34, 64, or 100, (iv) SEQ ID NO: 13, 35, 65, or 101, and (v) SEQ ID NO: 18, 38, 70, or 105 V having an amino acid sequence selected from the group consisting of H CDR1; (2): The following (i) SEQ ID NO: 2, 28, 54, or 79, (ii) SEQ ID NO: 8, 60, or 82, (iii) SEQ ID NO: 14, 66, or 84, (iv) SEQ ID NO: 19, 71, or 87, and (v) SEQ ID NO: 24, 76, or 90 V having an amino acid sequence selected from the group consisting of H CDR2; and (3): The following (i) SEQ ID NO: 3, 29, 55, 80, or 94, (ii) SEQ ID NO: 9, 32, 61, 83, or 98, (iii) SEQ ID NO: 15, 36, 67, 85, or 102, and (iv) SEQ ID NO: 20, 39, 72, 88, or 106 V having an amino acid sequence selected from the group consisting of H CDR3 Heavy chain variable (V H )region The antibody or fragment thereof comprising: 4. An antibody or fragment thereof that binds to α5β1 integrin, comprising: (1): The following (i) SEQ ID NO: 4, 30, 44, 56, or 95, (ii) SEQ ID NO: 10, 33, 46, 62, or 99, (iii) SEQ ID NO: 16, 37, 47, 68, or 103, and (iv) SEQ ID NO: 21, 40, 49, 73, or 107 V having an amino acid sequence selected from the group consisting of L CDR1; (2): The following (i) SEQ ID NO: 5 or 57, (ii) SEQ ID NO: 11 or 63, and (iii) SEQ ID NO: 22, 41, or 74 V having an amino acid sequence selected from the group consisting of L CDR2; and (3): The following (i) SEQ ID NO: 6, 45, 58, 81, or 96, (ii) SEQ ID NO: 17, 48, 69, 86, or 104, and (iii) SEQ ID NO: 23, 50, 75, 89, or 108 V having an amino acid sequence selected from the group consisting of L CDR3 Light chain variable (V L )region The antibody or fragment thereof comprising: 5. An antibody designated A-15B08, comprising a VH sequence that is SEQ ID NO:25 and a VL sequence that is SEQ ID NO:26; An antibody designated A-15B08-T62A comprising a VH sequence that is SEQ ID NO:135 and a VL sequence that is SEQ ID NO:26; An antibody designated A-15B08_Low, comprising a VH sequence of SEQ ID NO: 136 and a VL sequence of SEQ ID NO: 137; An antibody designated A-15B08_Low+Mod, comprising a VH sequence of SEQ ID NO: 138 and a VL sequence of SEQ ID NO: 139; An antibody designated A2-3B06, comprising a VH sequence that is SEQ ID NO:42 and a VL sequence that is SEQ ID NO:43; An antibody designated A2-5D10, comprising a VH sequence that is SEQ ID NO:51 and a VL sequence that is SEQ ID NO:52; An antibody designated A2-7A05, comprising a VH sequence that is SEQ ID NO: 77 and a VL sequence that is SEQ ID NO: 78; An antibody designated A2-7A05_Low, comprising a VH sequence of SEQ ID NO: 140 and a VL sequence of SEQ ID NO: 141; An antibody designated A2-7A05_Low+Mod, comprising a VH sequence of SEQ ID NO: 142 and a VL sequence of SEQ ID NO: 143; An antibody designated A2-7F01, comprising a VH sequence that is SEQ ID NO:91 and a VL sequence that is SEQ ID NO:92; an antibody designated C-14D12, comprising a VH sequence that is SEQ ID NO: 109 and a VL sequence that is SEQ ID NO: 110; An antibody designated C-14D12_Low, comprising a VH sequence of SEQ ID NO: 144 and a VL sequence of SEQ ID NO: 145; or An antibody designated C-14D12_Low+Mod, comprising the VH sequence of SEQ ID NO: 146 and the VL sequence of SEQ ID NO: 147. An antibody or fragment thereof that binds to α5β1 integrin, comprising all three heavy chain complementarity determining regions (CDRs) or all three light chain CDRs derived from said antibody or fragment thereof. 6. An antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated A-15B08. 7. An antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated A2-3B06. 8. An antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated A2-5D10. 9. The antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated A2-7A05. 10. The antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated A2-7F01. 11. The antibody or fragment thereof described in embodiment 5, comprising all three heavy chain CDRs and all three light chain CDRs from the antibody designated C-14D12. 12. An antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody is: (a) V listed in Tables 1 to 6 H CDR1, V H CDR2, and V H Heavy chain variable (V H ) area; or (b) V listed in Tables 1 to 6 L CDR1, V L CDR2, and V L The light chain variable (V L )region The antibody or fragment thereof comprising: 13. The antibody comprising: (a) V listed in Tables 1 to 6 H CDR1, V H CDR2, and V H Heavy chain variable (V H ) area; and (b) V listed in Tables 1 to 6 L CDR1, V L CDR2, and V L The light chain variable (V L )region 13. The antibody or fragment thereof of embodiment 12, comprising: 14. The antibody is selected from the group consisting of V and VL2 antibodies listed in Tables 1 to 6. H CDR1, V H CDR2, and V H Heavy chain variable (V H 13. The antibody or fragment thereof of embodiment 12, comprising a .DELTA.A-.DELTA.A-.DELTA.I ... 