Anti-alpha5 integrin antibodies and uses thereof

Abstract

The present disclosure provides antibodies or antigen-binding fragments thereof that bind to alpha5 integrin (e.g., human alpha5 integrin), and uses thereof. In certain embodiments, the disclosed antibodies are humanized antibodies.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 424,820, filed November 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a computer-readable Sequence Listing in XML file format submitted herewith, the entire contents of which are incorporated herein by reference. The Sequence Listing XML file submitted herewith is named "14763-004-228_SEQ_LISTING.xml," was created on November 7, 2023, and is 58,582 bytes in size.

[0003] 1.Technical Field The present disclosure generally relates to antibodies or antigen-binding fragments thereof that bind to alpha5 integrin (e.g., human alpha5 integrin), and uses thereof. In certain embodiments, the disclosed antibodies are humanized antibodies. [Background technology]

[0004] 2.Background Integrins are transmembrane proteins that bind to components of the extracellular matrix (ECM) and regulate cell adhesion, migration, and activation. Each integrin is composed of α and β transmembrane integrin subunits. The human genome contains 18 α integrin subunits and 8 β integrin subunits, which combine to generate 24 unique heterodimeric integrins. These heterodimers regulate cell behavior through mechanisms known as "inside-out" and "outside-in" signaling. In the former, intracellular proteins bind to the cytoplasmic domain of the integrin, stabilizing a conformation that binds extracellular ligands with high affinity. In the latter, the ligand-bound integrin then stimulates intracellular signaling cascades that regulate cell behavior.

[0005] The α5β1 integrin is known as the FN receptor due to its high affinity for fibronectin (FN) in the extracellular matrix (ECM). This binding is mediated by a ligand-binding site at the interface between the α and β subunits within the α5β1 head and an arginine-glycine-aspartic acid (RGD) peptide motif in the type III repeats of FN. α5β1 integrin binds to additional 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 the ligand, α5β1 is expressed by various cell types, including endothelial cells, mast cells, and macrophage lineages in peripheral tissues and the central nervous system (CNS) (e.g., microglia and perivascular macrophages).

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

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

[0008] 3. Summary of the Invention The present disclosure provides an antibody or antigen-binding fragment thereof that binds to alpha5 integrin (e.g., human alpha5 integrin). In certain embodiments, the anti-alpha5 integrin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31.

[0009] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0010] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0011] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34.

[0012] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, and (b) a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 34. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 34.

[0013] In certain embodiments, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 31, and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0014] In certain embodiments, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 31, and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0015] In certain embodiments, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 32, and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0016] In certain embodiments, (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 32, and (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and (b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0017] In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antigen-binding fragment is a Fab, Fab', F(ab')2, variable fragment (Fv), or single-chain variable fragment (scFv).

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof binds to α5β1 integrin. In certain embodiments, the antibody or antigen-binding fragment thereof inhibits binding of α5β1 integrin to fibronectin.

[0019] The present disclosure further provides antibodies or antigen-binding fragments thereof that compete with any of the disclosed antibodies or antigen-binding fragments thereof for binding to alpha5 integrin.

[0020] The present disclosure further provides antibodies or antigen-binding fragments thereof that bind to substantially the same epitope region with respect to binding to alpha5 integrin as any of the disclosed antibodies or antigen-binding fragments thereof.

[0021] Also provided are compositions comprising the disclosed antibodies or antigen-binding fragments thereof. In certain embodiments, the compositions are pharmaceutical compositions that include a pharmaceutically acceptable carrier.

[0022] The present disclosure further provides a conjugate comprising the disclosed antibody or antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is linked to a therapeutic agent, a detectable agent, or a diagnostic agent. In certain embodiments, the conjugate is an immunoconjugate. In certain embodiments, the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug. Also provided is a composition comprising the disclosed conjugate. In certain embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0023] The present disclosure further provides multispecific molecules comprising the disclosed antibodies or antigen-binding fragments thereof linked to a second functional moiety. In certain embodiments, the second functional moiety has a different binding specificity than the antibody or antigen-binding fragment thereof. Also provided are compositions comprising the disclosed multispecific molecules. In certain embodiments, the composition is a pharmaceutical composition that includes a pharmaceutically acceptable carrier.

