Alpha5beta1 integrin binding agents and uses thereof

Abstract

The present disclosure provides alpha5beta1 integrin binding agents (e.g., antibodies, including multispecific antibodies, such as dual specific antibodies) and uses thereof.

Description

[0001] Related applications

[0002] This application is a divisional application of the Chinese invention patent application filed on May 10, 2022, with the invention name “α5β1 integrin binders and their uses” and application number 202280049271.9.

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 187,371, filed on May 11, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] The present application incorporates by reference the sequence listing submitted with the present application as a text file entitled "14700-001-228_SEQ_LISTING.txt" created on May 8, 2022 and having a size of 142,533 bytes. Technical Field

[0006] The present disclosure generally relates to binding agents, such as antibodies, that bind to α5β1 (α5β1) integrin, including human α5β1 integrin, and methods of their use. Background Art

[0007] Integrins are transmembrane proteins that bind to extracellular matrix (ECM) components and regulate cell adhesion, migration and activation. Each integrin is composed of α and β transmembrane integrin subunits. There are 18 α integrin subunits and 8 β integrin subunits in the human genome, and they combine to produce 23 unique heterodimeric integrins. These heterodimers regulate cell behavior through mechanisms known as "inside-out" and "outside-in" signal transduction. In the former, intracellular proteins bind to the integrin cytoplasmic domain and stabilize a conformation that binds to extracellular ligands with high affinity. Then, through "outside-in" signal transduction, the ligand-bound integrin stimulates the intracellular signal transduction cascade that regulates cell behavior.

[0008] The α5β1 integrin is known as the fibronectin (FN) receptor due to its high affinity for 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 α5β1 headpiece and the arginine-glycine-aspartate (RGD) peptide motif in the type III repeats of FN. The α5β1 integrin binds other RGD-containing proteins, such as osteopontin and fibrillin, as well as proteins lacking the RGD motif, including CD40L, IL-1b, and the TNF-α-converting enzyme ADAM-17. Consistent with the tissue distribution of its ligand, α5β1 is expressed by multiple cell types in peripheral tissues and the central nervous system (CNS), including endothelial cells, mast cells, and the macrophage lineage (e.g., microglia and perivascular macrophages).

[0009] There is a well-established link between the α5β1 integrin and tumor angiogenesis. In addition, α5β1 has been shown to be present on tumor cells. Antibodies that bind to α5β1 have been shown not only to inhibit angiogenesis but also to help kill α5β1-expressing tumor cells. A link has also been demonstrated between the α5β1 integrin and neuroinflammatory diseases, including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Antibodies that bind to α5β1 have been shown to improve the experimental autoimmune encephalitis (EAE) model of MS and SOD1 in ALS. G93A Although the expression of α5β1 would appear to make it a potential target in antiangiogenic and cancer therapies, as well as in the treatment of neuroinflammatory diseases, clinical success using antibodies targeting α5 integrin has not yet been achieved.

[0010] Thus, there remains a need in the art for agents that can target α5β1 integrin to treat, prevent, or ameliorate α5-mediated diseases, disorders, or conditions, including those involving cells expressing α5β1, such as tumor cells and macrophages. SUMMARY OF THE INVENTION

[0011] The present invention discloses α5β1 integrin binding agents, including human α5β1 integrin binding agents. These agents include antibodies that bind to α5β1 integrin, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to α5β1 integrin. In some embodiments, these antibodies compete with antibodies having heavy chain variable regions and light chain variable regions as described herein (e.g., Tables 1-6) for binding to human α5β1 integrin.

[0012] The present disclosure also provides compositions comprising α5β1 integrin binding agents. In some embodiments, these compositions include antibodies that bind to α5β1 integrin, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to α5β1 integrin. In some embodiments, these compositions include antibodies that compete with antibodies having heavy chain variable regions and light chain variable regions described herein (e.g., Tables 1-6) for binding to human α5β1 integrin.

[0013] The present disclosure also provides methods for treating, preventing, or alleviating α5β1 integrin-mediated diseases, disorders, or conditions, including methods for treating, preventing, or alleviating one or more symptoms of the disease, disorder, or condition with an α5β1 integrin binding agent, or a composition comprising the agent, including an α5β1 integrin binding agent, or a composition comprising the agent. These compositions include antibodies that bind to α5β1 integrin, for example, monospecific or multispecific (e.g., bispecific) antibodies that bind to α5β1 integrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Exemplary results of a fibronectin (FN) inhibition assay, described further in Example 5, are shown.

[0015] Figures 2A-2B Shown is a sequence alignment of exemplary consensus sequences 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 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The boundaries of the CDRs are indicated according to Kabat, AbM, Chothia, Contact, and IMGT (see also, e.g., Tables 1-6).

[0016] Figures 2C-2D Shown are 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. The boundaries of the CDRs are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0017] Figures 2E-2F 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 are shown. The boundaries of the CDRs are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0018] Figures 2G-2H Sequence alignments of the heavy and light chain variable regions of C-14D12 and exemplary human engineered C-14D12_Low and C-14D12_Low+Mod are shown. The boundaries of the CDRs are indicated according to Kabat, AbM, Chothia, Contact, and IMGT.

[0019] Figure 3 Shown are sequence alignments of exemplary Fc sequences, including variant Fc sequences.

[0020] Figure 4A and 4B Exemplary results are shown for antibodies provided herein that disrupt the α5β1-FN integrin-ligand complex, as further described in Example 7.

[0021] Figure 5 Exemplary results of the α5β1 integrin FN blocking assay described further in Example 8 are shown.

[0022] Figure 6 Exemplary results are shown for chimera efficacy compared to hybridomas as further described in Example 8.

[0023] Figure 7 Exemplary SDS-PAGE results of various IgG4 chimeric antibodies further described in Example 8 are shown.

[0024] Figure 8 Exemplary results of the conformational assay described further in Example 9 are shown.

[0025] Figure 9 Exemplary results of a cell adhesion assay described further in Example 10 are shown. Detailed Description of the Invention

[0026] The present invention discloses α5β1 integrin binding agents. These agents include antibodies that bind to α5β1 integrin (e.g., monospecific or multispecific, including dual specificity), including antibodies that bind to human α5β1 integrin. These binding agents are useful in compositions and in methods of treating, preventing or alleviating α5β1 integrin-mediated diseases, disorders or conditions, including one or more symptoms of the disease, disorder or condition. α5β1 integrin-mediated diseases, disorders or conditions include cancer, angiogenesis-mediated diseases (e.g., diseases with 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 the binding of α5β1 integrin to fibronectin, (ii) inhibiting angiogenesis and / or (iii) treating or alleviating one or more symptoms of (i) cancer, (ii) angiogenesis-mediated diseases, disorders or conditions, or (iii) inflammatory diseases, disorders or conditions. The α5β1 integrin binding agents described herein, such as α5β1 integrin binding antibodies (e.g., monospecific or multispecific, including bispecific) are useful in compositions and in methods of treating α5β1-mediated diseases, disorders or conditions.

[0027] Unless otherwise indicated, the terms "α5 integrin," "CD49e," or "α5 integrin polypeptide," and similar terms refer to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native α5 integrin from any vertebrate source, including mammals, such as primates (e.g., humans, cynomolgus monkeys (cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). 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., having seven 60-amino acid FG-GAP repeats), a thigh, genu, calf1, calf2, transmembrane and intracellular domains, and four calcium-binding sites. The term α5 integrin encompasses "full-length," unprocessed α5 integrin, as well as any form of α5 integrin produced by processing in cells, 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, which binds to β1 as a heterodimer, is known in the art to interact with some ligands (e.g., fibronectin), and this interaction leads to changes in protein conformation and signal transduction, thereby causing changes in cellular activities such as cell adhesion, proliferation, apoptosis, migration, and phagocytosis. The full-length amino acid sequence of human α5 integrin is provided below (exemplary signal sequence = italicized text; exemplary extracellular domain = underlined text):

[0028]

[0029]

[0030] The full-length amino acid sequence of mouse α5 integrin is provided below (exemplary signal sequence = italicized text; exemplary extracellular domain = underlined text):

[0031]

[0032] Other related α5 integrin polypeptides also encompassed by the term α5 integrin include fragments, derivatives (e.g., substitutions, deletions, truncations, 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 will be appreciated by those skilled in the art, the α5 integrin binders (e.g., antibodies) described herein can bind to α5 integrin polypeptides, α5 integrin polypeptide fragments, α5 integrin antigens, and / or α5 integrin epitopes. An epitope can be part of a larger α5 integrin antigen, which can be part of a larger α5 integrin polypeptide fragment, which in turn can be part of a larger α5 integrin polypeptide. α5 integrin can exist in natural or denatured form. The α5 integrin polypeptides described herein can be isolated from a variety of sources, such as human tissue types or another source, or prepared by recombinant or synthetic methods. The α5 integrin polypeptide can comprise a polypeptide having the same amino acid sequence as a corresponding α5 integrin polypeptide of natural origin. Orthologs of α5 integrin polypeptides are also well known in the art.

[0033] Unless otherwise indicated, the terms "β1 integrin," "CD29," or "β1 integrin polypeptide," and similar terms refer to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native β1 integrin from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). β1 integrin, also known in the art as integrin β-1, ITGB1 protein, CD29 antigen, fibronectin receptor subunit β, and glycoprotein IIa, has multiple domains, including a β1 helmet, a hybrid and plexin-semaphoring-integrin (PSI) domains, four integrin-epidermal growth factor domains (I-EGF1, I-EGF2, I-EGF3, I-EGF4), a β-tail, a transmembrane domain, and an intracellular domain. The term β1 integrin encompasses "full-length" unprocessed β1 integrin, as well as any form of β1 integrin or any fragment thereof produced by processing in the cell. The term β1 integrin also encompasses naturally occurring variants of β1 integrin, such as SNP variants, splice variants, and allelic variants. β1 integrin, which binds to α5 as a heterodimer, is known in the art to interact with some ligands (e.g., fibronectin), and this interaction results in protein conformational changes and signal transduction, leading to changes in cellular activities such as cell adhesion, proliferation, apoptosis, migration, and phagocytosis. The full-length amino acid sequence of human β1 integrin is provided below (exemplary signal sequence = italic text; exemplary extracellular domain = underlined text):

[0034]

[0035] The full-length amino acid sequence of mouse β1 integrin is provided below (exemplary signal sequence = italicized text; exemplary extracellular domain = underlined text):

[0036]

[0037] Unless otherwise indicated, the terms "α4 integrin," "CD49d," or "α4 integrin polypeptide," and similar terms refer to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native α4 integrin from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). 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., having seven 60-amino acid FG-GAP repeats), a thigh, genu, calf1, calf2, a transmembrane domain, and an intracellular domain and also has three calcium binding sites. The term α4 integrin encompasses "full-length," unprocessed α4 integrin, as well as any form of α4 integrin produced by processing in cells, 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. α4 integrin, which binds to β1 as a heterodimer, is known in the art to interact with some ligands (e.g., VCAM1, fibronectin), and this interaction leads to changes in protein conformation and signal transduction, thereby leading to changes in cellular activities such as cell adhesion, proliferation, migration, and phagocytosis. The full-length amino acid sequence of human α4 integrin is provided below (exemplary signal sequence = italicized text; exemplary extracellular domain = underlined text):

[0038]

[0039] Unless otherwise indicated, the term "fibronectin" or "FN" and similar terms refer to polypeptides ("polypeptide" and "protein" are used interchangeably herein) or any native fibronectin from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). Fibronectin exists as a dimer or multimer linked by disulfide bonds and has a multimodular structure consisting primarily of three different repeats, referred to as 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 to 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 a major 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 provided below (exemplary signal sequence = italic text):

[0040]

[0041]

[0042] 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.

[0043] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and in the broadest sense and specifically cover, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length monoclonal antibodies), antibody compositions with poly-epitopic or mono-epitopic specificity, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human forms of antibodies having full-length heavy and / or light chains. The present disclosure also encompasses antibody fragments (and / or polypeptides comprising antibody fragments) that retain α5 integrin binding characteristics. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, e.g., 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 by antibody fragments, F(ab)2, Fd, Fc, diabodies, two-diabodies, disulfide-linked Fv (dsFv), single domain antibodies (e.g., VHH, nanobodies) or other fragments (e.g., a fragment consisting of the variable regions of heavy and light chains coupled by non-covalent bonds). 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 and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the variable region domain can be dimeric and contain a VH-VH, VH-VL or VL-VL dimer that binds to α5 integrin. If desired, the VH and VL chains can be covalently coupled directly or via a linker to form a single-chain Fv (scFv). For ease of reference, the scFv protein is referred to herein as being included in the class of "antibody fragments". Another form of an antibody fragment is a peptide comprising one or more complementary determining regions (CDRs) of an antibody. CDRs (also referred to as "minimal recognition units" or "hypervariable regions") can be obtained by constructing a polynucleotide encoding the CDRs of interest.For example, these polynucleotides are prepared by synthesizing the variable region using mRNA from antibody-producing cells as a template by using the polymerase chain reaction (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies." in Monoclonal Antibodies Production. Engineering and Clinical Application, Ritter et al. (Eds.), p. 166, Cambridge University Press (1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (Eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be introduced into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of novel antigen receptors (v-NARs), and bi-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1 126-1136, 2005). In some embodiments, the binding agent contains a light chain and / or heavy chain constant region, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, the antibody can include any of the above-mentioned epitope-binding fragments. The antibodies described herein can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an α5β1 binding antibody, including an antagonist antibody or an agonist antibody.

[0044] As used herein, the term "monospecific" when used to refer to a binding agent (eg, an antibody) refers to a binding agent that has one or more binding sites that each bind to the same epitope of the same antigen.

[0045] When used to refer to a binding agent (e.g., an antibody), the term "dual specificity" means that the binding agent is capable of specifically binding to at least two different antigenic determinants, for example, two binding sites formed by a pair of antibody heavy chain variable domains (VH) and an antibody light chain variable domain (VL) that bind to different antigens or bind to different epitopes on the same antigen, respectively. Such a dual specific binding agent (e.g., an antibody) can have a 1+1 format. Other dual specific binding agent (e.g., antibody) formats can be a 2+1 or 1+2 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a dual specific binding agent (e.g., an antibody) comprises two antigen binding sites, each can bind to a different antigenic determinant. Such a dual specific binding agent (e.g., an antibody) can bind to two different epitopes on the same antigen (e.g., an epitope on α5β1 integrin) or on different antigens (e.g., an epitope on α5β1 integrin and an epitope on αvβ1 integrin).

[0046] In the context of two or more nucleic acids or polypeptides, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues, when compared and aligned for maximum correspondence (introducing gaps, if necessary), without taking into account any 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. A variety of algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, the GCG Wisconsin software package, and variations thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical if 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%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, there is identity in the amino acid sequence region of at least about 10 residues in length, at least about 20 residues, at least about 40-60 residues, at least about 60-860 residues or any integer value therebetween. In some embodiments, there is identity in the region greater than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequence is basically identical on the total length of the sequence compared (such as the coding region of the target protein or antibody). In some embodiments, there is identity in the nucleotide sequence region of at least about 10 bases in length, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases or any integer value therebetween. In some embodiments, there is identity in the region greater than 60-80 bases, such as at least about 80-1000 bases or more bases, and in some embodiments, the sequence is basically identical on the total length of the sequence compared (such as the nucleotide sequence of the protein of interest).

[0047] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by another amino acid residue with a side chain having similar chemical properties. 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the replacement of phenylalanine with tyrosine is a conservative substitution. Generally, conservative substitutions in the polypeptides, soluble proteins, and / or antibody sequences disclosed herein do not eliminate the binding of the polypeptides, soluble proteins, and / or antibodies containing the amino acid sequence to the target binding site. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0048] 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 include non-amino acids (e.g., interrupted by non-amino acids). The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as (directly or indirectly) connection to or conjugation to a moiety, such as a tag component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art are also included in this definition. It should be understood that since the polypeptides disclosed herein may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides may exist as single chains.

[0049] As used herein, an "antigen" is a moiety or molecule containing an epitope to which a binding agent (e.g., an antibody) can bind. As such, an antigen can be bound by an antibody. In some embodiments, the antigen to which a binding agent (e.g., an antibody) described herein binds is α5β1 integrin (e.g., human α5β1 integrin) or a fragment thereof.

[0050] As used herein, "epitope" is a term in the art and refers to a local 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. With respect to polypeptide antigens, for example, an epitope can be contiguous amino acids of a polypeptide (a "linear" epitope) or an epitope can comprise amino acids from two or more contiguous regions of a polypeptide (a "conformational", "non-linear" or "discontinuous" epitope), for example, human α5β1 integrin. Those skilled in the art will appreciate that generally, linear epitopes 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 they are folded in a native three-dimensional protein structure. In other embodiments, in order to recognize and bind to the epitope, the antibody requires that the amino acid residues that make up the epitope display a specific conformation (e.g., bend, twist, turn or fold).

[0051] When two antibodies recognize the same, overlapping, or adjacent epitopes in three-dimensional space, the antibodies bind to an "epitope" or "substantially the same epitope" or "the same epitope" as a 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 competition assay, which can be configured in a number of different formats, for example, using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate or expressed on the surface of cells, and the ability of the unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive, fluorescent, or enzymatic tag.

[0052] "Epitope binning" is a method for grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems that use, for example, competition assays to distinguish the epitope recognition properties of different antibodies. These assays can be combined with computational processes that cluster antibodies based on their epitope recognition properties and identify antibodies with different binding specificities.

[0053] In the context of antibodies, as used herein, the terms "specifically binds," "specifically recognizes," "immunospecifically binds," "selectively binds," "immunospecifically recognizes," and "immunospecific" are analogous terms and refer to molecules that bind to an antigen (e.g., an epitope) as those skilled in the art understand binding. In some embodiments, "specifically binds" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or with some combination of the foregoing, than an alternative substance, including related or unrelated proteins. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity, as determined by, for example, immunoassays, Biacore TMIn some embodiments, the binding degree of antibody or antigen-binding domains to " non-target " protein is determined by using experimental techniques, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when it is bound to antigen with an affinity higher than any cross-reactive antigen, antibody or antigen-binding domains are bound to or specifically bound to antigen. Generally, specificity or selective reaction will be at least twice of background signal or noise and can be greater than 10 times of background. For discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed. 2, 1989). In some embodiments, the binding degree of antibody or antigen-binding domains to " non-target " protein is less than about 10% of the combination of the antibody or antigen-binding domains and its specific target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. In some embodiments, a molecule that specifically binds to an antigen binds to the antigen with a Ka that is at least 2 log, 2.5 log, 3 log, 4 log, or more greater than the Ka when the molecule binds to another antigen. In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins. In some embodiments, a molecule that specifically binds to an antigen does not cross-react with other non-α5β1 integrin proteins. In some embodiments, "specific binding" refers to, for example, a polypeptide or molecule that binds to an antigen with a Ka that is at least 0.1 mM or less. D , but more typically less than about 1 μM K D In some embodiments, "specific binding" refers to a polypeptide or molecule that binds to a protein or target with a K of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. DBinding to a target. Due to the sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets in more than one species. Similarly, due to the homology within certain regions of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize more than one protein or target. It should be 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. As such, "specific binding" does not necessarily require (although it can include) exclusive binding, for example, binding to a single target. Therefore, in some embodiments, a polypeptide or molecule can specifically bind to more than one target. In some embodiments, multiple targets can be bound by the same antigen binding site on the polypeptide or molecule. For example, in some cases, an antibody can include two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins. In some alternative embodiments, an antibody can be dual specific and include at least two antigen binding sites with different specificities. Generally, but not necessarily, reference to "binding" means "specific binding."