15. The antibody is selected from the group consisting of V and VL2 antibodies listed in Tables 1 to 6.L CDR1, V L CDR2, and V L The light chain variable (V L 13. The antibody or fragment thereof of embodiment 12, comprising a .DELTA.A-.DELTA.A-.DELTA.I ... 16. The antibody comprising: (a) Below: (1) SEQ ID NO: V having an amino acid sequence selected from the group consisting of 1, 7, 12, 13, and 18 H CDR1; (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 14, 19, and 24 H CDR2; and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 9, 15, and 20 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, 16, and 21 L CDR1; (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 22 L CDR2; and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23 L CDR3 Light chain variable (V L )region 13. The antibody or fragment thereof of embodiment 12, comprising: 17. The antibody comprising: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 1 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 2 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 The light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 18. The antibody comprising: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 7 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 8 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 9 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 10 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 The light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 19. The antibody: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 12 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 2 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 Light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 20. The antibody comprising: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 13 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 14 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 15 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 16 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 11 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 17 L CDR3 Light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 21. The antibody: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 18 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 19 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 20 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 21 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 22 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 23 L CDR3 The light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 22. The antibody comprising: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 1 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 24 H CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 3 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having the amino acid sequence of SEQ ID NO: 4 L CDR1; (2) V having the amino acid sequence of SEQ ID NO: 5 L CDR2; and (3) V having the amino acid sequence of SEQ ID NO: 6 L CDR3 The light chain variable (V L )region 17. The antibody or fragment thereof of embodiment 16, comprising: 23. The antibody comprising: (a) Below: (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 31, 34, 35, and 38. H CDR1; (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 19, 24, and 28 H CDR2; and (3) SEQ ID NO: V having an amino acid sequence selected from the group consisting of 29, 32, 36, and 39 H CDR3 Heavy chain variable (V H ) area; and (b) Below: (1) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 33, 37, and 40 L CDR1; (2) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 11, and 41 L CDR2, and (3) V having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 17, and 23 L CDR3 The light chain variable (V L )region 13. The antibody or fragment thereof of embodiment 12, comprising: 24. The antibody comprising: (a) Below: (1) V having the amino acid sequence of SEQ ID NO: 27 H CDR1; (2) V having the amino acid sequence of SEQ ID NO: 28 H CDR2; and (3) V having the ami...