[0024] Also provided are nucleic acids encoding the disclosed antibodies or antigen-binding fragments thereof. In certain embodiments, the nucleic acids comprise a V sequence comprising the amino acid sequence set forth in SEQ ID NO:31 or SEQ ID NO:32. H and / or a V comprising the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 34. L In certain embodiments, the first polynucleotide and the second polynucleotide encode: (a) a V comprising the amino acid sequence set forth in SEQ ID NO: 31; H a first polynucleotide encoding a V comprising the amino acid sequence set forth in SEQ ID NO: 33; La second polynucleotide encoding (b) a V comprising the amino acid sequence set forth in SEQ ID NO: 31; H a first polynucleotide encoding a V comprising the amino acid sequence set forth in SEQ ID NO: 34; L a second polynucleotide encoding (c) V comprising the amino acid sequence set forth in SEQ ID NO: 32 H a first polynucleotide encoding a V comprising the amino acid sequence set forth in SEQ ID NO: 33; L a second polynucleotide encoding (d) V comprising the amino acid sequence set forth in SEQ ID NO: 32 H a first polynucleotide encoding a V comprising the amino acid sequence set forth in SEQ ID NO: 34; L a second polynucleotide encoding the

[0025] Also provided are vectors comprising the disclosed nucleic acids. In certain embodiments, the vectors are expression vectors. Also provided are host cells comprising the disclosed vectors.

[0026] The present disclosure also provides methods for producing the disclosed anti-α5 integrin antibodies or antigen-binding fragments thereof. In certain embodiments, the methods comprise culturing a host cell disclosed herein under conditions that induce expression of the antibody or antigen-binding fragment thereof from the host cell.

[0027] The present disclosure further provides a method for detecting alpha5 integrin in whole cells or tissues. In certain embodiments, the method includes contacting a cell or tissue with the disclosed antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a detectable label, and determining the amount of labeled antibody or antigen-binding fragment thereof bound to the cell or tissue by measuring the amount of detectable label associated with the cell or tissue, where the amount of bound antibody or antigen-binding fragment thereof indicates the amount of alpha5 integrin in the cell or tissue.

[0028] The present disclosure further provides a method for treating an alpha5 integrin-related disease, disorder, or condition in a subject. In certain embodiments, the method comprises administering to the subject an antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein. The present disclosure further provides an antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein for use in treating an alpha5 integrin-related disease, disorder, or condition in a subject.

[0029] In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is associated with alpha5beta1. In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is a tumor. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a cancer. In certain embodiments, the cancer is selected from the group consisting of breast cancer, bladder cancer, melanoma, prostate cancer, mesothelioma, lung cancer, brain tumor, ovarian cancer, colon cancer, testicular cancer, thyroid cancer, squamous cell carcinoma, glioblastoma, neuroblastoma, neurofibromatosis, uterine cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

[0030] In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is associated with abnormal angiogenesis. In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is an ocular disease. In certain embodiments, the ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and uveitis.

[0031] In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is an inflammatory disease, disorder, disease, or condition. In certain embodiments, the inflammatory disease, disorder, or condition is a neuroinflammatory disease, disorder, or condition. In certain embodiments, the inflammatory disease, disorder, or condition is selected from the group consisting of macrophage-mediated innate immune disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and atherosclerosis.

[0032] The present disclosure further provides a method of inhibiting abnormal angiogenesis in a subject. In certain embodiments, the method comprises administering to the subject an antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein. The present disclosure further provides an antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein for use in inhibiting abnormal angiogenesis in a subject. In certain embodiments, the subject is afflicted with a tumor.

[0033] For the various methods disclosed herein, in certain embodiments, the subject is a human. DETAILED DESCRIPTION OF THE INVENTION

[0034] 4. Detailed Description The present disclosure provides an antibody or antigen-binding fragment thereof that binds to α5 integrin (e.g., human α5 integrin). In certain embodiments, the disclosed antibody is a humanized antibody. In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein can inhibit the binding of α5β1 integrin to fibronectin.

[0035] The present disclosure further provides compositions comprising the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the compositions are pharmaceutical compositions comprising a pharmaceutically acceptable carrier. Also provided are multispecific molecules comprising the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein. In addition, the present disclosure provides conjugates comprising the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein.

[0036] The present disclosure also provides methods for treating α5 integrin-associated diseases, disorders, or conditions using the anti-α5 integrin antibodies or antigen-binding fragments thereof, multispecific molecules, compositions, or conjugates disclosed herein. In certain embodiments, the disease, disorder, or condition is associated with α5β1 integrin (referred to as "α5β1 integrin-associated diseases, disorders, and conditions"). Examples of α5β1 integrin-associated diseases, disorders, and conditions include, but are not limited to, cancer, angiogenesis-related diseases (e.g., diseases characterized by abnormal angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases).

[0037] Non-limiting embodiments of the present disclosure are described herein and in the examples.