[0054] "Binding affinity" generally refers to the total strength 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, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Generally, the affinity of a binding pair can be expressed by the dissociation constant (K D ) represents the affinity of binding molecule X for its binding partner Y. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigen more rapidly and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes disclosed herein. In one embodiment, "K" can be measured by biomembrane interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA). D ” or “K D Alternatively, K can also be measured in a radiolabeled antigen binding assay (RIA). DThe "on-rate," "rate of association," or "association rate" can be determined, for example, using a Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881) or by Biacore using surface plasmon resonance (SPR) measurements using, for example, a BIAcore™-2000 or BIAcore™-3000 BIAcore, Inc., Piscataway, NJ. The "on-rate," "rate of association," or "association rate" can also be determined by the same SPR or BLI techniques described above using, for example, an Octet QK384 system (ForteBio, Menlo Park, CA) or a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), respectively. on and “off-rate”, “rate of dissociation” or “dissociation rate” or “k off ”.

[0055] When used in the context of α5β1 integrin binding agents (e.g., antibodies), the term "competes" refers to binding agents that compete for the same epitope or binding site on the target, including competition between such binding agents, as determined by an assay in which the binding agent under investigation 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). Various 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 that can be used 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 labeled assays, solid phase direct labeled sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1996). Harbor Press); solid phase direct labeling RIA using I-125 labeling (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 labeling 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 cell with 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 the solid surface or cell in the presence of the test antigen binding protein. Typically, the test antigen-binding protein is present in excess. Antibodies identified by competition 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 epitopes or overlapping epitopes) that are sufficiently adjacent to the epitope bound by the reference antibody for steric hindrance to occur. 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 a 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.

[0056] As used herein, the term "constant region" or "constant domain" is an antibody term well known in the art and refers to the portion of an antibody, e.g., the carboxyl-terminal portion of a light chain and / or heavy chain that is not directly involved in binding the antibody to an antigen, but that can exhibit various effector functions, such as interacting with Fc receptors. The term includes a portion of an immunoglobulin molecule that generally has a more conserved amino acid sequence than the variable domain of an immunoglobulin.

[0057] Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (e.g., a native sequence Fc region or amino acid sequence variant Fc region) and vary 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0058] In this article, the term "Fc region" is used to define the C-terminal region of a heavy chain immunoglobulin, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc regions of heavy chain immunoglobulins may be different, human IgG heavy chain Fc regions are generally defined to extend to their carboxyl terminus from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system). For example, the C-terminal lysine (according to the residue 447 of the EU numbering system) in the Fc region can be removed during the production or purification of an antibody or by recombinant engineering design of a nucleic acid encoding an antibody heavy chain. Exemplary Fc region sequences (CH2 domains = bold text; CH3 domains = underlined text) are provided below:

[0059]

[0060] 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), etc. These effector functions generally require the Fc region to be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be evaluated using a variety of assays as disclosed.

[0061] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, included, or combined with other sequences, such as variable region sequences). 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 and naturally occurring variants thereof. Figure 3 Exemplary IgG1 and IgG4 Fc sequences are shown in .

[0062] " Variant Fc region " comprises an amino acid sequence that is different from that of a native sequence Fc region by means of at least one amino acid modification (e.g., replacement, addition or deletion), preferably one or more amino acid replacements. In some embodiments, the variant Fc region has at least one amino acid replacement in the native sequence Fc region or in the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid replacements, and preferably about 1 to about 5 amino acid replacements, compared to the Fc region of the native sequence Fc region or the parent polypeptide. The variant Fc region described herein can have at least about 80% homology with the Fc region of the native sequence Fc region and / or the parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. In this article, the variant Fc region described herein can have an effector function loss (e.g., silent Fc). Exemplary variant Fc region ("silent Fc") sequences are provided below (CH2 domain = bold text, wherein amino acid changes are underlined; CH3 domain = underlined text):

[0063]

[0064] Figure 3 Exemplary variant Fc sequences are shown in , 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).

[0065] When used to refer to an antibody, as used herein, the term "heavy chain" refers to a polypeptide chain of about 50-70 kD, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxyl-terminal portion includes one or more constant regions. Based on the constant domain amino acid sequence, a "heavy chain" can refer to any of the different types, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including IgG subclasses, for example, IgG1, IgG2, IgG3, and IgG4.

[0066] When used to represent an antibody, as used herein, the term "light chain" can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl-terminal portion includes 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 different types, for example, kappa or lambda. Light chain amino acid sequences are well known in the art.

[0067] The terms "antigen binding fragment," "antigen binding domain," "antigen binding region," and similar terms refer to the portion of an antibody (e.g., CDR) that comprises the amino acid residues that interact with an antigen and confer specificity and affinity to the binding fragment, domain, or region for the antigen. "Antigen binding fragment," as used herein, includes "antibody fragments" that comprise a portion of an antibody that includes one or more CDRs, such as an antigen binding or variable region of an antibody.

[0068] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the foregoing.

[0069] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, including molecules that contain one or more antigen binding sites that bind to an α5β1 integrin antigen.

[0070] The 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 antibody described herein is an IgG antibody (e.g., human IgG), or its class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass.

[0071] In some embodiments, the antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein 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, for example, a human constant region. In some embodiments, the L chain constant region of these antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of these antibodies comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In some embodiments, these antibodies comprise an IgG constant region, for example, a human IgG constant region (e.g., IgG1, IgG2, IgG3 and / or IgG4 constant region).

[0072] The antibody or fragment thereof can preferentially bind to α5β1 integrin, such as human α5β1 integrin, meaning that the antibody or fragment thereof binds to α5β1 integrin with an affinity greater than that when it binds to an unrelated control protein, and / or binds to human α5β1 integrin with an affinity greater than that when it binds to an unrelated control protein. For example, the antibody or fragment thereof can specifically recognize and bind to α5β1 integrin or a portion thereof. "Specific binding" means 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 the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the antibody or fragment thereof can substantially bind only to α5β1 integrin (e.g., capable of distinguishing α5β1 integrin from other known polypeptides by, for example, measurable differences in binding affinity). In some embodiments, an α5β1 integrin binding agent (eg, an antibody) can react with an α5β1 integrin sequence other than a human α5β1 integrin sequence (eg, a cynomolgus monkey α5β1 integrin sequence).

[0073] The term "variable region" or "variable domain" refers to the amino-terminal portion of the antibody that is generally located at the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each specific antibody for its specific antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region are widely different in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a specific antibody for its specific antigen. However, the degree of difference is not evenly distributed within the approximately 110 amino acid intervals of the variable region. On the contrary, the V region is composed of a less variable (e.g., relatively unchanged) extension of about 15-30 amino acids called framework regions (FRs) separated by a shorter region with a greater degree of difference (e.g., extremely great difference) called a "hypervariable region" or alternatively a "complementarity determining region". The variable regions of the heavy and light chains respectively include four FRs (FR1, FR2, FR3, and FR4) that are primarily in a β-sheet configuration and are connected by three hypervariable regions, forming a loop that connects the β-sheet structure and, in some cases, constitutes a part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, through the hypervariable regions from the other chains, 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 edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequence of the variable region is widely different between different antibodies. The variation in sequence is concentrated in the CDRs, and the less variable part of the variable region is called the framework region (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In a specific embodiment, the variable region is a human variable region.

[0074] As used herein, the terms "hypervariable region", "HVR", "HV", "complementarity determining region" or "CDR" refer to the regions of an antibody variable region whose sequence is highly variable and forms structurally defined loops. Typically, an antibody comprises six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Several hypervariable region diagrams are used and are encompassed herein. Kabat CDRs are based on sequence divergence 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)). Alternatively, Chothia refers to the position of the 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 based on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the end of the loop is at 32; if only 35A is present, the end of the loop is at 33; if both 35A and 35B are present, the end of the loop is at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer-Verlag). The "contact" hypervariable regions are based on analysis of available complex crystal structures. The residues from each of these hypervariable regions or CDRs are indicated below.

[0075] A universal numbering system has been developed and widely used, the ImMunoGeneTics (IMGT) Information (Lefranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)). IMGT is a comprehensive information system specifically for immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of humans and other vertebrates. In this article, CDRs are referred to both in terms of amino acid sequence and position within the light chain or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved between species and is present in structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used for the transplantation and replacement of CDR residues from an immunoglobulin from one species to an acceptor framework, typically from a human antibody. Other numbering systems (AHon) have been developed by Honegger and Plückthun, J. Mol. Biol. 309: 657-670 (2001). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also described below. The exemplary system shown herein combines Kabat and Chothia.

[0076] Exemplary IMGT Kabat AbM Chothia Contact <![CDATA[V H CDR1]]> 26-35 27-38 31-35 26-35 26-32 30-35 <![CDATA[V H CDR2]]> 50-65 56-65 50-65 50-58 52a / 53-55 47-58 <![CDATA[V H CDR3]]> 95-102 105-117 95-102 95-102 96-101 93-101 <![CDATA[V L CDR1]]> 24-34 27-38 24-34 24-34 26-32 30-36 <![CDATA[V L CDR2]]> 50-56 56-65 50-56 50-56 50-52 46-55 <![CDATA[V L CDR3]]> 89-97 105-117 89-97 89-97 91-96 89-96

[0077] The hypervariable region may comprise an "extended hypervariable region" as follows: 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.

[0078] The term "vector" refers to a substance used to carry or include a nucleic acid sequence, including, for example, a material for introducing a nucleic acid sequence into a host cell. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include operable selection sequences or markers for stable integration into the host cell chromosome. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes that may be included, for example, provide antibiotic or toxin resistance, supplement nutritional deficiencies, or provide key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., as known in the art. When co-expressing two or more nucleic acid molecules (for example, both antibody heavy and light chains or both antibody VH and VL), the two nucleic acid molecules may be inserted, for example, into a single expression vector or different expression vectors. For single vector expression, the encoding nucleic acid may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art may be used to confirm the introduction of nucleic acid molecules into the host cell. These methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods to test for expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will appreciate that the nucleic acid molecule should be expressed in sufficient amounts to produce the desired product (e.g., an α5β1 integrin binding agent as described herein), and that expression levels can be optimized using methods well known in the art to achieve sufficient expression.

[0079] "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 caused in whole or in part by or as a result of α5β1 integrin or the interaction of α5β1 integrin with fibronectin and / or alternatively, any disease, disorder or condition in which inhibition of the in vivo effects of the interaction of α5β1 integrin with fibronectin is desired. α5β1 integrin-mediated diseases, disorders or conditions include cancer, angiogenesis-mediated diseases (e.g., diseases with abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases, including 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 characterized by or associated with abnormally elevated angiogenic activity in cells (e.g., tumor cells). In some embodiments, α5β1 integrin-mediated diseases are diseases, disorders or conditions specifically associated with abnormal angiogenesis (e.g., eye diseases, diabetic retinopathy or age-related macular degeneration). In some embodiments, α5β1 integrin-mediated diseases include diseases, disorders or conditions that are inflammatory diseases characterized by or associated with an inflammatory immune response (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).

[0080] 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 potential causes, prevent the occurrence of one or more symptoms and / or their potential causes, and / or ameliorate or treat damage resulting from or associated with the disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0081] As used herein, the term "therapeutically effective amount" 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, including a therapeutic agent, can be an amount necessary to (i) reduce or ameliorate the progression or development 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) disclosed herein 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 the therapeutically beneficial effects of the substance / molecule / agent outweigh any toxic or adverse effects. In some embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (eg, or drug) effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0082] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of a disease, disorder, or condition, or reducing the likelihood of the onset (or recurrence) of a disease, disorder, or condition, or associated symptoms. A complete therapeutic or prophylactic effect does not necessarily occur with the administration of one dose, and may occur only after a series of doses. Thus, a therapeutic or prophylactic effective amount may be administered in one or more administrations.

[0083] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0084] As used herein, "carrier" includes a carrier, excipient, or stabilizer that is nontoxic to the cells or mammals to which it is exposed at the dosages and concentrations used. Typically, the carrier is an aqueous pH buffered solution. Examples of carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including 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 including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salts forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM . The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered. These carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous glucose and glycerol solutions can also be used 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. The composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions, including pharmaceutical compounds, can contain a prophylactically or therapeutically effective amount of an α5β1 integrin binding agent (e.g., an antibody), for example, in an isolated or purified form, together with an appropriate amount of carrier to provide a form suitable for administration to a subject (e.g., a patient). The formulation should be suitable for the form of administration.

[0085] In some embodiments, the present disclosure provides α5β1 integrin binding agents that can be used as therapeutic agents herein. These agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to α5β1 integrin. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, and variants thereof with increased or decreased affinity or other properties.

[0086] In some embodiments, described herein are α5β1 integrin binding agents (e.g., antibodies) that bind to α5β1 integrin, including 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, including 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 mammalian (e.g., human) cells, including α5β1 integrin-expressing tumor cells. In some embodiments, the α5β1 integrin binding agent (e.g., an antibody) binds to an extracellular epitope of α5β1 integrin (e.g., an α5β1 integrin epitope) exposed on cells, such as tumor cells. In some embodiments, described herein are α5β1 integrin binding agents (e.g., antibodies) that bind to α5β1 integrin, such as human α5β1 integrin or a portion thereof. 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.

[0087] In some embodiments, an 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 amino acid sequence of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VLCDR3 of any one of the antibodies described herein, such as a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VLCDR3 shown in Tables 1-6. Thus, in some embodiments, an 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 1, 2, and / or 3 VH CDRs and / or 1, 2, and / or 3 VL CDRs from: (a) the antibody represented by A-15B08; (b) the antibody represented by A2-3B06; (c) the antibody represented by A2-5D10; (d) the antibody represented by A2-7A05; (e) the antibody represented by A2-7F01; or (f) the antibody represented by C-14D12, as shown in Tables 1-6. In some embodiments, an 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 1, 2, and / or 3 VH CDRs and 1, 2, and / or 3 VL CDRs from: a) the antibody represented by A-15B08; (b) the antibody represented by A2-3B06; (c) the antibody represented by A2-5D10; (d) the antibody represented by A2-7A05; (e) the antibody represented by A2-7F01; or (f) the antibody represented by C-14D12, as shown in Tables 1-6. 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., antibody) comprising a VH region and a VL region from: a) an antibody represented by A-15B08; (b) an antibody represented by A2-3B06; (c) an antibody represented by A2-5D10; (d) an antibody represented by A2-7A05; (e) an antibody represented by A2-7F01, or (f) an antibody represented by C-14D12, as shown in Tables 1-6.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., antibody) comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 25, or a humanized variant thereof, and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or a humanized variant thereof; (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 42, or a humanized variant thereof, and a VL domain comprising the amino acid sequence of SEQ ID NO: 43, or a humanized variant thereof; (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 51, or a humanized variant thereof, and a VL domain comprising the amino acid sequence of SEQ ID NO: 52, or a humanized variant thereof; (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 77, or a humanized variant thereof, and a VL domain comprising the amino acid sequence of SEQ ID NO: 78, or a humanized variant thereof; (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 80, or a humanized variant thereof (f) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 109, or a humanized variant thereof, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 110, or a humanized variant thereof.

[0088] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein comprise the VH region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 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 shown in Tables 1-6. Thus, in some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein comprise 1, 2, and / or 3 heavy chain CDRs and / or 1, 2, and / or 3 light chain CDRs from: (a) the antibody represented by A-15B08; (b) the antibody represented by A2-3B06; (c) the antibody represented by A2-5D10; (d) the antibody represented by A2-7A05; (e) the antibody represented by A2-7F01; or (f) the antibody represented by C-14D12, as shown in Tables 1-6. In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein comprise 1, 2, and 3 heavy chain CDRs and 1, 2, and 3 light chain CDRs from: (a) the antibody represented by A-15B08; (b) the antibody represented by A2-3B06; (c) the antibody represented by A2-5D10; (d) the antibody represented by A2-7A05; (e) the antibody represented by A2-7F01; or (f) the antibody represented by C-14D12, as shown in Tables 1-6.

[0089] In some embodiments, the α5β1 integrin binding agent (e.g., antibody) comprises a VH region comprising VH CDR1, VH CDR2, and / or VH CDR3, and a VL region comprising VL CDR1, VL CDR2, and / or VL 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). Thus, in some embodiments, the α5β1 integrin binding agent (e.g., antibody) described herein comprises 1, 2, and / or 3 heavy chain CDRs (e.g., VHCDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 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 1, 2, and / or 3 heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 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 1, 2, and / or 3 heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 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 1, 2, and / or 3 heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 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 1, 2, and / or 3 heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 5. In some embodiments, an α5β1 integrin-binding agent described herein comprises 1, 2, and / or 3 heavy chain CDRs (e.g., VH CDR1, VH CDR2, and / or VH CDR3) and / or 1, 2, and / or 3 light chain CDRs (e.g., VL CDR1, VL CDR2, and / or VL CDR3) from Table 6.In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein are multispecific (e.g., bispecific) and comprise a first binding domain comprising 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. CDR3), and a second binding domain comprising 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 other than 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).

[0090] 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 the VH sequence shown in SEQ ID NO: 25 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 26 or a humanized variant thereof.

[0091] 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 the VH sequence shown in SEQ ID NO: 42 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 43 or a humanized variant thereof.

[0092] The antibody denoted 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 the VH sequence shown in SEQ ID NO: 51 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 52 or a humanized variant thereof.

[0093] The antibody designated A2-7A05 comprises the CDR sequences according to Kabat and / or Chothia, AbM, Contact or IMGT as shown in Table 4, and in some embodiments, may comprise the VH sequence shown in SEQ ID NO: 77 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 78 or a humanized variant thereof.

[0094] 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 the VH sequence shown in SEQ ID NO: 91 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 92 or a humanized variant thereof.

[0095] 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 the VH sequence shown in SEQ ID NO: 109 or a humanized variant thereof and the VL sequence shown in SEQ ID NO: 110 or a humanized variant thereof.

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] In some embodiments, the α5β1 integrin binders described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binders, comprise a VH region or a VH domain. In other embodiments, the α5β1 integrin binders described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binders, comprise a VL region or a VL domain. In some embodiments, the α5β1 integrin binders described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binders, have the following combinations: (i) a VH domain or a VH region; and / or (ii) a VL domain or a VL region.

[0103] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise a heavy chain having the following combination: (i) a VH domain comprising the CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT described 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 VH domain as described herein and the following CH1, hinge, CH2, and CH3 amino acid sequences: Another exemplary IgG heavy chain comprises any VH domain having CDRs as described herein and followed by CH1, hinge, CH2, and CH3 amino acid sequences:

[0104] Figure 3 An exemplary Fc sequence is shown in .