Claims

1. Binds to alpha 5 (α5) integrin, and: a) a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 136, and / or a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 139; b) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 136, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 137; c) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 138, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 139; d) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in SEQ ID NO: 138, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in SEQ ID NO: 137; e) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 135, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 26; f) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 25, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 26; g) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 42, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 43; h) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 51, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 52; i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 77, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 78; j) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 91, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 92; k) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 109, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 110; l) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 140, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 141; m) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 142, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 143; n) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in the VH comprising the amino acid sequence of SEQ ID NO: 144, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in the VL comprising the amino acid sequence of SEQ ID NO: 145; or o) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in SEQ ID NO: 146, and / or a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in SEQ ID NO: 147, An antibody or an antigen-binding fragment thereof comprising:

2. a) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 set forth in VH comprising the amino acid sequence of SEQ ID NO: 136, and / or the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 set forth in VL comprising the amino acid sequence of SEQ ID NO: 139; b) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 set forth in VH comprising the amino acid sequence of SEQ ID NO: 136, and / or the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 set forth in VL comprising the amino acid sequence of SEQ ID NO: 137; c) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 set forth in VH comprising the amino acid sequence of SEQ ID NO: 138, and / or the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 set forth in VL comprising the amino acid sequence of SEQ ID NO: 139; or d) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in VH comprising the amino acid sequence of SEQ ID NO: 138, and / or the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in VL comprising the amino acid sequence of SEQ ID NO: 137; The antibody or antigen-binding fragment thereof according to claim 1.

3. a) the VH is: (i)(1) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (2) VH CDR2 including the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSALKS set forth in SEQ ID NO: 28; and (3) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; or (ii)(1) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (2) VH CDR2 including the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSSLKS; and (3) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and b) the VL is: (i)(1) a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH; (2) VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAT, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (3) a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; or (ii)(1) a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH; (2) VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAS, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (3) A VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. a) the VH is (i) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSALKS set forth in SEQ ID NO: 28, and (iii) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and b) the VL is (i) a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH: (ii) a VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAT, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (iii) a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

5. (i) according to the Kabat numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence SYGVH set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSALKS set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAT, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (ii) according to the AbM numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAT, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (iii) according to the Chothia numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence SDG set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence YLRSPP set forth in SEQ ID NO: 17; (iv) according to the Contact numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence TSYGVH set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence LWIYSTSNRA, and a VL CDR3 comprising the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; or (v) according to the IMGT numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYG set forth in SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO:9; and the VL comprises a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO:

6. The antibody or antigen-binding fragment thereof according to claim 4.

6. a) the VH is (i) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSALKS set forth in SEQ ID NO: 28, and (iii) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and b) the VL is (i) VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH: (ii) a VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAS, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (iii) a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. (i) according to the Kabat numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence SYGVH set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSALKS set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAS, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (ii) according to the AbM numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAS, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (iii) according to the Chothia numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence SDG set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence YLRSPP set forth in SEQ ID NO: 17; (iv) according to the Contact numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence TSYGVH set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence LWIYSTSNRA, and a VL CDR3 comprising the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; or (v) according to the IMGT numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYG set forth in SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO:9; and the VL comprises a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO:

6. The antibody or antigen-binding fragment thereof according to claim 6.

8. a) the VH is (i) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSSLKS; and (iii) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and b) the VL is (i) a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH; (ii) a VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAT, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (iii) a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

9. (i) according to the Kabat numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence SYGVH set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSSLKS, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAT, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (ii) according to the AbM numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAT, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (iii) according to the Chothia numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence SDG set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence YLRSPP set forth in SEQ ID NO: 17; (iv) according to the Contact numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence TSYGVH set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence LWIYSTSNRA, and a VL CDR3 comprising the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; or (v) according to the IMGT numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYG set forth in SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO:9; and the VL comprises a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO:

6. The antibody or antigen-binding fragment thereof according to claim 8.

10. a) the VH is (i) a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, the amino acid sequence GFSLTSYG set forth in SEQ ID NO: 7, the amino acid sequence SYGVH set forth in SEQ ID NO: 12, the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, or the amino acid sequence TSYGVH set forth in SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO: 8, the amino acid sequence SDG set forth in SEQ ID NO: 14, the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, or the amino acid sequence VIWSDGSTTYNSSLKS; and (iii) a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3, the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO: 9, the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15, or the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and b) the VL is (i) VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO: 10, the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, or the amino acid sequence RASSRVSSNSLH: (ii) a VL CDR2 comprising the amino acid sequence STS, the amino acid sequence STSNRAS, or the amino acid sequence LWIYSTSNRA set forth in SEQ ID NO: 11; and (iii) a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6, the amino acid sequence YLRSPP set forth in SEQ ID NO: 17, or the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