[0038] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0039] 4.1.Definition

[0040] Integrin

[0041] 4.3. Anti-α5 integrin antibodies and antigen-binding fragments thereof

[0042] 4.4. Antibody-Encoding Nucleic Acids and Vectors

[0043] 4.5.Detection Methods

[0044] 4.6. Compositions and Formulations

[0045] 4.7. Methods of treatment, and

[0046] 4.8.Kit

[0047] 4.1.Definition The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense to specifically include, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies with full-length heavy and / or light chains. The present disclosure also includes antibody fragments (and / or polypeptides comprising antibody fragments) that retain α5 integrin-binding property. Non-limiting examples of antibody fragments include the antigen-binding region of the antibody and / or the effector region of the antibody, 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 from antibody fragments, F(ab)2, Fd, Fc, diabodies, di-diabodies, disulfide-linked Fvs (dsFv), single domain antibodies (e.g., VHHs, nanobodies), or other fragments (e.g., fragments consisting of non-covalently linked heavy and light chain variable regions). Generally, the variable region domain is a fragment of an immunoglobulin heavy chain (VH). H ) and / or light chain (V L ) may be in any suitable configuration of the variable regions. For example, in this disclosure, antibodies also include tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, for example, the variable region may be dimeric, and may include a V that binds to α5. H -VH , V H -V L , or V L -V L Dimers are included. If necessary, V H Chain and V LThe chains can be covalently linked directly or via a linker to form a single-chain Fv (scFv). For ease of understanding, scFv proteins are referred to herein as being included in the category of "antibody fragments." Another form of antibody fragment is a peptide comprising one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also known as "minimal recognition units" or "hypervariable regions") can be obtained by constructing polynucleotides encoding the desired CDRs. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from antibody-producing cells as a template (see, for example, 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.), page 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.), page 137, Wiley-Liss, Inc. (1995)). Antibody fragments can be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable domains of novel antigen receptors (v-NARs), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005).An antibody or antigen-binding fragment thereof, in certain embodiments, comprises one or more constant regions, such as a light chain and / or heavy chain constant region, e.g., one or more IgG1, IgG2, IgG3, and / or IgG4 constant region. In certain embodiments, an antibody can comprise an epitope-binding fragment of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibody can be an antagonist or agonist antibody.

[0048] As used herein, the term "monospecific antibody" refers to an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen, e.g., alpha5 integrin (e.g., human alpha5 integrin).

[0049] As used herein, the term "bispecific molecule" refers to a molecule that binds to at least two distinct antigenic determinants, e.g., different antigens or different epitopes on the same antigen, each of which binds to an antibody heavy chain variable domain (V H ) and antibody light chain variable domain (V L Bispecific bispecific molecules refer to molecules (e.g., antibodies) that can bind to two binding sites formed by a pair of antigens (e.g., an α5 integrin) or a non-α5 integrin. Such multispecific molecules can be in a 1+1 format. Other bispecific molecule formats can be in 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 bispecific molecule contains two antigen-binding sites, each can bind to a different antigenic determinant. Such multispecific molecules can bind to two different epitopes on the same antigen (e.g., an epitope on an α5 integrin) or different antigens (e.g., an epitope on an α5 integrin and an epitope on a non-α5 integrin).

[0050] The terms "identical" or "percent identity," in the context of two or more nucleic acids or polynucleotides, refer to two or more sequences or subsequences that are the same or that have a specified percentage of the same nucleotides or amino acid residues when compared and aligned (introducing gaps, if necessary) for maximum correspondence without considering 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. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In certain embodiments, two nucleic acids or polypeptides are substantially identical when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or visual inspection, meaning that they share at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and in certain embodiments, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity. In certain embodiments, identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length, or any integer value therebetween. In certain embodiments, identity exists over a region longer than 60-80 residues, e.g., at least about 80-100 residues, and in certain embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies. In certain embodiments, identity exists over a region of nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length, or any integer value therebetween.In certain embodiments, the identity exists over a region longer than 60-80 bases, e.g., at least about 80-1000 bases or more, and in certain embodiments, the sequences are substantially identical over the entire length of the sequence being compared, e.g., the nucleotide sequence encoding a protein of interest.

[0051] As used herein, the term "conservative modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antigen-binding fragments thereof disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0052] Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are generally defined in the art, and include 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), nonpolar 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). Amino acid substitutions are introduced into the antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. For example, substituting tyrosine for phenylalanine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the present disclosure do not inhibit binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence to a target binding site. Methods for identifying conservative amino acid substitutions that do not abolish binding are well known in the art. In certain embodiments, conservative substitutions in the amino acid sequences disclosed herein, e.g., CDR sequences, heavy chain variable region (V H ) sequence, or the light chain variable region (V L ) The sequence can have at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, or at most about 10 amino acid residues modified and / or substituted.