[0105] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise a light chain having the following combination: (i) a VL domain comprising the CDRs described in any one of Tables 1-6; and (ii) a light chain constant domain (CL). Exemplary light chains (e.g., light chains for pairing with IgG heavy chains) comprise any VL domain having CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT as described herein and followed by a CL amino acid sequence:

[0106]

[0107] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise (a) a heavy chain having the following combination: (i) a VH domain having CDRs according to Kabat and / or Chothia, AbM, Contact or IMGT described in any one of Tables 1-6 and (ii) one or more heavy chain constant domains (e.g., CH1, hinge, CH2 and CH3); and (a) a light chain having the following combination: (i) a VL domain having CDRs according to Kabat and / or Chothia, AbM, Contact or IMGT described in any one of Tables 1-6 and (ii) a light chain constant domain (CL).

[0108] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 1. In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 2. In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 3. In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and / or VL CDR3 identified in Table 4. In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 5. In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise one or more CDRs, including six CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Table 6. In some embodiments, the α5β1 integrin-binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin-binding agents, comprise one or more CDRs, including 6 CDRs, e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 identified in Tables 1, 2, 3, 4, 5, and / or 6.

[0109] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 1 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 1 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0110] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 2 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 2 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0111] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 3 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 3 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0112] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 4 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 4 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0113] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 5 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 5 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0114] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein, including human α5β1 integrin-binding agents, comprise 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 other embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies), including human α5β1 integrin binding agents, comprise one or more CDRs, including three VH CDRs, e.g., VH CDR1, VH CDR2, and / or VH CDR3 listed in Table 6 according to Kabat and / or Chothia, AbM, Contact, or IMGT, and one or more CDRs, including three VL CDRs, e.g., VL CDR1, VL CDR2, and / or VLCDR3 listed in Table 6 according to Kabat and / or Chothia, AbM, Contact, or IMGT.

[0115] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies) 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 described herein (e.g., antibodies, such as bispecific antibodies) 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 described herein (e.g., antibodies, such as bispecific antibodies) 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 described herein (e.g., antibodies, such as bispecific antibodies) 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 described herein (e.g., antibodies, such as bispecific antibodies) 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 described herein (e.g., antibodies, such as bispecific antibodies) 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.

[0116] In some embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., 1, 2, or 3) VH CDRs listed in Tables 1-6. In other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., 1, 2, or 3) VL CDRs listed in Tables 1-6. In yet other embodiments, the α5β1 integrin-binding agents (e.g., antibodies, such as bispecific antibodies) described herein comprise one or more (e.g., 1, 2, or 3) VH CDRs listed in Tables 1-6 and one or more VL CDRs listed in Tables 1-6. Thus, in some embodiments, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH CDR1 having an amino acid sequence as set forth in 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, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH CDR2 having an amino acid sequence as set forth in 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, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VH CDR3 having an amino acid sequence as set forth in 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, an 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 set forth in any one of the amino acid sequences set forth in Tables 1-6. In some embodiments, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VL CDR1 having an amino acid sequence as set forth in 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, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VL CDR2 having an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 11, 22, 41, 57, 63, and 74.In some embodiments, an α5β1 integrin-binding agent (e.g., an antibody, such as a bispecific antibody) described herein comprises a VL CDR3 having an amino acid sequence as set forth in 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, an 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, VL CDR3 set forth in any one of the amino acid sequences set forth in Tables 1-6.

[0117] In some embodiments, the α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody) comprises a heavy chain variable (VH) region 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; and (2) a VH CDR2 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: 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; and / or a light chain variable (VL) region 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: 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.

[0118] In some embodiments, the α5β1 integrin binding agent described herein (e.g., an antibody, such as a bispecific antibody) comprises a heavy chain variable (VH) region 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; and (2) a VH CDR2 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: 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.

[0119] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies) comprise a light chain variable (VL) region 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 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: 5 or 57, (ii) SEQ ID NO: 11 or 63, and (iii) SEQ ID NO: 22, 41, or 74. 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.

[0120] In some embodiments, the present invention provides an antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises: (a) a heavy chain variable (VH) region 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 CDR2 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: 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; and / or a light chain variable (VL) region 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: 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 VLCDR3 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.

[0121] In some embodiments, the present invention provides an antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises a heavy chain variable (VH) region 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 CDR2 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,

[0122] or 90 and (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.

[0123] In some embodiments, the present invention provides an antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises a light chain variable (VL) region 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 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: 5 or 57, (ii) SEQ ID NO: 11 or 63, and (iii) SEQ ID NO: 22, 41, or 74. 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.

[0124] In some embodiments, described herein are antibodies or fragments thereof that bind to α5β1 integrin, comprising all three heavy chain complementarity determining regions (CDRs) and / or all three light chain CDRs from: (i) an antibody represented by A-15B08, comprising a VH sequence of SEQ ID NO: 25 or a humanized variant thereof and a VL sequence of SEQ ID NO: 26 or a humanized variant thereof; (ii) an antibody represented by A2-3B06, comprising a VH sequence of SEQ ID NO: 42 or a humanized variant thereof and a VL sequence of SEQ ID NO: 43 or a humanized variant thereof; (iii) an antibody represented by A2-5D10, comprising a VH sequence of SEQ ID NO: 51 or a humanized variant thereof and a VL sequence of SEQ ID NO: 52 or a humanized variant thereof; (iv) an antibody represented by A2-7A05, comprising a VH sequence of SEQ ID NO: 77 or a humanized variant thereof and a VL sequence of SEQ ID NO: 80 or a humanized variant thereof; In some embodiments, the antibody or fragment thereof comprises the VH sequence of SEQ ID NO: 78 or a humanized variant thereof; (v) an antibody denoted A2-7F01 comprising the VH sequence of SEQ ID NO: 91 or a humanized variant thereof and the VL sequence of SEQ ID NO: 92 or a humanized variant thereof; or (vi) an antibody denoted C-14D12 comprising the VH sequence of SEQ ID NO: 109 or a humanized variant thereof and the 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 (according to Kabat and / or Chothia, AbM, Contact or IMGT) from the antibody denoted A-15B08. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs (according to Kabat and / or Chothia, AbM, Contact or IMGT) from the antibody denoted A2-3B06. In some embodiments, the antibody or fragment thereof comprises all three heavy chain CDRs and / or all three light chain CDRs from the antibody denoted A2-5D10 (according to 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 denoted A2-7A05 (according to 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 denoted A2-7F01 (according to 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 C-14D12 (according to Kabat and / or Chothia, AbM, Contact or IMGT). In some embodiments, the antibody or fragment thereof competes for binding with an antibody or fragment thereof comprising: (i) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A-15B08 (see, e.g., Table 1), (ii) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-3B06 (see, e.g., Table 2), (iii) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-5D10 (see, e.g., Table 3), or (iv) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated C-14D12 (see, e.g., Table 6). In some embodiments, the antibody or fragment thereof competes for binding with an antibody or fragment thereof comprising: (i) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-7A05 (see, e.g., Table 4) or (ii) all three heavy chain CDRs and / or all three light chain CDRs from the antibody designated A2-7F01 (see, e.g., Table 5).

[0125] In some embodiments, the present invention describes an antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody comprises: (a) a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences as shown in Tables 1-6; and / or (b) a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences as shown in Tables 1-6. In some embodiments, the antibody comprises a heavy chain variable (VH) region comprising the VH CDR1, VH CDR2, and VHCDR3 amino acid sequences as shown in Tables 1-6. In some embodiments, the antibody comprises a light chain variable (VL) region comprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences as shown in Tables 1-6.

[0126] 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.

[0127] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 4; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0128] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 7; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 8; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 9; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 10; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0129] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 12; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 4; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0130] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 13; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 15; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 16; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0131] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 18; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 19; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 21; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 22; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 23.

[0132] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 24; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 4; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0133] 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.

[0134] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 27; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 30; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0135] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 31; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 8; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 32; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 33; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0136] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 34; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 30; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0137] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 36; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 37; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0138] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 38; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 19; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 39; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 40; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 41; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 23.

[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 set forth in SEQ ID NO: 27; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 24; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 29; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 30; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 6,

[0140] 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.

[0141] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 44; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0142] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 7; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 8; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 9; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 46; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0143] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 12; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 2; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 44; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0144] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 13; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 15; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 47; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 48.

[0145] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 18; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 19; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 49; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 22; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 50.

[0146] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 1; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 24; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 3; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 44; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0147] 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 VLCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 69, and 75.

[0148] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 53; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 54; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 55; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 58.

[0149] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 59; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 60; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 61; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 62; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 63; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 58.

[0150] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 64; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 54; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 55; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 58.

[0151] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 65; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 66; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 67; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 68; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 63; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 69.

[0152] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 71; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 72; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 73; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 75.

[0153] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 53; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 76; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 55; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 58.

[0154] 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 VLCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 86, and 89.

[0155] 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 set forth in SEQ ID NO: 53; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 79; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 80; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 81,

[0156] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 59; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 82; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 83; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 62; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 63; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 81.

[0157] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 64; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 79; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 80; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 81.

[0158] 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 set forth in SEQ ID NO: 65; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 84; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 85; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 68; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 63; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 86.

[0159] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 70; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 87; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 88; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 73; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 74; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 89.

[0160] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 53; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 90; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 80; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 56; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 57; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 81.

[0161] In some embodiments, the present invention provides an antibody 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. CDR3.

[0162] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 93; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 95; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 96.

[0163] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 97; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 8; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 98; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 99; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 96.

[0164] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 100; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 95; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 96.

[0165] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 101; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 14; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 102; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 103; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 11; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 104.

[0166] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 105; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 19; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 106; and (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 107; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 22; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 108.

[0167] In some embodiments, the present invention provides an antibody comprising: (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 93; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 24; and (3) a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; and (b) a light chain variable (VL) region comprising: (1) a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 95; (2) a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 5; and (3) a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 96.

[0168] In some embodiments, described herein are antibodies comprising the VH and / or VL regions described herein that further comprise 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.

[0169] In some embodiments, the antibodies described herein are monoclonal antibodies. In some embodiments, the monoclonal antibodies are humanized antibodies, human antibodies, 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 by antibody fragments.

[0170] In some embodiments, the CDRs disclosed herein include consensus sequences derived from a group of related antibodies (see, e.g., Tables 1-6). As described herein, a "consensus sequence" refers to an amino acid sequence having conserved amino acids that are common among some sequences and variable amino acids that vary 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 for CDRs of α5β1 integrin binding agents (e.g., antibodies, such as bispecific antibodies) are as follows: Figure 2A and 2B In some embodiments, the CDRs disclosed herein include exemplary consensus sequences derived from groups of related antibodies (see, e.g., Tables 1-6, including, e.g., a first exemplary group from Tables 1, 2, 3, and 6 and a second exemplary group from Tables 4 and 5). Figure 2A and 2B 1 integrin binding agent (e.g., antibody, such as a bispecific antibody) are shown in . Thus, in some embodiments, the α5β1 integrin binding agent (e.g., antibody, such as a bispecific antibody) described herein comprises (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having an amino acid sequence of GFSLTX1YGVH (SEQ ID NO: 120), wherein X1 is a naturally occurring amino acid (e.g., S, T, or D); (2) a VH CDR2 having an amino acid sequence of VIWSDGSTTYX1SX2LKS (SEQ ID NO: 121), wherein X1 and / or X2 are each (or either) independently a naturally occurring amino acid (e.g., X1 is N, Q, S, or A and / or X2 is A or T); and (3) a VH CDR3 having an amino acid sequence of X1X2X3X4X5X6X7X8X9X 10Y (SEQ ID NO: 122) or HX1X2X3X4X5X6X7X8X9RYDALX10Y (SEQ ID NO: 123), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9 and / or X 10 Each (or any one) 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 10is D or A; and / or (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of TASSX1VX2SX3X4X5H (SEQ ID NO: 124), wherein X1, X2, X3, X4 and / or X5 are each (or any one of) 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 CDR2 having the amino acid sequence of STSNLAS (SEQ ID NO: 125); and (3) a VL CDR3 having the amino acid sequence of HQYX1RSPPT (SEQ ID NO: 126), wherein X1 is a naturally occurring amino acid (e.g., L, Y or H). Thus, in some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies, such as bispecific antibodies) comprise (a) a heavy chain variable (VH) region comprising: (1) a VH CDR1 having the amino acid sequence GYTFTIYWIN (SEQ ID NO: 127); (2) a VH CDR2 having the amino acid sequence X1IYPGSX2STX3YNEKFKX4 (SEQ ID NO: 128), wherein X1, X2, X3, and / or X4 are each (or any one of) naturally occurring amino acids (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 the amino acid sequence TGTGGX1AY (SEQ ID NO: 129). CDR3, wherein X1 is a naturally occurring amino acid (e.g., X1 is absent, L or F); and / or (b) a light chain variable (VL) region comprising: (1) a VL CDR1 having the amino acid sequence of RASSSVNYMY (SEQ ID NO: 130); (2) a VL CDR2 having the amino acid sequence of FTSSLAP (SEQ ID NO: 131); and (3) a VL CDR3 having the amino acid sequence of QQX1TX2SPFT (SEQ ID NO: 132), wherein X1 and / or X2, respectively (or either one) is a naturally occurring amino acid (e.g., X1 is F or L and / or X2 is T or G).

[0171] In some embodiments, described herein are binding agents (e.g., antibodies) that bind to substantially the same epitope as any of the antibodies described herein or fragments thereof. In some embodiments, described herein are binding agents (e.g., antibodies) that compete with any of the antibodies described herein or fragments thereof for binding to human α5β1 integrin. In some embodiments, the binding agent is an antibody or fragment thereof.

[0172] In certain aspects, the CDRs of α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, can be determined using 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., USDepartment of Health and Human Services, NIH Publication No. 91-3242).

[0173] In certain aspects, the CDRs of α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, can be determined according to the Chothia system (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).

[0174] In certain aspects, α5β1 integrin binding agents (e.g., antibodies), including CDRs of human α5β1 integrin binding agents, which will be referred to herein as "IMGT CDRs," can be determined according to 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.

[0175] In certain aspects, the CDRs of α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, which will be referred to herein as "AbM CDRs," can be determined according to the AbM system, e.g., as described in MacCallum et al., 1996, J. Mol. Biol., 262: 732-745. See also, e.g., Martin, A., "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

[0176] In certain aspects, the CDRs of α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, which will be referred to herein as "Contact CDRs," can be determined according to the Contact system (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5:732-745). Contact CDRs are based on analysis of available complex crystal structures.

[0177] In some embodiments, the position 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 the α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents described herein (see, e.g., CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT in Tables 1-6) can be altered by 1, 2, 3, 4, 5, or 6 amino acid positions (e.g., one or more amino acid modifications) as 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 position of a CDR defining any one of Tables 1, 2, 3, 4, 5, or 6 (according to Kabat and / or Chothia, AbM, Contact, or IMGT) can be altered by shifting the N-terminal and / or C-terminal boundaries of the CDR by 1, 2, 3, 4, 5, or 6 amino acids relative 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 other embodiments, 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 the α5β1 integrin-binding agents (e.g., antibodies), including human α5β1 integrin-binding agents described herein (see, e.g., CDRs according to Kabat and / or Chothia, AbM, Contact, or IMGT in Tables 1-6) can be altered by 1, 2, 3, 4, 5, or more amino acids (e.g., shorter or longer), 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 VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be 1, 2, 3, 4, 5, or more amino acids shorter than one or more CDRs described in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (according to 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 other embodiments, the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more CDRs described in SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90 and 93-108 (according to 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 other embodiments, the amino terminus of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (according to 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 other embodiments, the carboxyl terminus of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be extended by 1, 2, 3, 4, 5 or more carboxyl amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90 and 93-108 (according to 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 other embodiments, the amino termini of the VH and / or VL CDR1, CDR2 and / or CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (according to Kabat and / or Chothia, AbM, Contact, or IMGT), as 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 some embodiments, the carboxyl terminus of the VH and / or VL CDR1, CDR2, and / or CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more CDRs described by SEQ ID NOs: 1-24, 27-41, 44-50, 53-76, 79-90, and 93-108 (according to Kabat and / or Chothia, AbM, Contact, or IMGT), as 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 can be used to determine whether binding to α5β1 integrin (e.g., human α5β1 integrin) is maintained, for example, the binding assays and conditions described in the "Examples" section described herein. For example: Example 2 described herein describes an assay for measuring binding to α5β1 integrin (eg, human α5β1 integrin).

[0178] In other embodiments, the α5β1 integrin binding agents (e.g., antibodies) provided herein that bind to α5β1 integrin, including human α5β1 integrin binding agents, comprise conservative sequence modifications (e.g., modifications of one or more amino acids in one or more CDRs as described above). For polypeptides that are α5β1 integrin binding agents (e.g., antibodies), such as human α5β1 integrin binding agents, conservative sequence modifications include conservative amino acid substitutions, including those in which the amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains are 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, a predicted nonessential 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 eliminate 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, nucleotide and amino acid sequence modifications refer to up to 1, 2, 3, 4, 5, or 6 amino acid substitutions of the CDRs described in Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, e.g., up to (no more than) 4 conservative amino acid substitutions, e.g., up to (no more than) 3 conservative amino acid substitutions, e.g., up to (no more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution.

[0179] 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 an exemplary antibody variant. A-15B08-T62A was generated by replacing the threonine residue at position 62 in CDRH2 of antibody A-15B08 with alanine to remove the putative N-glycosylation site. Figure 2C and 2DThe VH, VL, and CDR sequences according to various numbering schemes for A-15B08-T62A are shown in More specifically, the antibody designated A-15B08-T62A comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 135 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 26. Figure 2C and 2D The six CDR sequences of A-15B08-T62A according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0180] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 135. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 135; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. 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.

[0181] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 135, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0182] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 135, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0183] If desired, the α5β1 integrin binding agent (e.g., antibody), including human α5β1 integrin binding agent (directly or indirectly) is linked or conjugated to a portion having an effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or a portion having immune recruitment activity to form an antibody-drug conjugate (ADC). The portion 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., antibody), including human α5β1 integrin binding agent, is optionally linked or conjugated (directly or indirectly) to a portion that assists in separation from the mixture (e.g., a label) or a portion that has reporter molecule activity (e.g., a detection label or a reporter protein). It will be understood that the features of the α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents described herein also extend to polypeptides comprising α5β1 integrin binding agent fragments.

[0184] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein, including human α5β1 integrin binding agents, are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or to a diagnostic agent or detectable agent (e.g., a labeled agent, including a labeled antibody). The conjugated or recombinantly linked antibodies can be useful, for example, for diagnosing, treating, and / or preventing α5β1 integrin-mediated diseases, disorders, and conditions, including cancer (e.g., cancers associated with or characterized by tumor cells expressing or overexpressing α5β1 integrin), angiogenesis-mediated diseases (e.g., diseases associated with or characterized by abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases, including MS and ALS).