11. (i) according to the Kabat numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence SYGVH set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence VIWSDGSTTYNSSLKS, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAS, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (ii) according to the AbM numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYGVH set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence VIWSDGSTT set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence HYDYDGDWFAY set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence RASSRVSSNSLH, a VL CDR2 comprising the amino acid sequence STSNRAS, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO: 6; (iii) according to the Chothia numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSY set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence SDG set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence YDYDGDWFA set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence SSRVSSNS set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence YLRSPP set forth in SEQ ID NO: 17; (iv) according to the Contact numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence TSYGVH set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence WLVVIWSDGSTT set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFA set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence SSNSLHWY set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence LWIYSTSNRA, and a VL CDR3 comprising the amino acid sequence HQYLRSPP set forth in SEQ ID NO: 23; or (v) according to the IMGT numbering scheme, the VH comprises a VH CDR1 comprising the amino acid sequence GFSLTSYG set forth in SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence IWSDGST set forth in SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence ARHYDYDGDWFAY set forth in SEQ ID NO:9; and the VL comprises a VL CDR1 comprising the amino acid sequence SRVSSNS set forth in SEQ ID NO:10, a VL CDR2 comprising the amino acid sequence STS set forth in SEQ ID NO:11, and a VL CDR3 comprising the amino acid sequence HQYLRSPPT set forth in SEQ ID NO:

6. The antibody or antigen-binding fragment thereof according to claim 10.

12. (A) the VH is: (a)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; (b)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:24; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; (c)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:38; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:36, or SEQ ID NO:39; (d)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:24; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; (e)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:70; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:71, or SEQ ID NO:76; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, or SEQ ID NO:72; (f)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:70; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:87, or SEQ ID NO:90; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:88; or (g)(i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:101, or SEQ ID NO:105; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, or SEQ ID NO:106; and (B) the VL is: (a)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:21; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:17, or SEQ ID NO:23; (b)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:37, or SEQ ID NO:40; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:41; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:17, or SEQ ID NO:23; (c)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:49; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45, SEQ ID NO: 48, or SEQ ID NO: 50; (d)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, or SEQ ID NO:73; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:57, SEQ ID NO:63, or SEQ ID NO:74; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:75; (e)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68, or SEQ ID NO:73; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:57, SEQ ID NO:63, or SEQ ID NO:74; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81, SEQ ID NO: 86, or SEQ ID NO: 89; or (f)(i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, or SEQ ID NO: 107; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96, SEQ ID NO: 104, or SEQ ID NO: 108; The antibody or antigen-binding fragment thereof according to claim 1.

13. (A)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28, and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, or SEQ ID NO: 21; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22, and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:17, or SEQ ID NO:23; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (B)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:24, and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, or SEQ ID NO: 21; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:17, or SEQ ID NO:23; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (C)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 38; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28, and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:36, or SEQ ID NO:39; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 37, or SEQ ID NO: 40; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:41; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:17, or SEQ ID NO:23; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (D)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 18; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, or SEQ ID NO:24, and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:20; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 49; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:45, SEQ ID NO:48, or SEQ ID NO:50; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (E)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 70; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:71, or SEQ ID NO:76; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, or SEQ ID NO:72; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, SEQ ID NO: 62, SEQ ID NO: 68, or SEQ ID NO: 73; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 63, or SEQ ID NO: 74; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:75; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 60, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 61; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 70, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 71, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 72; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58; (F)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 70; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:87, or SEQ ID NO:90; and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:88; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, SEQ ID NO: 62, SEQ ID NO: 68, or SEQ ID NO: 73; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 63, or SEQ ID NO: 74; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:81, SEQ ID NO:86, or SEQ ID NO:89; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 59, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 62, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 63, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 70, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 87, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 88; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89; or (7) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; or (G)(1) the VH is (i) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 105; (ii) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:24, or SEQ ID NO:28, and (iii) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102, or SEQ ID NO:106; and (i) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, or SEQ ID NO: 107; (ii) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:11, or SEQ ID NO:22; and (iii) comprises a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:96, SEQ ID NO:104, or SEQ ID NO:108; (2) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96; (3) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 98; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 99, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96; (4) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 96; (5) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104; (6) the VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108; or (7) The VH comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94; and the VL comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

96.