[0185] Such diagnosis and / or detection, including the use of diagnostic and / or detectable agents, can be accomplished by coupling the α5β1 integrin binding agent (e.g., an antibody) to a detectable substance (e.g., a labeled agent, including a labeled antibody), including, for example, an enzyme, including but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; a prosthetic group, including but not limited to, streptavidin / biotin or avidin / biotin; a fluorescent material, including but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent material, including but not limited to, luminol; a bioluminescent material, including but not limited to, luciferase, luciferin, or aequorin; a chemiluminescent material, including but not limited to, an acridinium-based compound or HALOTAG; a radioactive material, including but not limited to, (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, 14 9Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn or 117Sn; positron emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0186] Labeled reagents (e.g., labeled antibodies) that specifically bind to α5β1 integrin can be used for diagnostic purposes to detect, diagnose, or monitor α5β1 integrin-mediated diseases, disorders, or conditions. Methods for detecting α5β1 integrin-mediated diseases, disorders, or conditions are described herein, comprising: (a) determining 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) as described herein that specifically bind to α5β1 integrin; and (b) comparing the level of α5β1 integrin to a control level (e.g., such as a level in a normal tissue sample from a patient who does not have an α5β1 integrin-mediated disease, disorder, or condition or from the same patient before the onset of the disease), wherein an increase in the measured level of α5β1 integrin compared to the control level of α5β1 integrin indicates an α5β1 integrin-mediated disease, disorder, or condition. Also described herein are diagnostic assays for diagnosing an α5β1 integrin-mediated disease, disorder, or condition comprising: (a) determining the level of α5β1 integrin in a cell or tissue sample of an individual using one or more α5β1 integrin binding agents (e.g., antibodies) as described herein that specifically bind to α5β1 integrin; and (b) comparing the level of α5β1 integrin to a control level (e.g., the level in a normal tissue sample), wherein an increase in the determined level of α5β1 integrin compared to the control level of α5β1 integrin is indicative of an α5β1 integrin-mediated disease, disorder, or condition. In certain embodiments, described herein are methods of treating an α5β1 integrin-mediated disease, disorder, or condition in a subject, comprising: (a) determining the level of α5β1 integrin in a cell or tissue sample of the subject using one or more α5β1 integrin binding agents (e.g., antibodies) as described herein that specifically bind to α5β1 integrin; and (b) comparing the level of α5β1 integrin to a control level (e.g., a level in a normal tissue sample), wherein an increase in the determined level of α5β1 integrin 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 an α5β1 integrin binding agent (e.g., antibody) herein to a subject identified as having an α5β1 integrin-mediated disease, disorder, or condition. A more definitive diagnosis of an α5β1 integrin-mediated disease, disorder, or condition may allow medical professionals to employ preventative measures or aggressive treatments earlier, thereby preventing the onset or further development of the α5β1 integrin-mediated disease, disorder, or condition.

[0187] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) described herein, including human α5β1 integrin binding agents, are components of a kit. In some embodiments, the kit includes an α5β1 integrin binding agent (e.g., antibody) or a composition (e.g., pharmaceutical composition) comprising an α5β1 integrin binding agent (e.g., antibody) packaged in appropriate packaging materials. The kit optionally includes a label or package insert that includes a description of the components, or instructions for in vitro, in vivo, or ex vivo use of the components.

[0188] Also described herein are α5β1 integrin-binding agents (e.g., antibodies) recombinantly linked or conjugated (covalently or non-covalently, directly or indirectly) to a heterologous protein or polypeptide (or fragment thereof, e.g., to a polypeptide (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to produce a fusion protein, and their uses. Specifically, described herein are fusion proteins comprising an α5β1 integrin-binding agent (e.g., antibody) described herein, including an antigen-binding fragment of a human α5β1 integrin-binding agent (e.g., comprising CDR1, CDR2, and / or CDR3 of VH and / or VL) 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., antibody) is linked is useful for targeting the α5β1 integrin-binding agent to specific cells (e.g., α5β1 integrin-expressing cells, including tumor cells).

[0189] In addition, the α5β1 integrin binding agents (e.g., antibodies), including 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 marker or tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine provides for convenient purification of fusion proteins. Other peptide tags used 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.

[0190] Methods for linking or conjugating moieties, including polypeptides, to antibodies (directly or indirectly) are well known in the art, any of which can be used to prepare the antibody-drug conjugates or fusion proteins described herein.

[0191] In some embodiments, the α5β1 integrin binding agent (e.g., antibody) described herein is a fusion protein. As used herein, the term "fusion protein" refers to a polypeptide comprising the amino acid sequence of a binding agent (e.g., an antibody) and the amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein that is not typically a part of an antibody (e.g., a non-α5β1 integrin binding antibody)). In some embodiments, the fusion protein retains the biological activity of the α5β1 integrin binding agent. In some embodiments, the fusion protein comprises an α5β1 integrin antibody VH region, a VL region, a VH CDR (1, 2, or 3 VH CDRs) and / or a VLCDR (1, 2, or 3 VL CDRs), wherein the fusion protein binds to an α5β1 integrin epitope, an α5β1 integrin fragment, and / or an α5β1 integrin polypeptide.

[0192] Fusion proteins can be produced, 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 used to alter the activity of α5β1 integrin binders (e.g., antibodies) as described herein, including human α5β1 integrin binders, including, for example, α5β1 integrin binders with high affinity and low dissociation rates. In some embodiments, α5β1 integrin binders, including human α5β1 integrin binders, can be altered prior to recombination by random mutagenesis performed via error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding the α5β1 integrin binders described herein can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0193] The α5β1 integrin binding agents (e.g., antibodies) described herein, including human α5β1 integrin binding agents, can also be attached to solid supports useful for immunoassays or purification of target antigens. These solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0194] The α5β1 integrin binding agents (eg, antibodies) described herein, including human α5β1 integrin binding agents, can also be linked or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0195] The linker can be a "cleavable moiety" that facilitates release of the linked or conjugated agent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates disclosed herein (e.g., antibody-drug conjugates) include, without limitation, acid-labile linkers (e.g., hydrazone linkers), disulfide-bond-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, e.g., valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance.

[0196] Conjugates of antibodies and agents can be prepared using a variety of bifunctional protein coupling agents, including those in which the agent is a drug for preparing ADCs, 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). The present disclosure also contemplates that conjugates of antibodies and agents can be prepared using any suitable method as disclosed in the art, including those in which the agent is a drug for preparing ADCs (see, e.g., Bioconjugate Techniques (Ed. Hermanson, 2nd ed. 2008)).

[0197] Conventional conjugation strategies for antibodies and reagents, including situations where the reagent is a drug for the preparation of ADCs, have been based on random conjugation chemistry involving the ε amino group of a Lys residue or the thiol group of a Cys residue, which has resulted in heterogeneous conjugates. Recent advances in technology allow site-specific conjugation to antibodies, resulting in uniform loading and avoiding conjugate subgroups with altered antigen-binding or pharmacokinetics. These include the engineering of "thiol monoclonal antibodies (thiomabs)", which include cysteine ​​replacements at positions providing reactive thiol groups on heavy and light chains, and which do not destroy immunoglobulin folding and assembly or alter antigens. In another approach, selenocysteine ​​is co-translationally inserted into the antibody sequence by inserting the terminal recoding stop codon UGA from selenocysteine, thereby allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids.

[0198] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies), including human α5β1 integrin binding agents, are conjugated to cytotoxic agents. In some embodiments, the α5β1 integrin binding agents disclosed herein (e.g., antibodies), including human α5β1 integrin binding agents, can be optionally conjugated to one or more cytotoxic agents disclosed herein or known in the art to produce ADCs. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including 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 or fragment thereof of bacterial, fungal, plant, or animal origin, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curculin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the cytotoxic agent is a radioactive isotope to produce a radioconjugate or a radioconjugated agent. A variety of radionuclides are available for the production of radioconjugated reagents, including but not limited to 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re, and 212Bi. Conjugates of polypeptides or molecules and one or more small molecule toxins, such as calicheamicin, maytansine, trichothecenes, and CC1065, as well as derivatives of these toxins having toxin activity, can also be used. Conjugates of polypeptides or molecules and cytotoxic agents are prepared using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0199] In some embodiments, the α5β1 integrin binding agents described herein (e.g., antibodies), including human α5β1 integrin binding agents, are conjugated to drugs such as signal transduction regulators, pro-apoptotic agents, mitotic inhibitors, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormone agents, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors. In some embodiments, the mitotic inhibitor is Aplysia, Auristatin, Maytansine, and a plant alkaloid. In some embodiments, the drug is Aplysia, Auristatin, Maytansine, and a plant alkaloid. Examples of Auristatin are monomethyl Auristatin F (MMAF) or monomethyl Auristatin E (MMAE). Examples of Maytansine 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 antibiotics, calicheamicin, and pyrotoxin. In some embodiments, the actinomycin is a pyrrolobenzodiazepine (PBD).

[0200] As described herein, α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, can be monospecific, bispecific, trispecific, or have more specificity. These agents can include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have 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 against α5β1 integrin, which has 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, multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of the antibodies described herein, for example, the CDR sequences in Table 1, Table 2, Table 3, Table 4, Table 5, and / or Table 6. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse, chimeric, human, or humanized antibodies. In some embodiments, one of the binding specificities of a multispecific antibody is for α5β1 integrin and the other for any other target (e.g., αvβ3 integrin). In some embodiments, a multispecific (e.g., bispecific) antibody can comprise more than one target (e.g., antigen) binding domain, wherein different binding domains are specific for different 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, a 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).

[0201] Methods for preparing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann, ed., 2011).

[0202] 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. 6795-106.

[0203] For example, bispecific antibody molecules can be divided into different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen-binding moieties attached; (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, Kλ-body, orthogonal Fab.

[0204] In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knob-in-hole" strategy, a SEED platform, common heavy chains (e.g., in κλ-bodies), and heterodimeric Fc regions. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art and include knob-in-hole, duobody, azymetric, charge pairs, HA-TF, SEEDbody, and differential protein A affinity.

[0205] Another form of dual-specific antibodies is IgG with other antigen-binding moieties. For example, by attaching other antigen-binding units to monospecific IgG (for example, at the N-terminus or C-terminus of any one of heavy chain or light chain), monospecific IgG can be engineered to have dual specificity. Exemplary other antigen-binding units include single domain antibodies (for example, variable heavy chain or variable light chain), engineered protein skeletons and paired antibody variable domains (for example, single chain variable fragments or variable fragments). The non-limiting examples of additional IgG forms 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, exemplary antibody formats are monospecific or multispecific (e.g., bispecific antibodies) B-body formats as described in, for example, International Patent Application Publication No. WO 2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0206] Dual-specific antibody fragments (BsAbs) are dual-specific antibody molecule formats that lack some or all of the constant domains of an antibody. For example, some BsAbs lack an Fc region. In some embodiments, a dual-specific antibody fragment comprises a heavy chain and a light chain region connected by a peptide linker that allows the BsAb to be effectively expressed in a single host cell. Non-limiting examples of dual-specific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scdiabodies, scdiabodies-CH3, diabodies-CH3, three antibodies (triple bodies), minibodies, 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 HCAbs, scdiabodies-Fc, diabodies-Fc, tandem scFv-Fc, and intrabodies.

[0207] Dual specific fusion protein comprises the antibody fragment that is connected to other protein.For example, dual specific fusion protein can be connected to other protein to increase other specificity and / or functionality.In some embodiments, docking lock (dock-and-lock, DNL) method can be used to produce the dual specific antibody molecule with higher titer.In addition, the serum half-life of antibody fragment can be extended with the dual specific antibody fusion protein of albumin binding protein or human serum albumin.In some embodiments, chemical conjugation, for example, the chemical conjugation of antibody and / or antibody fragment can be used to produce BsAb molecule.Exemplary dual specific antibody conjugate comprises CovX body form, wherein low molecular weight drug and each Fab arm or antibody or its fragment single reactive lysine site specificity are puted together.In some embodiments, puting together improves serum half-life.

[0208] Multispecific antibodies, including methods for producing bispecific antibodies, are known in the art. For example, multispecific antibodies, including bispecifics, 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 various methods known in the art, including affinity chromatography.

[0209] In some embodiments, the α5β1 integrin binding agents (e.g., antibodies) disclosed herein, including human α5β1 integrin binding agents, can be provided in any antibody form disclosed herein or known in the art. As a non-limiting example, in some embodiments, the α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, can be selected from Fab tandem 1g (FIT-1g); DVD-1g; hybrid hybridoma (quadroma or tetradoma); anti-carrier protein platform (Pieris); double-chain antibody; single-chain double-chain antibody; tandem single-chain Fv fragment; TandAbs, triple-specific Ab (Affimed); Darts dual-affinity retargeting agent (Macrogenics); dual-specific Xmabs (Xencor); dual-specific T cell engagement agent (Bites; Amgen; 55 kDa); triabody; triabody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (Creative Biolabs); Duobody platform (Genmab); dock-and-lock platform; knob-in-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).

[0210] In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain and one or more other binding domains that bind to one or more targets other than α5β1 integrin (e.g., αv integrin). In some embodiments, the multispecific (e.g., bispecific) antibodies disclosed herein comprise an α5β1 integrin binding domain comprising 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 comprising 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 comprising 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 comprising 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 comprising 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) antibodies disclosed herein comprise 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).

[0211] In some embodiments, described herein are multispecific (e.g., bispecific) antibodies comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs shown in Table 1 (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 comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs shown in Table 2 (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 comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs shown in Table 3 (e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, 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 comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs shown in Table 4 (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 comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs as shown 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 comprising a binding domain that binds to an α5β1 integrin comprising the VH and VL CDRs as shown 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).

[0212] Antibodies that bind to α5β1 integrin can be obtained by any suitable method, such as, but not limited to, immunization with whole tumor cells containing α5β1 integrin and a collection of antibodies, recombinant techniques, or screening libraries using antibodies or antibody fragments that target epitopes in the extracellular domain of α5β1 integrin. Monoclonal antibodies can be produced 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); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980); Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988); and Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd ed., Glover et al. (eds.), p. 93 (Oxford University Press 1995)). For example, an exemplary technique for producing monoclonal antibodies includes immunizing an animal with a human α5β1 integrin antigen and generating hybridomas from spleen cells collected from the animal. The hybridomas can produce monoclonal antibodies or antibody fragments that bind to α5β1 integrin.

[0213] In some embodiments, monoclonal antibodies or antibody fragments can be separated from antibody phage libraries, including antibody phage libraries as described herein. In some embodiments, antibody phage libraries can be produced using the technology described in (for example) Antibody Phage Display: Methods and Protocols, PMO'Brien and R.Aitken, eds., Humana Press, Totawa NJ, 2002. In some embodiments, antibody clones can be selected by screening phage libraries. Phage libraries can contain phages displaying a variety of fragments (for example, Fab, scFv) of antibody variable regions (Fv) fused to phage coat proteins. These phage libraries are screened for antibodies against desired antigens. Clones expressing Fv fragments (for example, Fab, scFv) that can be bound to the desired antigen are adsorbed to the antigen and therefore separated from the non-binding clones in the library. Then, the binding clones are eluted from the antigen and further enriched by other antigen adsorption / elution cycles.

[0214] The variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked by a short, flexible peptide, or as Fab fragments, in which they are separately fused to constant domains and interact non-covalently, as described, for example, in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).

[0215] The VH and VL genomic repertoires can be individually cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be searched for antigen-binding clones, as described, for example, by Winter et al., supra. Libraries from immune sources provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned, thereby providing a single source of human antibodies against a wide range of non-self and 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 prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences that encode highly variable CDR3 regions and achieve in vitro rearrangement, as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0216] Library screening can be achieved by a variety of 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 fixed to the adsorption plate or used in cell sorting, or conjugated to biotin for capture by streptavidin-coated beads, or used in any other method of panning display libraries. Antibody selection by slow lysis kinetics (e.g., excellent binding affinity) can be facilitated by using long washes and monovalent phage display, as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92 / 09690, and low antigen coating density, as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[0217] α5β1 integrin binding agents (e.g., antibodies) can be obtained by designing an appropriate antigen screening program to select phage clones of interest, and then using the VH and / or VL sequences (e.g., Fv sequences) from the phage clones 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 to construct full-length α5β1 integrin binding agent (e.g., antibody) clones.

[0218] Likewise, human antibodies that bind to α5β1 integrin can be produced by any of several 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 as known in the art and based on the disclosure herein. Methods for obtaining human antibodies from transgenic animals are further described in, for example, Bruggemann et al., Curr. Opin. Biotechnol., 8:45558, 1997; Jakobovits et al., Ann. NY Acad Sci., 764:52535, 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.

[0219] 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, wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. Transgenic non-primate mammalian hosts capable of generating an immune response to an immunogen have also been described, wherein the antibodies have primate constant and / or variable regions, and wherein the endogenous immunoglobulin encoding loci are replaced or inactivated. For example, International Patent Publication No. WO 96 / 30498 discloses the use of a Cre / Lox system to modify immunoglobulin loci in mammals, thereby replacing all or part of the constant or variable regions to form modified antibody molecules. For example, International Patent Publication No. WO 94 / 02602 discloses a non-human mammalian host with an inactivated endogenous Ig locus and a functional human Ig locus. For example, U.S. Patent No. 5,939,598 discloses a method for preparing transgenic mice, wherein the mice lack endogenous heavy chains and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions. Using transgenic animals, such as transgenic animals described herein, an immune response can be produced to selected antigenic molecules, and antibody-producing cells can be removed from animals and used to produce hybridomas of monoclonal antibodies secreting people-derived. Immunity protocols, adjuvants, etc. are known in the art and are used for, for example, the immunity of transgenic mice described in, for example, International Patent Publication No. WO 96 / 33735. Monoclonal antibodies can be tested to suppress or neutralize the biological activity or physiological effects of the corresponding protein.

[0220] The present invention discloses humanized antibodies that bind to α5β1 integrin, including human α5β1 integrin. Various 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 therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically collected from an "import" variable domain. Humanized antibodies that bind to α5β1 integrin can be generated using techniques known to those skilled 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. Patent Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0221] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six complementary determining regions (CDRs) of a parental non-human antibody (e.g., rodent) are grafted into 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 referred to these as "specificity determining residues" or SDRs. In the SDR grafting technique, only the SDR residues are grafted into the human antibody framework (see, e.g., Kashmiri et al., Methods 36: 25-34, 2005).

[0222] The selection of human variable domains (both light and heavy chains) for making humanized antibodies can be important for reducing antigenicity. For example, according to the so-called "best-fit" approach, the variable domain sequence of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to that of a rodent can be selected as the human framework for the humanized antibody (see, e.g., Sims et al. (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mol. Biol. 196: 901. Another approach 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 can be used for 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 derived from the most abundant human subgroup, V L 6Subgroup I(V L 6I) and V H Subgroup III (V H In another approach, human germline genes are used as the source of the framework regions.

[0223] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. The method involves comparing non-human sequences with a functional human germline genomic library. Then, genes encoding them or canonical structures closely related to murine sequences are selected. Then, within genes that share a canonical structure with non-human antibodies, those with the highest homology within the CDR are selected as FR donors. Ultimately, non-human CDRs are transplanted onto these FRs (see, e.g., Tan et al., J. Immunol. 169: 1119-1125, 2002).

[0224] Generally, it is also desirable to humanize antibodies while retaining their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one approach, humanized antibodies are prepared by a parental sequence analysis method and a variety of conceptual humanization products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible 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 SwissPDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays allows analysis of the likely role of the residues in the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind 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 significantly involved in influencing antigen binding.