13. The antibody or antigen-binding fragment thereof according to claim 12.

14. (i) the VH comprises the amino acid sequence set forth in SEQ ID NO: 136, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 139; (ii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 136, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 137; (iii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 138, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 139; (iv) the VH comprises the amino acid sequence set forth in SEQ ID NO: 138, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 137; (v) the VH comprises the amino acid sequence set forth in SEQ ID NO: 135, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 26; (vi) the VH comprises the amino acid sequence set forth in SEQ ID NO:25, and the VL comprises the amino acid sequence set forth in SEQ ID NO:26; (vii) the VH comprises the amino acid sequence set forth in SEQ ID NO:42, and the VL comprises the amino acid sequence set forth in SEQ ID NO:43; (viii) the VH comprises the amino acid sequence set forth in SEQ ID NO:51, and the VL comprises the amino acid sequence set forth in SEQ ID NO:52; (ix) the VH comprises the amino acid sequence set forth in SEQ ID NO: 109, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 110; (x) the VH comprises the amino acid sequence set forth in SEQ ID NO: 144, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 145; (xi) the VH comprises the amino acid sequence set forth in SEQ ID NO: 146, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 147; (xii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 77, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 78; (xiii) the VH comprises the amino acid sequence set forth in SEQ ID NO: 91, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 92; (xiv) the VH comprises the amino acid sequence set forth in SEQ ID NO: 140, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 141; or (xv) the VH comprises the amino acid sequence set forth in SEQ ID NO: 142, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 143; The antibody or antigen-binding fragment thereof according to claim 1.

15. The antibody or antigen-binding fragment thereof of claim 1, 2, or 12, wherein the VH or VL further comprises a human framework sequence.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the VH and VL further comprise human framework sequences.

17. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, or 12, wherein the VH or VL further comprises framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and / or framework 4 (FR4) sequences.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein the VH and VL further comprise framework 1 (FR1), framework 2 (FR2), framework 3 (FR3), and framework 4 (FR4) sequences.

19. 13. The antibody or antigen-binding fragment thereof of claim 1, 2, or 12, wherein the antibody is a monoclonal antibody, and optionally the monoclonal antibody is a humanized antibody, a human antibody, or a chimeric antibody.

20. Fab, Fab', F(ab') 2 , Fv, scFv, (scFv) 2 13. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, or 12, which is a multispecific antibody formed from a single chain antibody molecule, a dual variable region antibody, a single variable region antibody, a linear antibody, a V region, or an antibody fragment.

21. The antibody or antigen-binding fragment thereof of claim 1, 2, or 12, wherein the alpha5 integrin is human alpha5 integrin.

22. 15. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 12 or 14 conjugated or recombinantly fused to a diagnostic agent, a detectable agent, or a therapeutic agent, which may optionally be a chemotherapeutic agent, a cytotoxin, or a drug.

23. A binding agent that binds to essentially the same epitope as the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 12 or 14.

24. 24. The binding agent of claim 23, which is an antibody or an antigen-binding fragment thereof.

25. 24. The binding agent of claim 23, comprising a non-antibody protein scaffold.

26. 26. The binding agent of claim 25, wherein the non-antibody protein scaffold comprises a fibronectin scaffold, anticalin, adnectin, affibody, DARPin, finomer, affitin, affilin, avimer, cysteine-rich knottin peptide, or an engineered Kunitz-type inhibitor.

27. A binding agent that competes with the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 12 or 14 for binding to alpha5 integrin.

28. 28. The binding agent of claim 27 which is an antibody or an antigen-binding fragment thereof.

29. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 12 or 14.

30. 30. A vector comprising the polynucleotide of claim 29.

31. A cell comprising the vector of claim 30.

32. 15. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 12 or 14, and optionally further comprising a pharma- ceutically acceptable carrier.

33. 23. A composition comprising the conjugate of claim 22, and optionally further comprising a pharma- ceutically acceptable carrier.

34. a) a medicament for treating an alpha5 integrin-mediated disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an α5 integrin-mediated disease, disorder, or condition in a subject; Use of the antibody or antigen-binding fragment thereof described in any one of claims 1, 2, 12 or 14 in the manufacture of a therapeutic agent.