[0225] Another method for antibody humanization is based on a measure of antibody humanity called human sequence content (HSC). This method compares the mouse sequence to the human germline genomic library and scores the differences as HSC. Then, by maximizing its HSC, rather than using a global identity measure, the target sequence is humanized to generate a variety of different humanized variants. (See, e.g., Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

[0226] In addition to the methods described above, empirical methods can be used to produce and select humanized antibodies. These methods include those based on the generation of large humanized variant libraries and the selection of the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome and yeast display libraries and by colony screening (see, e.g., 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).

[0227] In the FR library approach, a series of residue variants are introduced at specific positions in the FRs, and the library is then screened to select the FRs that best support the grafted CDRs. The residues to be replaced may 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 a more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0228] In FR rearrangement, the complete FR is combined with the non-human CDR, rather than generating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36:43-60, 2005). The library can be screened for binding in a two-step selection method in which the first step is to humanize the VL, followed by the VH. Alternatively, a one-step FR rearrangement method can be used. This method has been shown to be more efficient than the two-step screening because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0229] The "humanization" method is based on the experimental identification of the necessary minimum specificity determinants (MSDs) and is based on the sequential replacement and binding evaluation of non-human fragments into human FR libraries. It starts in the region of the CDR3 of the non-human VH and VL chains and gradually replaces the other regions of the non-human antibody with human FRs, including CDR1 and CDR2 of both VH and VL. This method generally results in epitope retention and identification of antibodies from multiple subclasses with different human V-segment CDRs. Humanization 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).

[0230] " Human engineering " method comprises by making specific changes to antibody amino acid sequence to produce the modified antibody that immunogenicity reduces in people while still retaining the desired binding property of original non-human antibody, changes non-human antibody or antibody fragment, as mouse or chimeric antibody or antibody fragment.Usually, this technology comprises the amino acid residue of non-human (for example, mouse) antibody is classified as " low risk ", " medium risk " or " high risk " residue.Use and classify by the overall risk / reward calculation that the predicted benefit (for example, for immunogenicity in people) of making specific replacement with respect to replacement will affect the risk assessment of gained antibody folding and / or replacement with human residue.Can select to replace specific people amino acid residue at the given position (for example, low or medium risk) of non-human (for example, mouse) antibody sequence by comparing with the corresponding region of specific or total human antibody sequence.According to comparison, the amino acid residue at low (" low ") or medium (" medium ") risk position in non-human sequence can be replaced with the corresponding residue in human antibody sequence. Techniques for preparing human engineered proteins are described in more detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Patents 5,766,886, 5,770,196, 5,821,123 and 5,869,619, and PCT Patent Application Publication No. WO 93 / 11794.

[0231] Provided herein are exemplary humanized antibodies generated based on the above-mentioned human engineering methods, including humanized antibodies represented as A-15B08_LoW, A-15B08_Low+Mod, A2-7A05_Low, A2-7A05_Low+Mod, C-14D12_Low, and C-14D12_Low+Mod. Figure 2C 、 2D 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 2E, 2F, 2G, and 2H.

[0232] 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. Figure 2C and 2D The six CDR sequences of A-15B08_Low according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0233] More specifically, 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. Figure 2C and 2D The six CDR sequences of A-15B08_LoW+Mod according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0234] More specifically, 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. Figure 2E and 2F The six CDR sequences of A2-7A05_Low according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0235] More specifically, 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. Figure 2E and 2F The six CDR sequences of A2-7A05_Low+Mod according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0236] More specifically, 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. Figure 2G and 2H The six CDR sequences of C-14D12_Low according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0237] More specifically, the antibody denoted 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. Figure 2G and 2H The six CDR sequences of C-14D12_Low+Mod according to Kabat, AbM, Chothia, Contact and IMGT are shown in FIG.

[0238] In some embodiments, provided herein are α5β1 integrin binding agents (e.g., antibodies or fragments thereof that bind 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, e.g. Figure 2C 、 2D , 2E, 2F, 2G and 2H.

[0239] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 136; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0240] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 139. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 138; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 139. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0241] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 140. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 141. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 140; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 141. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0242] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 143. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO: 142; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO: 143. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0243] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 144. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 144; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0244] In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 146. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 147. In some embodiments, the α5β1 integrin-binding agents provided herein comprise a VH comprising the VH CDR1, VH CDR2, and VH CDR3 as set forth in the VH comprising the amino acid sequence set forth in SEQ ID NO: 146; and a VL comprising the VL CDR1, VL CDR2, and VL CDR3 as set forth in the VL comprising the amino acid sequence set forth in SEQ ID NO: 147. In some embodiments, the CDRs are numbered according to Kabat. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the CDRs are numbered according to Chothia. In some embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to a combination of two or more numbering schemes selected from Kabat, AbM, Chothia, Contact, and IMGT.

[0245] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0246] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 138, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0247] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0248] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 142, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0249] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 144, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0250] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 146, and a VL comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical 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%).

[0251] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 136, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 137.

[0252] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 138, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 139.

[0253] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 140, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 141.

[0254] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 142, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 143.

[0255] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 144, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 145.

[0256] In some embodiments, the antibodies or fragments thereof provided herein comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 146, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 147.

[0257] 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, anticalin, fibronectin (adnectin), affibody (affibody), DARPin, fynomer, affitin, affilin, high affinity polymer (avimer), cysteine-rich knottin (knottin) peptide or engineered Kunitz-type inhibitor. Methods for producing such non-antibody protein scaffolds are well known in the art, any of which can be used to produce an α5β1 integrin binding agent comprising a non-antibody protein scaffold (see, e.g., 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)).

[0258] Also provided are materials for producing α5β1 integrin binding agents, including human α5β1 integrin binding agents and fragments thereof. For example, isolated cells (e.g., hybridomas, transformed or transfected cells) can produce α5β1 integrin binding agents (e.g., antibodies or antibody fragments). In this regard, the cell (e.g., an isolated cell) can produce an antibody or fragment thereof comprising a VH and a 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 Tables 1, 2, 3, 4, 5, or 6 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. Figures 2C-2HIn some embodiments, the polynucleotides described herein may comprise one or more nucleic acid sequences encoding an α5β1 integrin binding agent (e.g., an antibody or antibody fragment). In some embodiments, the polynucleotides are isolated and / or recombinant polynucleotides. In various aspects, the isolated polynucleotides comprise sequences encoding an antibody heavy chain variable region (VH) and / or an antibody light chain variable region (VL). L ) nucleotide sequence, wherein V H and V L Containing the CDRs shown in Table 1, the CDRs shown in Table 2, the CDRs shown in Table 3, the CDRs shown in Table 4, the CDRs shown in Table 5, the CDRs shown in Table 6, or the CDRs shown in Table 7. Figures 2C-2H The CDRs shown are identical CDRs (according to Kabat and / or Chothia, AbM, Contact or IMGT).

[0259] In some embodiments, one or more vectors (e.g., expression vectors) can contain one or more polynucleotides for expression of one or more polynucleotides in a suitable host cell. These vectors are useful, for example, for amplifying polynucleotides in host cells to produce useful quantities thereof, and for expressing binding agents, such as antibodies or antibody fragments, using recombinant techniques.

[0260] In some embodiments, one or more vectors are expression vectors, wherein the one or more polynucleotides encoding the antibody sequence are operably connected to one or more polynucleotides comprising expression control sequences. Specifically contemplated are the autonomous replication recombinant expression constructs of one or more polynucleotides encoding the antibody sequence binding α5β1 integrin, such as plasmids and viral DNA vectors. The expression control DNA sequence includes a promoter, an enhancer, and an operator, and is typically selected based on the expression system in which the expression construct (e.g., an expression vector) will be used. Promoter and enhancer sequences are typically selected for the ability to improve gene expression, while operator sequences are typically selected for the ability to regulate gene expression. An expression construct (e.g., an expression vector) may also include sequences encoding one or more selectable markers that allow identification of a host cell with the construct. An expression construct (e.g., an expression vector) may also include sequences that contribute to, and preferably promote, homologous recombination in a host cell. In some embodiments, an expression construct (e.g., an expression vector) may also include sequences necessary for replication in a host cell.

[0261] Exemplary expression control sequences include promoter / enhancer sequences, including, for example, cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993); Tie promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995); simian virus 40 promoter; DRA (downregulated in adenomas; Alrefai et al., Am. J. Physiol. Gastrointest. Liverpool Physiol., 293:G923-G934, 2007); MCT1 (monocarboxylate transporter 1; Cuff et al., Am. J. Physiol. Gastrointet. Liverpool Physiol., G977-G979.2005); and Math1 (mouse amodulin 1; Shroyer et al., Gastroenterology, 132:2477-2478, 2007). 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 cell-specific promoter or an endothelial cell-specific promoter. The polynucleotide may also optionally include a suitable polyadenylation sequence (e.g., SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g., 3') of the polypeptide coding sequence.

[0262] If desired, the one or more polynucleotides optionally further comprise a nucleotide sequence encoding a secretory signal peptide fused in frame to the polypeptide sequence. The secretory signal peptide directs secretion of the antibody polypeptide by cells expressing the one or more polynucleotides and is cleaved from the secreted polypeptide by the cells. The one or more polynucleotides may also optionally comprise a sequence whose only intended function is to aid in large-scale production of the vector. Polynucleotides for gene therapy can be produced and administered using procedures that have been described in the literature for a variety of transgenics. See, for example, Isner et al., Circulation, 91: 2687-2692, 1995; and Isner et al., Human Gene Therapy, 7: 989-1011, 1996.

[0263] In some embodiments, the polynucleotides may also comprise other sequences to facilitate uptake by the host cell and expression of the antibody or fragment thereof (and / or any other peptide). In some embodiments, a "naked" transgene encoding an antibody or fragment thereof as described herein (e.g., a transgene without virus, liposome or other vector-assisted transfection) is used.

[0264] Any suitable vector can be used to introduce one or more polynucleotides encoding antibodies or fragments thereof into a host. Exemplary vectors that have been described include replication-defective retroviral vectors, including but not limited to lentiviral vectors (see, e.g., Kim et al., J. Virol., 72(1): 811-816, 1998; Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46); parvoviral vectors, such as adeno-associated virus (AAV) vectors (U.S. Pat. No. 5,474,934); 51; 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., 45:87-98, 1997); adenovirus (AV) vectors (see, e.g., U.S. Pat. Nos. 5,792,453; 5,82 4,544; 5,707,618; 5,693,509; 5,670,488; 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, 6 8: 143-155, 1992); adenovirus adeno-associated virus chimeric vectors (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); liposome 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 vector is rendered replication-defective by, for example, deleting or disrupting a selectable gene required for viral replication.

[0265] Other non-viral delivery mechanisms contemplated 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, 1984), direct microinjection (Harland and

[0015] The present invention relates to the use of liposomes loaded with DNA (Weintraub, J. Cell Biol., 101: 1094-1099, 1985), liposomes loaded with DNA (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: 8463-8467, 1987), gene bombardment using high-speed microprojectiles (Yang et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467, 1987), and the use of liposomes loaded with DNA (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84: 8463-8467, 1987). 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).

[0266] The expression vector (or the antibody or fragment thereof described herein) can be entrapped in liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases. Targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C, eds., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275(5301): 810-814, 1997). Various commercial methods are also contemplated, including "lipofection" technology. In some embodiments, the liposomes can be complexed with hemagglutinating virus (HVJ). This has been shown to facilitate fusion with cell membranes and promote cellular entry of liposome-encapsulated DNA (see, for example, Kaneda et al., Science, 243: 375-378, 1989). In some embodiments, the liposomes are complexed with or used in conjunction with a nuclear non-histone sex chromosome protein (HMG-1) (see, e.g., Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In some embodiments, the liposomes are complexed with or used in conjunction with both HVJ and HMG-1. These expression constructs have been successfully used for the transfer and expression of nucleic acids in vitro and in vivo. In some embodiments, an α5β1 integrin binding agent (e.g., an antibody), including a human α5β1 integrin binding agent, is included in the liposomes to target the liposomes to cells that express α5β1 integrin on their surface (e.g., tumor cells).

[0267] The cell may comprise one or more polynucleotides or one or more vectors, for example, the cell is transformed or transfected with one or more polynucleotides encoding an α5β1 integrin binder (e.g., an antibody), including a human α5β1 integrin binder, or one or more vectors comprising the one or more polynucleotides. In some embodiments, the cell expresses and produces an α5β1 integrin binder (e.g., an antibody), including a human α5β1 integrin binder, containing one or more, including six, CDRs that are at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 100%) identical to the CDRs of A-15B08 (see, e.g., Table 1). In some embodiments, the cell expresses and produces an α5β1 integrin binder (e.g., an antibody), including a human α5β1 integrin binder, containing V sequences comprising the same CDRs as those of A2-3B06. H and V L(See, e.g., Table 2.) In some embodiments, the cells express and produce α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, comprising V sequences comprising the same CDRs as those of A2-5D10. H and V L (See, e.g., Table 3.) In some embodiments, the cells express and produce α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, comprising V sequences comprising the same CDRs as those of A2-7A05. H and V L (See, e.g., Table 4.) In some embodiments, the cells express and produce α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, comprising V sequences comprising the same CDRs as those of A2-7F01. H and V L (See, e.g., Table 5.) In some embodiments, the cells express and produce α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, comprising V sequences comprising the same CDRs as those of C-14D12. H and V L (See, e.g., Table 6). The cell can be a prokaryotic cell, such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or a eukaryotic cell, such as an animal cell (e.g., a myeloma cell, a Chinese hamster ovary (CHO) cell, or a hybridoma cell), a yeast (e.g., Saccharomyces cerevisiae), or a plant cell (e.g., a tobacco, corn, soybean, or rice cell). The use of mammalian host cells can provide translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that may be desirable to confer optimal biological activity on the recombinant expression product. Similarly, polypeptides (e.g., α5β1 integrin binding agents, such as antibodies), including human α5β1 integrin binding agents) can be glycosylated or non-glycosylated and / or covalently modified to include one or more water-soluble polymer linkages, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0268] Methods for introducing DNA or RNA into host cells are well known and include transformation, transfection, electroporation, nuclear injection, or fusion with vectors such as liposomes, micelles, ghost cells, and protoplasts. These host cells are useful for amplifying polynucleotides and expressing polypeptides encoded by the polynucleotides. In this regard, a method for producing an α5β1 integrin binding agent (e.g., an antibody) may comprise culturing host cells and isolating the α5β1 integrin binding agent. Transfer of naked DNA expression constructs into cells can be achieved using particle bombardment, which is based on the ability to accelerate DNA-coated microprojectiles to high speeds, thereby allowing them to pierce cell membranes and enter cells without killing them (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 a high-voltage discharge to generate an electric current, which in turn provides power (see, e.g., Yang et al., Proc. Natl. Acad Sci USA, 87: 9568-9572, 1990). The microprojectiles used are composed of biologically inert materials, such as tungsten or gold beads. The host cells can be isolated and / or purified. Host cells can also be cells transformed in vitro to induce transient or permanent expression of a polypeptide in vitro. Host cells can also be isolated cells transformed ex vivo and introduced post-transformation, for example, to produce a polypeptide in vivo for therapeutic purposes. The definition of host cell specifically excludes genetically modified humans.

[0269] The various methods for producing antibodies from polynucleotides are generally well known. For example, Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, New York, 1989 (also referring to Maniatis et al., 3rd edition, Cold Spring Harbor Laboratory, New York, 2001) describe basic molecular biology procedures. In addition, various publications describe techniques suitable for producing antibodies by DNA manipulation, expression vectors, and transformation and cultivation of suitable cells (see, for example, Mountain and Adair, Chapter 1, in Biotechnology and Genetic Engineering Reviews, Tombs, ed., Intercept, Andover, UK, 1992); and Current Protocols in Molecular Biology, Ausubel, ed., Wiley Interscience, New York, 1999).

[0270] α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, are produced using any suitable method, for example, by isolation from immunized animals, recombinant or synthetic production, or genetic engineering, including as described above. Antibody fragments derived from antibodies are obtained, for example, by proteolysis of the antibodies. For example, papain or pepsin digestion of intact antibodies yields a 5S fragment, or a monovalent Fab fragment, and an Fc fragment, respectively, referred to as F(ab')2. F(ab)2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described in, for example, 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; U.S. Pat. No. 4,331,647; and Andrews, S. M. and Titus, J. A. in Current Protocols in Immunology (Coligan et al., eds.), John Wiley & Sons, New York (2003), pp. 2.8.12.8.10 and 2.10A.12.10A.5.

[0271] α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, can be genetically engineered. For example, α5β1 integrin binding agents (e.g., antibodies), including human α5β1 integrin binding agents, comprise variable regions or variable domains produced, for example, by recombinant DNA engineering techniques. In this regard, the variable regions are optionally modified by insertions, deletions, or changes in the antibody amino acid sequence to produce the antibody of interest, including as described above. For example, polynucleotides encoding the complementarity determining regions (CDRs) of interest, including the CDRs listed in Tables 1-6, are prepared by synthesizing the variable region using mRNA from antibody-producing cells as a template using the polymerase chain reaction (PCR). See, for example, Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclona, ​​Antibodie: Production, Engineering and Clinical Application, Ritter et al. (Eds.), p. 166 (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (Eds.), p. 137 (Wiley Liss, Inc. 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106-110, 1991). Current antibody manipulation techniques allow the construction of engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remaining variable region domain from a second antibody. These techniques are used, for example, to humanize an antibody or to improve its affinity for binding to a target.

[0272] "Humanized antibody " is an antibody in which the CDRs of the variable heavy and light chains of non-human immunoglobulins are transferred to human variable domains. In some embodiments, constant regions do not need to exist, but if they exist, they are optionally substantially identical to human immunoglobulin constant regions, for example, at least about 85-90%, about 95%, 96%, 97%, 98%, 99% or more identical. Therefore, in some cases, except for possible CDRs, all parts of humanized immunoglobulins are substantially identical to the corresponding parts of natural human immunoglobulin sequences. For example, humanized antibodies are human immunoglobulins (for example, host antibodies), wherein the hypervariable region residues of host antibodies are replaced by non-human species (donor antibodies), such as mice, rats, rabbits or non-human primates with desired specificity, affinity and ability.

[0273] 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 a disease, disorder, or condition. In some embodiments, the subject is diagnosed with an α5β1 integrin-mediated disease, disorder, or condition. α5β1 integrin-mediated diseases, disorders, and conditions include, but are not limited to, cancer (e.g., cancers associated with or characterized by tumor cells expressing or overexpressing α5β1 integrin), angiogenesis-mediated diseases (e.g., diseases associated with or characterized by abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases, including MS and ALS).

[0274] In some embodiments, described herein are methods of treating 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. In some embodiments, the subject is diagnosed with cancer.

[0275] In some embodiments, described herein are methods of 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.

[0276] In some embodiments, described herein are methods of (i) treating angiogenesis-mediated diseases, disorders, or conditions or (ii) inhibiting angiogenesis in a subject (e.g., a subject with 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 angiogenesis-mediated diseases, disorders, or conditions.

[0277] In some embodiments, described herein are methods of 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.

[0278] In some embodiments, described herein are methods of treating an inflammatory disease, disorder, or condition in a subject, including a neuroinflammatory disease, disorder, or condition (e.g., MS, ALS), 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, including a neuroinflammatory disease, disorder, or condition (e.g., MS, ALS).

[0279] In some embodiments, described herein are methods of alleviating one or more symptoms associated with an inflammatory disease, including 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.