35. 35. The use of claim 34, wherein the subject is administered one or more therapeutic agents in combination with the antibody or antigen-binding fragment thereof.

36. a) a medicament for treating cancer or tumor in a subject; b) a pharmaceutical agent for alleviating one or more symptoms associated with a cancer or tumor in a subject; or c) a medicament for treating an angiogenesis-mediated disease, disorder, or condition in a subject; Use of the antibody or antigen-binding fragment thereof described in any one of claims 1, 2, 12 or 14 in the manufacture of a therapeutic agent.

37. 37. The use of claim 36, wherein the subject is administered one or more therapeutic agents in combination with the antibody or antigen-binding fragment thereof.

38. a) a medicament for treating an inflammatory disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an inflammatory disease, disorder, or condition in a subject. Use of the antibody or antigen-binding fragment thereof described in any one of claims 1, 2, 12 or 14 in the manufacture of a therapeutic agent.

39. 40. The use of claim 38, wherein the subject is administered one or more therapeutic agents in combination with the antibody or antigen-binding fragment thereof.

40. a) a medicament for treating an alpha5 integrin-mediated disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an α5 integrin-mediated disease, disorder, or condition in a subject; 23. Use of the conjugate of claim 22 in the manufacture of

41. 41. The use of claim 40, wherein the subject is administered one or more therapeutic agents in combination with the conjugate.

42. a) a medicament for treating cancer or tumor in a subject; b) a pharmaceutical agent for alleviating one or more symptoms associated with a cancer or tumor in a subject; or c) a medicament for treating an angiogenesis-mediated disease, disorder, or condition in a subject; 23. Use of the conjugate of claim 22 in the manufacture of

43. 43. The use of claim 42, wherein the subject is administered one or more therapeutic agents in combination with the conjugate.

44. a) a medicament for treating an inflammatory disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an inflammatory disease, disorder, or condition in a subject.

23. Use of the conjugate of claim 22 in the manufacture of

45. 45. The use of claim 44, wherein the subject is administered one or more therapeutic agents in combination with the conjugate.

46. a) a medicament for treating an alpha5 integrin-mediated disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an α5 integrin-mediated disease, disorder, or condition in a subject; 33. Use of the composition of claim 32 in the manufacture of

47. 47. The use of claim 46, wherein the subject is administered one or more therapeutic agents in combination with the composition.

48. a) a medicament for treating cancer or tumor in a subject; b) a pharmaceutical agent for alleviating one or more symptoms associated with a cancer or tumor in a subject; or c) a medicament for treating an angiogenesis-mediated disease, disorder, or condition in a subject; 33. Use of the composition of claim 32 in the manufacture of

49. 49. The use of claim 48, wherein the subject is administered one or more therapeutic agents in combination with the composition.

50. a) a medicament for treating an inflammatory disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an inflammatory disease, disorder, or condition in a subject.

33. Use of the composition of claim 32 in the manufacture of

51. 51. The use of claim 50, wherein the subject is administered one or more therapeutic agents in combination with the composition.

52. a) a medicament for treating an alpha5 integrin-mediated disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an α5 integrin-mediated disease, disorder, or condition in a subject; 34. Use of the composition of claim 33 in the manufacture of

53. 53. The use of claim 52, wherein the subject is administered one or more therapeutic agents in combination with the composition.

54. a) a medicament for treating cancer or tumor in a subject; b) a pharmaceutical agent for alleviating one or more symptoms associated with a cancer or tumor in a subject; or c) a medicament for treating an angiogenesis-mediated disease, disorder, or condition in a subject; 34. Use of the composition of claim 33 in the manufacture of

55. 55. The use of claim 54, wherein the subject is administered one or more therapeutic agents in combination with the composition.

56. a) a medicament for treating an inflammatory disease, disorder, or condition in a subject; or b) a pharmaceutical agent for alleviating one or more symptoms associated with an inflammatory disease, disorder, or condition in a subject.

34. Use of the composition of claim 33 in the manufacture of

57. 57. The use of claim 56, wherein the subject is administered one or more therapeutic agents in combination with the composition.

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