[0280] One or more therapeutic agents described herein can be administered to a subject of the methods described above in combination with an α5β1 integrin binding agent (eg, antibody) or fragment thereof described herein, or a pharmaceutical composition comprising a binding agent (eg, antibody) described herein.

[0281] In some embodiments, the antibody is a human antibody, including but not limited to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences, such as, for example, Kabat et al. (1991) Sequences of proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242. If the antibody contains a constant region, the constant region is also preferably derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, for example, to enhance antibody activity, but do not include CDRs from other species (e.g., mouse CDRs placed within human variable framework regions).

[0282] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody) binds to and kills tumor cells in cell culture. These cell cultures 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 cancer cells, glioblastoma cells, neuroblastoma cells, uterine cancer cells, colorectal cancer cells, gastric cancer cells, bladder cancer cells, and pancreatic cancer cells.

[0283] In some embodiments, described herein are methods of inhibiting abnormal angiogenesis in a subject (e.g., a subject with a tumor). For example, the methods comprise administering an α5β1 integrin binding agent (e.g., an antibody) described herein, such as a human α5β1 integrin binding agent, in an amount effective to inhibit abnormal angiogenesis. In some embodiments, the method comprises 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., Tables 1, 2, 3, 4, 5, and / or 6 for CDRs and VH / VL) for binding to human α5β1 integrin and / or binds to a region of α5β1 integrin recognized by antibody A-15B08, antibody A2-3B06, antibody A2-5D10, antibody A2-7A05, antibody A2-7F01, and / or antibody C-14D12 (see, e.g., Tables 1, 2, 3, 4, 5, and / or 6 for CDRs and VH / VL), thereby resulting in inhibition of aberrant angiogenesis, including α5β1 integrin binding agents. In some embodiments, one or more binding agents (eg, antibodies), polynucleotides, vectors, and / or cells as described above can be used in methods of inhibiting abnormal angiogenesis in vivo (eg, methods of treating cancer in a subject).

[0284] Also provided are methods for regulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject. For example, the method comprises administering to the subject a composition comprising an α5β1 integrin binding agent (e.g., an antibody) in an amount effective for regulating tumor growth in the subject. "Tumor" refers to any neoplastic cell growth or proliferation, whether malignant or benign, and refers to all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a mammalian neoplasm in the physiological condition described, that is, a neoplasm generally characterized by unrestricted or abnormal cell growth and including malignant neoplasms including (but not limited to): carcinomas, lymphomas, blastomas, sarcomas, and leukemias. 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, spleen cancer, prostate cancer, including hormone-refractory prostate cancer, liver cancer, head and neck cancer, stomach 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 of 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, alone or in combination with another agent, to a subject in need thereof.

[0285] "Inhibiting" abnormal angiogenesis does not require 100% inhibition. Any inhibition that reduces tumor growth and / or metastasis is contemplated. Similarly, "modulating" tumor growth refers to reducing tumor size, slowing the growth of a tumor, or inhibiting the size of an existing tumor. Complete elimination of the tumor is not required; any reduction in tumor size or reduction in tumor growth constitutes a beneficial biological effect in a subject. In this regard, tumor cell removal can be enhanced, for example, by at least about 5%, at least about 10%, or at least about 20%, compared to the level of removal observed in the absence of the method (e.g., in a biological control subject or sample not exposed to the agent of the method). For example, the effect is detected by a reduction in tumor size or a reduction in tumor metastasis, a reduction or maintenance of tumor marker levels, or a reduction or maintenance of tumor cell populations. In some embodiments, tumor cell removal is enhanced, for example, 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., antibody) of the method.

[0286] The specific administration regimen of the α5β1 integrin binding agent (e.g., antibody) for a particular subject will depend in part on the agent used, the amount of the agent administered, the route of administration, and the cause and extent of any side effects. The amount of the agent (e.g., antibody) administered to the subject (e.g., a mammal, such as a human) should be sufficient to produce the desired response within a reasonable time frame. Therefore, in some embodiments, the amount of the α5β1 integrin binding agent (e.g., antibody) or pharmaceutical composition described herein administered to the subject is an effective amount. In some embodiments, the amount of the α5β1 integrin binding agent (e.g., antibody) or pharmaceutical composition described herein administered to the subject is a therapeutically effective amount. In some aspects, the method comprises administering (e.g.,) from about 0.1 μg / kg to about 100 mg / kg or more. Some conditions or disease states require prolonged treatment, which may or may not require the administration of α5β1 integrin binding agents (e.g., antibodies), including doses of human α5β1 integrin binding agents (e.g., antibodies), over multiple uses.

[0287] Suitable routes of administration of compositions comprising α5β1 integrin binding agents (e.g., antibodies), such as human α5β1 integrin binding agents (e.g., antibodies) are well known in the art. Although more than one route can be used to administer an agent (e.g., an antibody), a particular route can provide a faster and more effective response than another route. Depending on the circumstances, the 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 membranes, ingested, inhaled, and / or introduced into the circulation. For example, it is desirable to deliver a composition comprising an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, by intravenous, subcutaneous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, ​​vaginal or rectal injection, by a sustained release system or by an implant device. If desired, the α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, is administered locally via intraarterial or intravenous administration to the area of ​​interest, for example, via the hepatic artery for delivery to the liver. Alternatively, the α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, is administered locally via implantation of a membrane, sponge or another suitable material onto which the binding agent is absorbed or encapsulated. When an implant device is used, in one aspect, the device is implanted into any suitable tissue or organ, and the α5β1 integrin binding agent (e.g., antibody), such as human α5β1 integrin binding agent, is delivered (e.g., via diffusion, timed-release bolus, or continuous administration. In other aspects, the α5β1 integrin binding agent (e.g., antibody) is administered directly to tissue exposed during tumor resection or other surgical procedures.

[0288] The present invention discloses compositions, such as pharmaceutical compositions, comprising an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, and a carrier (e.g., a pharmaceutically acceptable carrier). The specific carrier used may depend on chemical-physical considerations, such as solubility and lack of reactivity with the binding agent or co-therapy and by route of administration. Pharmaceutically acceptable carriers are well known in the art, and examples thereof are described herein. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Injectable formulations are further described in, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Co., Philadelphia. Pa., Banker and Chalmers., eds., pp. 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)). In one aspect, pharmaceutical compositions comprising an α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, are placed in a container along with packaging materials that provide instructions for use of these pharmaceutical compositions. Typically, these instructions include a description of the concentrations of reagents that may be necessary for reconstitution of the pharmaceutical composition, and in some embodiments, a clear expression of the relative amounts of excipient components or diluents (e.g., water, saline, or PBS).

[0289] In some aspects, the methods described herein further comprise administering one or more other agents, including therapeutic agents, which may be present in the composition or may be administered together with the α5β1 integrin binding agent (e.g., an antibody), such as a human α5β1 integrin binding agent, or provided in a separate composition using the same or different administration routes. The one or more other agents, including therapeutic agents, may be administered together with the α5β1 integrin binding agent (e.g., an antibody) (e.g., for combination therapy) or separately (e.g., simultaneously, alternatingly, sequentially). These other therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapies and immunotherapeutics, cytotoxic agents, chemotherapeutic agents, and inhibitors.

[0290] Therapeutic antibodies that can be used with an α5β1 integrin binding agent (e.g., an antibody) as described herein (e.g., for combination therapy) include, but are not limited to, 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., AMG820), C1Q-binding antibodies (ANX005), CD40L-binding antibodies (e.g., lulizumab), FGFR antibodies (e.g., bemarituzumab), 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 type I interferon (IFN) (e.g., sifalimumab).

[0291] Immunotherapies and immunotherapeutics that can be used (e.g., in combination therapy) with an α5β1 integrin binding agent (e.g., an antibody) as described herein include, but are not limited to, cytokines, interleukins, tumor necrosis factor, and combinations thereof. In some embodiments, the immunotherapy includes an immunotherapeutic agent that modulates an immune response, e.g., a checkpoint inhibitor or a checkpoint agonist. In some embodiments, the immunotherapeutic agent is an antibody modulator targeting the following as known in the art: 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, TGF β, OX40, 41BB, LIGHT, CD40, GITR, TGF-β, TIM-3, SIRP-α, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS and / or BTNL2 and other targets known in the art. In some embodiments, the immunotherapeutic agent is an agent that increases the activity of natural killer (NK) cells. In some embodiments, the immunotherapeutic agent is an agent that suppresses immune response suppression. In some embodiments, the immunotherapeutic agent is an agent that suppresses suppressive cells or suppressive 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 inhibitory immune checkpoint receptor activity.

[0292] In some embodiments, the immunotherapeutic agent includes T cell regulators, which are selected from agonists or activators of costimulatory molecules. In one embodiment, the agonist of the costimulatory molecules is selected from GITR, OX40, ICOS, SLAM (for example, SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3 or the agonist of CD83 parts (for example, agonistic antibodies or their antigen-binding fragments, or soluble fusion). In other embodiments, effector cell combination includes dual specific T cell engagement agents (for example, dual specific antibody molecules bound to CD3 and tumor antigens (for example, EGFR, PSCA, PSMA, EpCAM, HER2 etc.)).

[0293] Cytotoxic agents that can be used with the α5β1 integrin binding agents (e.g., antibodies) as described herein (e.g., for combination therapy) include substances that inhibit or prevent cellular function and / or cause cell death or destruction. Exemplary cytotoxic agents include, but are not limited to, 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.

[0294] Chemotherapeutic agents that can be used with the α5β1 integrin binding agents (e.g., antibodies) as described herein (e.g., for combination therapy) include compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; aziridines and methylmelamines, including hexamethylmelamine, trothamide, triethylenephosphamide, triethylenethiophosphamide, and trimethylolomelamine; polyacetyl groups (specifically, bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callistine ystatin); CC-1065 (including its synthetic analogs of adolesin, carzelesin and biszelesin); candidiasis (particularly candidiasis 1 and candidiasis 8); caudate tin; duodenal toxin (including synthetic analogs, KW-2189 and CB1-TM1); argentin; pancratistatin; sarcodictyin; sponge inhibitors; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholephosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride, melphalan, nembicin, cholesteryl phenylacetic acid mustard, prednimustine, tricholoma. phosphamide, uracil; nitrosoureas such as carmustine, pyroglurea, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, specifically calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycins in), including dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarcin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycinis, dactinomycin, daunorubicin, detoximum iodide, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenamide anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, azathioprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and azauridine; and androgens, such as carprofen Testosterone, drostanolone propionate, cyclopentolone, melastane, testosterone; adrenal inhibitors such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diacronitrile; eflornithine acid; elliptonium acetate; epothilone; etoglucagon; gallium nitrate; hydroxyurea; mushroom polysaccharides; lonidainine; maytansines, such as maytansine and ansamitocin; mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin; phenamet, pirarubicin; losoxantrone; podophyllic acid; 2-acetylhydrazine; procarbazine; PSK Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizolan; spirogermanamine; tricholomanic acid; triazoline; 2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, baculocin A, and anguidine); ethyl carbamate; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g., TAXOL Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE TMCremophor-free albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE Docetaxel ( Rorer, Antony, France); Chlorambucil; GEMZAR Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine Vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronic acid; irinotecan (Camptosar, CPT-11) (including regimens of irinotecan with 5-FU and folinic acid); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; folinic acid (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, and EGFR that reduce cell proliferation (e.g., erlotinib (Tarceva) TM )) and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are anti-hormonal agents that act to modulate or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX Tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifene, keoxifene, LY117018, onapristone, and FARESTON toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE Megestrol acetate, AROMASIN Exemestane, formestane, fadrozole, RIVISOR Folozole, FEMARA Letrozole and ARIMIDEX anastrozole; 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 signal transduction pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME ribozymes) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, for example, ALLOVECTIN Vaccines, LEUVECTIN Vaccines and VAXIDs Vaccine; PROLEUKIN rIL-2;LURTOTECAN Topoisomerase 1 inhibitor; ABARELIX rmRH; vinorelbine and esperamicin and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0295] Inhibitors that can be used with an α5β1 integrin binding agent (e.g., an antibody) as described herein (e.g., for combination therapy) include, but are not limited to, kinase inhibitors, such as FAK inhibitors (e.g., GSK2256098), MEK inhibitors (e.g., cobimetinib, trametinib, 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).

[0296] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody) as disclosed herein can be used in combination with an inhibitor of PD-1 or an inhibitor of PD-L1, e.g., an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody, e.g., nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, or avelumab.

[0297] In some embodiments, an α5β1 integrin binding agent (e.g., an antibody) as disclosed herein can be used in combination with a CTLA-4 inhibitor, e.g., an anti-CTLA-4 antibody, e.g., plimumab (Yervoy), or with an antibody to a cytokine, or with a bispecific antibody that binds to PD-L1 and CTLA-4 or PD-1 and CTLA-4, or with other anti-cancer agents.

[0298] The additional agent may be a pharmaceutically acceptable salt, ester, amide, hydrate and / or prodrug of any of the therapeutic agents described above or other agents.

[0299] The additional therapeutic agent may be a pharmaceutically acceptable salt, ester, amide, hydrate and / or prodrug of any of the therapeutic agents described above or other agents.

[0300] It is to be understood that variations that do not significantly affect the activity of the various embodiments described herein are also provided within the definitions and subject matter described herein. Accordingly, the following examples are intended to illustrate, but not to limit, the present disclosure. Example

[0301] Example 1: Antibody Generation and Initial Screening for α5 Integrin Binding

[0302] NZBW and CD-1 mice (4 each) 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. K562 cells (ATCC) that had been activated for 24 hours with 10 ng / mL PMA (phorbol 12-myristate 13-acetate; Sigma-Aldrich, St. Louis, MO; Catalog No. 5.00582) were analyzed by flow cytometry. CCL-243 TM ; Manassas, VA 20110) were positive, and approximately 4,000 hybridoma supernatants (2,500 from the NZBW strain and 1,500 from the CD-1 strain) were screened, stored frozen, and thawed immediately before use. Briefly, hybridoma supernatants were incubated with activated K562 cells for 20 minutes, washed, then incubated with a fluorescently conjugated detection antibody for 20 minutes, washed, resuspended in 7-aminoactinomycin D, and the mean fluorescence intensity (MFI) was measured on 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 with MFIs significantly higher (>1.5×) than those obtained from hybridoma culture medium alone (MFI of 125 for K562 cells).

[0303] Table 7

[0304]

[0305]

[0306]

[0307] Example 2: Screening for α5 binding specificity

[0308] The selected 249 positive hybridoma supernatants were screened for reactivity to rh-α5β1 in a plate-based ELISA as described in Example 1. Immulon4HBX ELISA 96-well plates (Thermo Fisher Scientific, Waltham, MA, Catalog No. 3855) were coated with 1 μg / mL of rh-α5β1 (R&D Systems, Minneapolis, MN 55413, Catalog No. 3230-A5) in PBS supplemented with 0.5 mM MgCl2, MnCl2, and CaCl2 and incubated overnight at 4°C. The plates were washed three times with wash buffer (1× Tris-buffered saline containing 0.05% Tween 20), blocked with 2% BSA in 1× TBS (prepared from 10× Thermo Scientific Blocker BSA (10×) in TBS; Thermo Fisher Scientific, Waltham, MA, catalog number No. 37520) for 2 hours at room temperature (RT), and then incubated with hybridoma supernatants diluted in standard diluent (2% BSA, 1× TBS, 0.05% Tween 20). After incubation at room temperature for 1 hour, the wells were washed three times, incubated with a 1:8000 dilution of a biotinylated goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA, catalog number 62-6540) for 1 hour, washed three times, and then incubated with poly-HRP streptavidin (ThermoFisher Scientific, Waltham, MA, catalog number N200) for 30 minutes. The wells were washed four times and incubated with TMB (Thermo Fisher Scientific, Waltham, MA, catalog number N301) for 5-10 minutes, followed by the addition of ELISA stop solution (Invitrogen, Carlsbad, CA, catalog number SS04). The absorbance at 450 nm was measured. The results are shown in Tables 8A and 8B.

[0309] Table 8A

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] Table 8B

[0316]

[0317]

[0318]

[0319] Concurrently, 249 hybridomas were screened for specificity for the human α5 subunit by testing reactivity against rh-α4β1 (R&D Systems, Minneapolis, MN 55413; Catalog Nos. 3230-A5 & 5668-A4). The results are shown in Tables 8A and 8B. Any hybridoma that reacts to both rh-α4β1 and rh-α5β1 is likely specific for the β1 subunit and is not considered α5 specific. Using the protocol described above, 29 hybridoma supernatants reacted strongly with rh-α4β1 (absorbance at 450 nm > 1.0) and were not considered α5 specific.

[0320] In addition, the hybridomas were tested for cross-reactivity against 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 (absorbance at 450 nm > 1.0) and strong reactivity to rh-α4β1.

[0321] Of the 220 hybridomas that showed specific binding to the α5 subunit, 28 were selected for further characterization and represented a range of anti-α5 binding antibodies with absorbance readings at 450 nm ranging from 0.32 to 2.3 that showed no cross-reactivity to rh-α4β1. In this 1:10 dilution single-point assay, none showed detectable cross-reactivity to rm-α5β1.

[0322] Example 3: Selection of antibodies with high α5 binding affinity

[0323] Kinetic analysis was performed on 28 hybridoma supernatants selected as described in Example 2 to calculate the equilibrium dissociation constant (K) of the antibody clones binding to 20 nM rh-α5β1 (Acro Biosystems, Newark, DE; Catalog No. IT1-H52W5) using a ForteBio Octet BMIA instrument (ForteBio, Fremont, CA). D =k off / kon). An anti-mouse IgG Fc biosensor was loaded with each clone supernatant. The assay steps were as follows: sensor check (30 s) -> Ab loading (700 s) -> Quenching (480 s) -> Baseline (480 s) -> Ab binding (600 s) -> Dissociation (600 s). Table 9 shows the kinetic data. 20 hybridomas had a K of less than 10 nanomolar (nM). D , which ranged from 0.4 to 7.8 nM and were selected for further characterization.

[0324] Table 9

[0325]

[0326] Example 4: Variable region sequencing to identify unique antibody sequences

[0327] 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 9 unique heavy and light chain pairs, which were assigned group numbers 1 to 9. One antibody clone was selected to represent each unique sequence group as shown in Table 10.

[0328] Table 10

[0329]

[0330] Example 5: Selection of antibodies that inhibit the binding of fibronectin to α5β1

[0331] 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 and assayed by BCA (Pierce TM Protein concentration was measured using a 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.

[0332] Immulon4 HBX ELISA 96-well plates were coated with FN by incubation overnight at 4° C. 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). The 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, 1×TBS 0.05% Tween 20) containing 0.1 μg / mL rh-α5β1-6×His-tagged protein (Acro Biosystems, Newark, DE, Catalog No. IT1-H52W5) to generate an 11-point 1:3 antibody dilution series ranging from 10,000 ng / mL to 0.17 ng / mL. An isotype control antibody (Control Ab; Ms IgG2a EMD Millipore Corp, Billerica, MA, Catalog No. PP102) was used to normalize data between different assay runs. For the assay, after removing the blocking solution and washing the wells three times, 100 μL of the antibody dilution series / His-tagged-α5β1 mixture was added to the wells. After 1 hour at room temperature, the wells were washed three times, incubated with biotinylated anti-6×His-tag Ab (Invitrogen, Carlsbad, CA, catalog number MAI-21315-BTIN) at 1:1000 in standard diluent for 1 hour, washed three times, incubated with poly-HRP streptavidin (Thermo Fisher Scientific, Waltham, MA, catalog number N200) for 30 minutes, washed four times, incubated with TMB substrate (Thermo Fisher Scientific, Waltham, MA, catalog number N301) for 2-5 minutes, and then ELISA stop solution (Invitrogen, Carlsbad, CA, catalog number SS04) was added. The absorbance at 450 nm was measured. The data points were normalized to the values ​​of the isotype control Ab at each concentration and reported as a percentage of the absorbance at 450 nm normalized to the control Ab. Nonlinear regression analysis was used to fit a curve (4-parameter) to the data using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA). Figure 1 The results are shown in Table 11.

[0333] Table 11

[0334] Antibody IC50 Maximum inhibition% A-10F05 N / A N / A A-15B08 0.0033 96 A2-3B06 0.0130 93 A2-5D10 0.1475 93 A2-7A05 0.0271 41 A2-7F01 0.0215 60 C-4A04 N / A N / A C-10F09 N / A N / A C-14D12 0.0028 96 Control Ab N / A 0

[0335] Three classes of antibodies were identified as defined by their ability to strongly inhibit α5β1 binding to FN (≥93% maximal inhibition), partially inhibit binding to FN (≤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.

[0336] Example 6: Competition binding studies for identifying epitope binding groups

[0337] Epitope analysis was performed on the six antibodies selected as described in Example 5 using biofilm interferometry (BLI) via competitive binding studies. 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 that had been pre-incubated with rh-α5β1 to determine whether the antibody-antigen binding prevented or permitted binding to the antibody on the sensor. The observed binding (response) amplitude was compared to that of the antigen alone under the same conditions. If the total response was greater than 120% of the antigen alone, the antibodies were compatible with each other. If the response was less than 80% of the antigen alone, the antibodies blocked each other. The protocol was as follows: The antibody panel was pre-incubated with the antigen (1 hour). Equilibrate sensor (30 seconds (s)) -> Load lead Ab onto sensor (700s) -> Quench (480s) -> Read baseline (480s) -> Measure binding of pre-incubated Ab+Ag to loaded sensor (600s). The results are shown in Tables 12A and 12B.

[0338] 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. When complexed with α5β1, A2-3B06 and C-14D12 were unable to bind to A-15B08 on the sensor, 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 two epitope groups represented by these six antibodies. A2-7A05 and A2-7F01 represent one group, while A-15B08, A2-3B06, A2-5D10, and C-14D12 represent the second group.

[0339] Table 12A

[0340] Immobilized mAb Second mAb* Response (nm) response% compete A-15B08 C-14D12 -0.15 -72.85％ + A-15B08 A-15B08 -0.15 -73.81％ + A-15B08 A2-3B06 0.10 50.05％ + A-15B08 No mAb 0.21 100.00％ comparison A-15B08 A2-5D10 0.22 106.82％ + A-15B08 A2-7F01 0.29 142.59％ - A-15B08 A2-7A05 0.31 150.63％ - A2-7A05 A2-7A05 -0.17 -451.35％ + A2-7A05 A2-7F01 -0.17 -472.70％ + A2-7A05 No mAb 0.04 100.00％ comparison A2-7A05 A2-3B06 0.07 198.65％ - A2-7A05 C-14D12 0.11 309.19％ - A2-7A05 A2-5D10 0.17 448.38％ - A2-7A05 A-15B08 0.25 666.22％ - C-14D12 C-14D12 -0.11 -50.49％ + C-14D12 A-15B08 -0.09 -38.40％ + C-14D12 A2-3B06 0.12 53.28％ + C-14D12 No mAb 0.22 100.00％ comparison C-14D12 A2-5D10 0.24 106.12％ + C-14D12 A2-7F01 0.30 133.09％ - C-14D12 A2-7A05 0.31 139.39％ -

[0341] *Premixed with 20nM rh-α5β1

[0342] Table 12B

[0343]

[0344] Example 7: Antibody Binding Disrupts the α5β1-FN Integrin-Ligand Complex

[0345] Surface plasmon resonance (SPR) and cell-based assays were used to test the effects of antibodies on the dissociation of rh-α5β1 protein bound to human FN protein. The antibodies tested represent two groups of antibodies identified in Example 6, which define two distinct epitope binding groups and have different ligand blocking properties. They are A-15B08, a strong 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 No. CP22175) was tested as a comparator for its ability to inhibit α5β1 integrin binding to FN by competing at the ligand binding pocket.

[0346] Description of the SPR method used: FN protein dissolved in water was manually printed onto bare gold-coated (47 nm thickness) PlexArray Nanocapture sensor chips (Plexera Bioscience, Seattle, WA) at 40% humidity. The chips were incubated overnight at 4°C and 80% humidity and rinsed with 10× PBST (0.1 M phosphate buffer, 1.54 M NaCl, pH 7.4, 0.5% Tween 20) for 10 minutes (min), 1× PBST (0.01 M phosphate buffer, pH 7.4, 0.154 M NaCl, 0.05% Tween 20) for 10 minutes, and two 10-minute rinses with deionized water. The chips were then blocked overnight with a solution of 5% (w / v) skim milk in water and washed with 10× PBST for 10 minutes, 1× PBST for 10 minutes, and two 10-minute rinses with deionized water, 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) was passed through a coupling prism, reflected from the SPR-active gold surface and received by a CCD camera. Buffer and sample were injected into a 30 μL flow cell mounted on a coupling prism by a non-pulsating piston pump. Each SPR measurement cycle comprises four steps: at a constant speed of 2 μL / second (s), washed with 1 × PBS running buffer to obtain a stable baseline, injected with 5 uL / s for 400 nM rh-α5β1300 s (to reach equilibrium) for binding to FN, then injected with a separate running buffer for 50 s to dissociate rh-α5β1 at 2 μL / s, and finally injected with 1 μM antibody for 250 s at 2 μL / s. All measurements were performed at 25 ° C. The SPR binding response (au) was recorded and plotted over time.

[0347] Both antibodies had a positive effect on the dissociation of rh-α5β1 protein bound to FN, but to different extents, whereas cRGD had no effect. Figure 4AThe superposition of the SPR responses with and without the addition of antibody or cRGD is shown. Injection of either antibody resulted in a transient increase in the SPR response, indicating that FN, rh-α5β1 protein, and the antibody form a ternary complex. Significantly, the A-15B08 antibody, previously shown by ELISA to be a strong blocker of FN binding, caused the FN-α5β1 complex to dissociate rapidly, as detected by a decrease in the SPR response close to baseline during the 250 s between the injection of the antibody and the measurement of the SPR response. This property of A-15B08 rapidly dissociating the complex contrasts with published results showing much slower dissociation rates by other anti-α5β1 antibodies under similar conditions (Mould et al., J Bio Chem., 30; 291(40): 20993-21007 (2016)). The A2-7A05 antibody, which was shown by ELISA to only partially block the binding of α5β1 to FN, did not cause such a rapid decrease in the SPR response, although it did cause partial dissociation at a slower rate, which is comparable to results previously published by Mould et al. (2016) for some other antibodies. In contrast, injection of the small molecule antagonist cRGD did not have any discernible effect on the dissociation rate of the complex. The two antibodies had similar binding affinities for the rh-α5β1 protein (K for A-15B08 and A2-7A05, 0.001, 0.002, and 0.006, respectively). D 9 and 1.31E-09, respectively, see also Table 9), indicating that the differences in off-rates elicited by these antibodies are 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 less effective than allosteric antagonist antibodies at dissociating ligand-integrin complexes, suggesting that the dissociative activity observed with the antibodies tested herein involves an allosteric mechanism.

[0348] The ability of the A-15B08 and A2-7A05 antibodies, as well as cRGD, to induce cellular α5β1 integrin dissociation from FN was also tested. U87MG cells (HTB-14) are known to be originally derived from glioblastoma tumors. TM , ATCC, Manassas, VA) express α5β1 integrin and adhere to FN-coated plates. After overnight incubation on FN-coated plates, U87MG cells form a loosely packed monolayer with an extended spindle shape. If cells are induced to detach from the plate, for example, by trypsin, the attachment points loosen and the cells become rounded. These morphological changes were used to evaluate whether antibodies and the small molecule inhibitor cRGD can induce U87MG cells to detach from FN-coated plates.

[0349] The following method was used to evaluate cell detachment activity: 96-well cell culture plates (Thermo Fisher Scientific, Waltham, MA, catalog No. 165306) were coated with FN (human fibronectin, R&D Systems, Minneapolis, MN 55413, catalog No. 1918-FN) by incubating 50 uL per well of 32 μg / mL FN in 1× PBS for 1 hour at 37° C., followed by 2× washing with EMEM medium (ATCC, Manassas, VA, catalog No. 30-2003) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, VA, catalog No. 30-2021). U87MG cells were plated at 20,000 cells per well in EMEM medium + 10% FBS overnight to allow for maximum attachment. The next day, the culture medium was replaced with fresh culture 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 concentrations of 20, 4, 0.8, and 0.16 μM. All tests were repeated twice. Cell morphology was evaluated and images were captured using an ECHO Rebel light microscope (ECHO, San Diego, CA) with a 10× objective.

[0350] The results showed that at an antibody concentration as low as 0.4 μg / mL, antibody A-15B08, a representative of strong FN-blocking antibodies, caused significant cell rounding due to cell contact detachment, which was observed at 1 hour and 3 hours, the latter being the time when images were acquired ( Figure 4B The partial antagonist A2-7A05 also showed cell detachment at 0.4 μg / mL antibody concentration, but at a much lower level and only evident at 3 hours. The weaker ability of this 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 concentration or time point tested ( Figure 4B The extent to which both antibodies detached cells from FN-coated wells was consistent with the SPR dissociation data generated by the purified proteins ( Figure 4A ), where strong antagonist antibodies cause rapid dissociation of integrin-FN complexes while partial antagonists only weakly cause dissociation.

[0351] Example 8: Generation of human IgG4 chimeras and removal of N-glycosylation sites in CDRH2 does not significantly affect the blocking activity of antibody FN

[0352] The antibody expression plasmid was constructed as a human IgG4 chimera with variable domains from antibodies A-15B08, C-14D12, and A2-7A05. In addition, the threonine residue at position 62 in CDRH2 of antibody A-15B08 was changed to alanine to remove the putative N-glycosylation site and designated as IgG4 clone A-15B08-T62A. Figure 2C and 2D The VH and VL sequences of A-15B08-T62A and 6 CDR sequences (according to various numbering schemes) are shown in Figure 2. The correctness of the sequence was verified using Sanger sequencing, and the plasmid concentration was determined by measuring the absorbance value at a wavelength of 260 nm. The expression clone was transfected into suspension-adapted CHO K1 cells and grown in animal component-free, serum-free medium. The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter). The supernatant was purified using MabSelect TM SuRe TM Antibodies were purified using Cytiva (Marlborough, MA). Purity was determined by analytical size exclusion chromatography with an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8×300 mm; Agilent Technologies, Inc., Santa Clara, CA) at 0.8 mL / min using PBS as the running buffer. Yield was 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 a 250 mL culture.

[0353] A small aliquot of each chimeric antibody was tested for freeze-thaw stability by storing overnight at -80°C and then thawing on ice three times. All four chimeric antibodies were stable to 3× freeze-thaw cycles compared to antibodies stored at 4°C alone, as judged by their ability to inhibit the binding of α5β1 integrin to fibronectin (FN). The ELISA method used was as described in Example 2. GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA) was used to analyze the ELISA data by nonlinear regression curve fitting (4-parameter) ( Figure 5 ) IC50 was calculated. The obtained IC50 indicated that 3× freeze-thaw (F / T) only minimally affected the potency of the antibody in the FN-blocking assay (Table 13). In addition, the potency of the IgG4 chimera was compared with that of a mouse hybridoma derived using the same FN-blocking ELISA and IC50 determination method. The results showed that in all cases, the potency of the chimera was slightly higher than that of the hybridoma by about 2-fold (see Figure 6 & Table 14), except for the case of A2-7A05 where the difference was smaller (IC50 0.115 vs. 0.130).

[0354] Table 13

[0355]

[0356] Table 14

[0357]

[0358] The glycosylation status of A-15B08 was determined by comparing the mobility of its heavy chain in 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. The glycosylation status of A-15B08 was determined using MOPS buffer (Invitrogen, Carlsbad, CA, Catalog No. B0001) with a Bolt TM 2 μg of each antibody was separated by SDS-PAGE using 4-12.5% ​​Tris-Bis Plus (Invitrogen, Carlsbad, CA, Catalog No. NW04120BOX) miniature protein gels. The mobility of the antibody heavy chain A-15B08-T62A with the mutated N-glycosylation site was similar to that of the other two antibody heavy chains from C-14D12 and A2-7A05 that do not contain the putative N-glycosylation site. Notably, the migration of the heavy chain of A-15B08 was slower than that of the other three antibodies, providing evidence that it is indeed glycosylated at this site ( Figure 7 Based on this data, it is expected that antibody A2-5D10 (not mutated and not tested by SDS-PAGE) will also be glycosylated at a similar NST sequence in its CDRH2.

[0359] Example 9: Binding of the antibody to α5β1 integrin changes its conformation to an inactive form

[0360] The ability of anti-5 antibodies to modulate integrin conformation from active to inactive was determined using 12G10 (mouse anti-human integrin β1 / CD29 antibody, Novus Biologicals, Littleton, CO, catalog No. NB100-63255), an antibody that preferentially binds to the 1-chain when integrin is in the active or open conformation. Nunc MaxiSorp flat-bottom 96-well plates (Invitrogen, Waltham, MA, catalog No. 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 No. 28382) by incubation overnight at 4°C. The plates were then washed three times with wash buffer (1× Tris-buffered saline containing 0.05% Tween 20) and then blocked with 2% BSA in 1× TBS for 2 hours at room temperature (RT). The human IgG4 chimeric form of the antibody was diluted in standard diluent (2% BSA, 1×TBS 0.05% Tween 20) containing 0.05 μg / mL rh-α5β1-plus 6×His-tagged protein (Acro Biosystems, Newark, DE, Catalog No. IT1-H52W5) and 0.5 mM MnC12 (TEKnova, Hollister, CA, Catalog No. M0350) to generate a 7-point 1:5 antibody dilution series ranging from 10,000 ng / mL to 0.64 ng / mL. In addition to human IgG4 chimeric antibodies A-15B08, C-14D12, and A2-7A05, three other antibodies tested included an IgG4 isotype control (human IgG4, K, anti-fluorescein Ab00102-13.0, AbsoluteAntibody, Wilton, UK), an anti-integrin α-5 clone SNAKA51 (MilliporeSigma, St. Louis, MO, catalog No. MABT201), known to induce integrin α-5 into an active conformation, and finally the 12G10 antibody itself, which can directly compete with 12G10 for binding to the MaxiSorp plate. For the assay, after removing the blocking solution and washing the wells three times, 100 μL of the antibody dilution series, His-tagged-α5β1 mixture was added to the wells.After 1 hour at RT, the wells were washed 3 times, incubated with biotinylated anti-6×His-tag Ab (Invitrogen, Carlsbad, CA, catalog number 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 number No. N200) for 30 minutes, washed 3 times, incubated with TMB substrate (Thermo Fisher Scientific, Waltham, MA, catalog number No. N301) for 2-5 minutes, and then ELISA stop solution (Invitrogen, Carlsbad, CA, catalog number No. SS04) was added. The absorbance at 450 nm was measured. For each dilution series, the data points were normalized to the absorbance of the wells without antibody and reported as percent binding. Nonlinear regression analysis was used to fit a curve (3-parameter) to the data using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA). Figure 8 The results are shown in .

[0361] The results showed 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 FN binding, did not reduce α5β1 integrin binding to 12G10. An IgG4 isotype control similarly did not reduce α5β1 integrin binding to 12G10. The SNAKA51 antibody, known to shift α5β1 integrin to an active conformation, also increased α5β1 integrin binding to 12G10. Without being bound by theory, antibodies that strongly inhibit α5β1 integrin binding to its primary ligand, FN, may achieve this in part by shifting the α5β1 integrin to an inactive conformation.

[0362] Example 10: Antibody Inhibition of Cell Adhesion to Fibronectin

[0363] The cell surface α5β1 integrin receptor interacts with FN to promote cell adhesion. To test whether human IgG4 chimeric anti-α5β1 antibodies inhibit adhesion, a plate-based cell adhesion assay was developed for the U87MG cell line (HTB-14) derived from a glioblastoma tumor and known to express α5β1 integrin. TMU87MG 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 the flask using 0.05% trypsin, 0.02% EDTA (Lifeline Cell Technology, Frederick, MD, catalog number CM0017), washed once with modified Dulbecco's 1× PBS (DPBS) containing calcium and magnesium (HyClone, Logan, UT, catalog number SH30028-02), and resuspended in unsupplemented EMEM medium at a cell concentration of 2,000,000 cells per mL. Cells were rested in an incubator for 30 minutes before being mixed with a 6-point 1:3 dilution series of antibodies ranging from 3 to 0.01 μg / mL in a 96-well polypropylene round-bottom plate. 100,000 resting cells were premixed with each antibody dilution to a final volume of 100 μL and then transferred to a human FN-coated 96-well plate (R&D Systems, Minneapolis, MN, Catalog No. CWP001). The cell-antibody mixture was incubated at 37°C, 5% CO₂ for 1 hour. Non-adherent cells were removed from the wells by inverting the plate on an absorbent pad and then washed 2× with 200 μL per well of 1× DPBS (EMD Millipore Corp, Billerica, MA, Catalog No. TMS-012-A) without calcium and magnesium. With the suggestion dilution (1: 2000), 100 μ L of 1 × DPBS containing Hoechst 33342 dye (Thermo Fisher Scientific, Waltham, MA, catalog number No. 62249) is added to each well to help cell counting to nuclear fluorescence staining. Image analysis software provided using ImageXpress Pico automatic cell imaging system (Molecular Devices, San Jose, CA) is used to count cells remaining adhered to the wells, based on fluorescence counting of stained nuclei, in this case using a DAPI filter to detect Hoechst nuclear staining. Using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA), nonlinear regression analysis is used for curve (4-parameter) fitting to data.

[0364] Figure 9Results are shown. The average number of U87MG cells adhered to FN-coated wells in the absence of antibody was 37,022, with a standard deviation (stdev) of 2,364 (n=16). Cell counts from wells without FN coating averaged 628 cells, with an stdev of 143 (n=12). For antibodies A-15B08, A-15B08-T62A, and C-14D12, which are strong blockers of FN binding, the highest concentration of antibody tested at 3 μg / mL inhibited adhesion in the range of 78% to 86%, while antibody A2-7A05, which was shown to be a partial blocker of FN binding, inhibited 25% (Table 15). Data from this cell adhesion blocking assay confirm the inhibitory effect of the antibodies of the present invention on the binding of α5β1 to Fn and on cell adhesion.

[0365] Table 15

[0366] Implementation Method

[0367] 1. An antibody or a fragment thereof that competitively binds to α5β1 integrin with an antibody, the antibody or the fragment thereof comprising:

[0368] (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: 26; (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 NO: 138, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 139; (viii) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 144, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 145; (ix) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 146, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 147; or

[0369] (B) (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 77, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 78; (ii) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 91, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 92; (iii) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 140, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 141; and / or (iv) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 142, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 143.

[0370] 2. An antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises:

[0371] (a) Heavy chain variable (V H ) area, which includes:

[0372] (1)V H CDR1 having an amino acid sequence selected from the group consisting of:

[0373] (i) SEQ ID NO: 1, 27, 53 or 93,

[0374] (ii) SEQ ID NO: 7, 31, 59 or 97,

[0375] (iii) SEQ ID NO: 12, 34, 64 or 100,

[0376] (iv) SEQ ID NO: 13, 35, 65 or 101, and

[0377] (v) SEQ ID NO: 18, 38, 70 or 105;

[0378] (2)V H CDR2 having an amino acid sequence selected from the group consisting of:

[0379] (i) SEQ ID NO: 2, 28, 54 or 79,

[0380] (ii) SEQ ID NO: 8, 60 or 82,

[0381] (iii) SEQ ID NO: 14, 66 or 84,

[0382] (iv) SEQ ID NO: 19, 71 or 87, and

[0383] (v) SEQ ID NO: 24, 76 or 90; and

[0384] (3)V H CDR3 having an amino acid sequence selected from the group consisting of:

[0385] (i) SEQ ID NO: 3, 29, 55, 80 or 94,

[0386] (ii) SEQ ID NO: 9, 32, 61, 83 or 98,

[0387] (iii) SEQ ID NO: 15, 36, 67, 85 or 102, and

[0388] (iv) SEQ ID NO: 20, 39, 72, 88 or 106;

[0389] and

[0390] (b) Light chain variable (V L ) area, which includes:

[0391] (1)V L CDR1 having an amino acid sequence selected from the group consisting of:

[0392] (i) SEQ ID NO: 4, 30, 44, 56 or 95,

[0393] (ii) SEQ ID NO: 10, 33, 46, 62 or 99,

[0394] (iii) SEQ ID NO: 16, 37, 47, 68 or 103, and

[0395] (iv) SEQ ID NO: 21, 40, 49, 73 or 107;

[0396] (2)V L CDR2 having an amino acid sequence selected from the group consisting of:

[0397] (i) SEQ ID NO: 5 or 57,

[0398] (ii) SEQ ID NO: 11 or 63, and

[0399] (iii) SEQ ID NO: 22, 41 or 74; and

[0400] (3)V L CDR3 having an amino acid sequence selected from the group consisting of:

[0401] (i) SEQ ID NO: 6, 45, 58, 81 or 96

[0402] (ii) SEQ ID NO: 17, 48, 69, 86 or 104, and

[0403] (iii) SEQ ID NO: 23, 50, 75, 89 or 108.

[0404] 3. An antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises a heavy chain variable (V H ) area, which includes:

[0405] (1)V H CDR1 having an amino acid sequence selected from the group consisting of:

[0406] (i) SEQ ID NO: 1, 27, 53 or 93,

[0407] (ii) SEQ ID NO: 7, 31, 59 or 97,

[0408] (iii) SEQ ID NO: 12, 34, 64 or 100,

[0409] (iv) SEQ ID NO: 13, 35, 65 or 101, and

[0410] (v) SEQ ID NO: 18, 38, 70 or 105;

[0411] (2)V H CDR2 having an amino acid sequence selected from the group consisting of:

[0412] (i) SEQ ID NO: 2, 28, 54 or 79,

[0413] (ii) SEQ ID NO: 8, 60 or 82,

[0414] (iii) SEQ ID NO: 14, 66 or 84,

[0415] (iv) SEQ ID NO: 19, 71 or 87, and

[0416] (v) SEQ ID NO: 24, 76 or 90; and

[0417] (3)V H CDR3 having an amino acid sequence selected from the group consisting of:

[0418] (i) SEQ ID NO: 3, 29, 55, 80 or 94,

[0419] (ii) SEQ ID NO: 9, 32, 61, 83 or 98,

[0420] (iii) SEQ ID NO: 15, 36, 67, 85 or 102, and

[0421] (iv) SEQ ID NO: 20, 39, 72, 88 or 106.

[0422] 4. An antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody or fragment thereof comprises a light chain variable (V L ) area, which includes:

[0423] (1)V L CDR1 having an amino acid sequence selected from the group consisting of:

[0424] (i) SEQ ID NO: 4, 30, 44, 56 or 95,

[0425] (ii) SEQ ID NO: 10, 33, 46, 62 or 99,

[0426] (iii) SEQ ID NO: 16, 37, 47, 68 or 103, and

[0427] (iv) SEQ ID NO: 21, 40, 49, 73 or 107;

[0428] (2)V L CDR2 having an amino acid sequence selected from the group consisting of:

[0429] (i) SEQ ID NO: 5 or 57,

[0430] (ii) SEQ ID NO: 11 or 63, and

[0431] (iii) SEQ ID NO: 22, 41 or 74; and

[0432] (3)V L CDR3 having an amino acid sequence selected from the group consisting of:

[0433] (i) SEQ ID NO: 6, 45, 58, 81 or 96,

[0434] (ii) SEQ ID NO: 17, 48, 69, 86 or 104, and

[0435] (iii) SEQ ID NO: 23, 50, 75, 89 or 108.

[0436] 5. 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 from:

[0437] an antibody denoted A-15B08 comprising the VH sequence as SEQ ID NO: 25 and the VL sequence as SEQ ID NO: 26;

[0438] the antibody denoted A-15B08-T62A, comprising the VH sequence as SEQ ID NO: 135 and the VL sequence as SEQ ID NO: 26;

[0439] the antibody denoted A-15B08_Low, comprising the VH sequence as SEQ ID NO: 136 and the VL sequence as SEQ ID NO: 137;

[0440] the antibody denoted A-15B08_LoW+Mod, comprising the VH sequence as SEQ ID NO: 138, and the VL sequence as SEQ ID NO: 139;

[0441] an antibody denoted A2-3B06 comprising the VH sequence as SEQ ID NO: 42, and the VL sequence as SEQ ID NO: 43;

[0442] an antibody denoted A2-5D10, comprising the VH sequence as SEQ ID NO: 51, and the VL sequence as SEQ ID NO: 52;

[0443] an antibody denoted A2-7A05, comprising the VH sequence as SEQ ID NO: 77, and the VL sequence as SEQ ID NO: 78;

[0444] the antibody denoted A2-7A05_Low, comprising the VH sequence as SEQ ID NO: 140, and the VL sequence as SEQ ID NO: 141;

[0445] the antibody denoted A2-7A05_Low+Mod, comprising the VH sequence as SEQ ID NO: 142, and the VL sequence as SEQ ID NO: 143;

[0446] an antibody denoted A2-7F01 comprising the VH sequence as SEQ ID NO: 91, and the VL sequence as SEQ ID NO: 92;

[0447] an antibody denoted C-14D12, comprising the VH sequence as SEQ ID NO: 109, and the VL sequence as SEQ ID NO: 110;

[0448] the antibody denoted C-14D12_Low, comprising the VH sequence of SEQ ID NO: 144, and the VL sequence of SEQ ID NO: 145; or

[0449] The antibody denoted C-14D12_Low+Mod comprises the VH sequence as SEQ ID NO: 146, and the VL sequence as SEQ ID NO: 147.

[0450] 6. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody designated A-15B08.

[0451] 7. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody denoted as A2-3B06.

[0452] 8. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody denoted as A2-5D10.

[0453] 9. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody denoted as A2-7A05.

[0454] 10. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody denoted as A2-7F01.

[0455] 11. The antibody or fragment thereof of embodiment 5, wherein the antibody or fragment thereof comprises all three heavy chain CDRs and all three light chain CDRs from the antibody denoted as C-14D12.

[0456] 12. An antibody or fragment thereof that binds to α5β1 integrin, wherein the antibody comprises:

[0457] (a) Heavy chain variable (V H ) region, which includes V as shown in Table 1-6 H CDR1, V H CDR2 and V H CDR3 amino acid sequence;

[0458] or

[0459] (b) Light chain variable (V L ) region, which includes V as shown in Table 1-6 L CDR1, V L CDR2 and V L CDR3 amino acid sequence.

[0460] 13. The antibody or fragment thereof according to embodiment 12, wherein the antibody comprises:

[0461] (a) Heavy chain variable (V H ) region, which includes V as shown in Table 1-6 H CDR1, V H CDR2 and V H CDR3 amino acid sequence;

[0462] and

[0463] (b) Light chain variable (V L ) region, which inc...

Claims

1. An antibody or antigen-binding fragment thereof that binds to integrin α5, wherein the antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 136 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 139; b) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 136 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 137; c) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 138 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 139; d) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 138 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 137; e) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 135 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 26; f) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 25 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO: 26; g) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 42 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO: 43; h) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 51 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO: 52; i) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 77 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 78; j) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO:91 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO:92; k) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 109 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 110; 1) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 140 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO: 141; m) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence set forth in SEQ ID NO: 142 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence set forth in SEQ ID NO: 143; n) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 144 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO: 145; or o) a heavy chain variable region (VH) comprising VHCDR1, VH CDR2 and VH CDR3 as shown in VH comprising the amino acid sequence shown in SEQ ID NO: 146 and / or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 as shown in VL comprising the amino acid sequence shown in SEQ ID NO:

147.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein a) the VH comprising i) (1) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 18; (2) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24 or SEQ ID NO: 28; and (3) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; or ii) (1) a VH CDR1 having 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; (2) a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24 or VIWSDGSTTYNSSLKS; and (3) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and b) the VL comprising (i) (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH; (2) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 11, STSNRAT or LWIYSTSNRA; and (3) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO: 23; or (ii) (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH; (2) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 10, STSNRAS or LWIYSTSNRA; and (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO:

23.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein a) the VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and b) the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, STSNRAT or LWIYSTSNRA and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO: 23; optionally, wherein i) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 12, a VH CDR2 having the amino acid sequence of SEQ ID NO: 28, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 3; and a VL CDR1 having the amino acid sequence of RASSRVSSNSLH, a VL CDR2 having the amino acid sequence of STSNRAT, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 6; ii) the VH comprising a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 24, and a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a VL CDR1 having the amino acid sequence shown in RASSRVSSNSLH, a VL CDR2 having the amino acid sequence shown in STSNRAT, and a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6; iii) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VLCDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 17; iv) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 18, a VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 20; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, a VL CDR2 having the amino acid sequence of LWIYSTSNRA, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 23; or v) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 9; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO:

6.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein a) the VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and b) the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, STSNRAS or LWIYSTSNRA, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO: 23; optionally, wherein i) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and a VL CDR1 having an amino acid sequence as shown in RASSRVSSNSLH, a VL CDR2 having an amino acid sequence as shown in STSNRAS, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; ii) the VH comprising a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 24, and a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a VL CDR1 having the amino acid sequence shown in RASSRVSSNSLH, a VL CDR2 having the amino acid sequence shown in STSNRAS, and a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6; iii) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 13, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 15; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 16, a VLCDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 17; iv) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 18, a VH CDR2 having the amino acid sequence of SEQ ID NO: 19, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 20; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 21, a VL CDR2 having the amino acid sequence of LWIYSTSNRA, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 23; or v) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 9; and a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO:

6.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein a) the VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24 or VIWSDGSTTYNSSLKS, and (iii) a VHCDR3 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, STSNRAT or LWIYSTSNRA, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: NO:6, the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:23; or b) the VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 24 or VIWSDGSTTYNSSLKS, and (iii) a VHCDR3 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 21 or RASSRVSSNSLH, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, STSNRAS or LWIYSTSNRA, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: NO:6, the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:23; optionally, wherein i) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in VIWSDGSTTYNSSLKS, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in RASSRVSSNSLH, a VL CDR2 having an amino acid sequence as shown in STSNRAT, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; ii) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in RASSRVSSNSLH, a VL CDR2 having an amino acid sequence as shown in STSNRAT, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; iii) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 16, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 17; iv) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 18, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 20; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a VL CDR2 having an amino acid sequence as shown in LWIYSTSNRA, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; v) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 7, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; vi) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in VIWSDGSTTYNSSLKS, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in RASSRVSSNSLH, a VL CDR2 having an amino acid sequence as shown in STSNRAS, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; vii) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in RASSRVSSNSLH, a VL CDR2 having an amino acid sequence as shown in STSNRAS, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; viii) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 16, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 17; ix) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 18, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 20; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a VL CDR2 having an amino acid sequence as shown in LWIYSTSNRA, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; or x) the VH comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, a VH CDR2 having the amino acid sequence of SEQ ID NO: 8, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 9; and the VL comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 10, a VL CDR2 having the amino acid sequence of SEQ ID NO: 11, and a VL CDR3 having the amino acid sequence of SEQ ID NO:

6.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein (A) the VH comprising (a)(i) VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; (b) (i) a VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; (c)(i) a VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 36 or SEQ ID NO: 39; (d)(i) VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; (e)(i) a VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67 or SEQ ID NO: 72; (f)(i) a VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 85 or SEQ ID NO: 88; or (g)(i) a VH CDR1 having the amino acid sequence shown 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 having the amino acid sequence shown 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 having the amino acid sequence shown in SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:102 or SEQ ID NO:106; and (B) the VL comprising (a)(i) VL CDR1 having the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16 or SEQ ID NO:21; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22; and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO: 23; (b)(i) VL CDR1 having the amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:37 or SEQ ID NO:40; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 41; and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID NO: 23; (c)(i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:49; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22; and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 48 or SEQ ID NO: 50; (d)(i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68 or SEQ ID NO:73; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 57, SEQ ID NO: 63 or SEQ ID NO: 74; and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 58, SEQ ID NO: 69 or SEQ ID NO: 75; (e)(i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:68 or SEQ ID NO:73; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 57, SEQ ID NO: 63 or SEQ ID NO: 74; and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 81, SEQ ID NO: 86 or SEQ ID NO: 89; (f)(i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:103 or SEQ ID NO:107; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22; and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 96, SEQ ID NO: 104 or SEQ ID NO: 108; optionally, wherein (1) the VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16 or SEQ ID NO: 21, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7 The amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 23; (2) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 6; (3) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 7, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 9; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 10, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 6; (4) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (5) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 15; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 17; (6) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 18, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 20; and the VL comprising a VLCDR1 having an amino acid sequence as set forth in SEQ ID NO: 21, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 22, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 23; (7) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (8) The VH, which comprises (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and the VL, which comprises (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16 or SEQ ID NO: 21, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7 The amino acid sequence shown in NO:17 or SEQ ID NO:23; (9) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (10) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 7, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 9; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 10, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 6; (11) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (12) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 16, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 17; (13) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 18, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 20; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 21, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; (14) The VH comprises a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprises a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (15) The VH, which comprises (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 32, SEQ ID NO: 36 or SEQ ID NO: 39; and the VL, which comprises (i) a VLCDR1 having an amino acid sequence as shown in SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 37 or SEQ ID NO: 40, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 41, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7 The amino acid sequence shown in NO:17 or SEQ ID NO:23; (16) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 27, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 29; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 30, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (17) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 31, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 32; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 33, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (18) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 34, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 29; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 30, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 6; (19) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 35, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 36; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 37, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 17; (20) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 38, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 39; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 40, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 41, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 23; (21) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 27, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 29; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 30, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 6; (22) The VH, which comprises (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO: 20; and the VL, which comprises (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 49, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22, and (iii) a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15 or SEQ ID NO:

20. The amino acid sequence shown in SEQ ID NO:45, SEQ ID NO:48 or SEQ ID NO:50; (23) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 44, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 45; (24) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 7, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 46, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 45; (25) the VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 12, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 44, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 45; (26) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 13, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 15; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 47, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 48; (27) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 18, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 20; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 49, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 22, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 50; (28) The VH comprising a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; and the VL comprising a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 44, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 45; (29) The VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67 or SEQ ID NO: 72; and the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 56, SEQ ID NO: 62, SEQ ID NO: 68 or SEQ ID NO: 73, (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO:57, SEQ ID NO:63 or SEQ ID NO:74, and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO:58, SEQ ID NO:69 or SEQ ID NO:75; (30) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 53, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 54, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 55; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 58; (31) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 59, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 60, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 61; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 62, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 58; (32) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 64, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 54, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 55; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 58; (33) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 65, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 66, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 67; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 68, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 69; (34) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 70, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 71, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 72; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 75; (35) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 53, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 76, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 55; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 58; (36) The VH comprising (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 85 or SEQ ID NO: 88; and the VL comprising (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 56, SEQ ID NO: 62, SEQ ID NO: 68 or SEQ ID NO: 73, (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO:57, SEQ ID NO:63 or SEQ ID NO:74, and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO:81, SEQ ID NO:86 or SEQ ID NO:89; (37) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 53, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 79, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 80; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 81; (38) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 59, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 82, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 83; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 62, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 81; (39) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 64, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 79, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 80; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 81; (40) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 65, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 84, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 85; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 68, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 86; (41) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 70, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 87, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 88; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 89; (42) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 53, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 90, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 80; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 81; (43) The VH, which comprises (i) a VH CDR1 having an amino acid sequence as shown 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 having an amino acid sequence as shown 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 having an amino acid sequence as shown in SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102 or SEQ ID NO: 106; and the VL, which comprises (i) a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103 or SEQ ID NO: 107, (ii) a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 11 or SEQ ID NO: 22, and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO:96, SEQ ID NO:104 or SEQ ID NO:108; (44) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 93, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 94; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 95, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 96; (45) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 97, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 8, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 98; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 99, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 96; (46) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 100, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 28, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 94; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 95, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 96; (47) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 101, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 102; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 103, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 104; (48) the VH comprising a VH CDR1 having an amino acid sequence as set forth in SEQ ID NO: 105, a VH CDR2 having an amino acid sequence as set forth in SEQ ID NO: 19, and a VH CDR3 having an amino acid sequence as set forth in SEQ ID NO: 106; and the VL comprising a VL CDR1 having an amino acid sequence as set forth in SEQ ID NO: 107, a VL CDR2 having an amino acid sequence as set forth in SEQ ID NO: 22, and a VL CDR3 having an amino acid sequence as set forth in SEQ ID NO: 108; (49) The VH comprises a VH CDR1 having an amino acid sequence as shown in SEQ ID NO: 93, a VH CDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 94; and the VL comprises a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 95, a VL CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO:

96.

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

143.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein (i) the VH region or VL region further comprises (a) a human framework sequence; and / or (b) a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence; (ii) the antibody is a monoclonal antibody; (iii) the antibody is a humanized antibody, a human antibody or a chimeric antibody; (iv) the antibody is a single-chain antibody molecule, a dual variable region antibody, a single variable region antibody, a linear antibody, a V region, or a multispecific antibody formed by antibody fragments; (v) the antibody is Fab, Fab', F(ab')2, Fv, single chain variable fragment scFv or (scFv)2; and / or (vi) The integrin α5 is human integrin α5.

9. A conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is conjugated or recombinantly fused to a diagnostic agent, a detectable agent or a therapeutic agent, wherein the therapeutic agent is optionally a chemotherapeutic agent, a cytotoxin or a drug.

10. A polynucleotide comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

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