[0053] The term "polypeptide" refers to a polymer of amino acids of any length. Polymers can be linear or branched, can contain modified amino acids, and can include non-amino acids (e.g., can be interrupted by non-amino acids). These terms also include amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage or conjugation (directly or indirectly) to a moiety, such as a labeling component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (e.g., a non-native amino acid), as well as other modifications known in the art. Because the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, the polypeptide can exist as a single chain.

[0054] As used herein, the term "epitope" refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be a contiguous stretch of amino acids in a polypeptide (a "linear" epitope), or it can include amino acids in two or more non-contiguous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope), such as human α5β1 integrin. A linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in certain embodiments, an antibody binds to a group of amino acids, regardless of whether they are folded into a native three-dimensional protein structure. In certain embodiments, antibodies require that the amino acid residues that make up the epitope exhibit a particular conformation (e.g., a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0055] An antibody or antigen-binding fragment thereof binds to an "epitope region," "epitope," "essentially the same epitope," or "the same epitope" as a reference antibody or reference antigen-binding fragment thereof if both recognize the same, overlapping, or adjacent epitopes or epitope regions in three-dimensional space. The most commonly used rapid method for determining whether two antibodies or two antigen-binding fragments bind to the same, overlapping, or adjacent epitopes or epitope regions in three-dimensional space is a competition assay, which can be configured in a variety of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and the ability of an unlabeled antibody to block the binding of the labeled antibody is measured using a radioactive, fluorescent, or enzymatic label.

[0056] "Epitope binning" is the process of grouping antibodies based on the epitopes they recognize. More specifically, epitope binning includes methods and systems for identifying the epitope recognition characteristics of different antibodies, for example, using competitive assays. Such assays can be combined with computational processes to cluster antibodies based on their epitope recognition characteristics and identify antibodies with different binding specificities.

[0057] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," "selectively bind," "immunospecifically recognize," and "immunospecific" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope), and such binding is understood by those of skill in the art. In certain embodiments, "specifically bind" means, for example, that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or some combination thereof, than alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen may generally bind other peptides or polypeptides with lower affinity, as determined, for example, by immunoassay, Biacore™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, an antibody or antigen-binding fragment thereof binds, or specifically binds, an antigen if it binds with higher affinity than any cross-reactive antigens, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the background signal or noise, and can be more than 10 times the background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antibody or antigen-binding domain to a "non-target" protein is less than about 10% of the binding of the antibody or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In certain embodiments, molecules that specifically bind to an antigen bind to the antigen with a K that is at least about 2 logs, at least about 2.5 logs, at least about 3 logs, at least about 4 logs, or greater than the K that these molecules use to bind to other antigens.In certain embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins. In certain embodiments, a molecule that specifically binds to an antigen does not cross-react with other non-α5β1 integrin proteins. In certain embodiments, "specifically binds" refers to, for example, a polypeptide or molecule that binds to a protein or target with a K of about 0.1 mM or less. D In certain embodiments, "specifically binds" means that the polypeptide or molecule binds 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. D Specific binding means binding to a target at a specific site. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets in multiple species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize multiple proteins or targets. In certain embodiments, it is understood that a polypeptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require (but can include) exclusive binding, e.g., binding to a single target. Thus, in certain embodiments, a polypeptide or molecule can specifically bind to multiple targets. In certain embodiments, multiple targets can bind to the same antigen-binding site on the polypeptide or molecule. For example, in certain embodiments, an antibody can contain 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 is bispecific, containing at least two antigen-binding sites with different specificities. Generally, although not necessarily, reference to "binding" means "specific binding."

[0058] "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 specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally tend to bind antigens more quickly and for a longer period of time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. In certain embodiments, "K" is used to measure affinity. D " or "K D The "K value" may be measured, for example, by biolayer interferometry (BLI) using an OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, or in addition, the K D can also be measured, for example, by a radiolabeled antigen binding assay (RIA) performed on the Fab version of the antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881), or by using a surface plasmon resonance (SPR) assay by Biacore, for example, using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Also referred to as "on-rate" or "rate of binding" or "binding rate" or "k on " and "off-rate" or "rate of dissociation" or "dissociation rate" or "k off" can also be determined by the same SPR or BLI techniques described above, using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) or the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), respectively.

[0059] The term "constant region" or "constant domain" as used herein is a term well known in the art and refers to the portion of an antibody, e.g., the carboxy-terminal portions of the light and / or heavy chains, that is not directly involved in binding the antibody to an antigen, but that may exhibit various effector functions, such as interacting with Fc receptors. This term includes portions of immunoglobulin molecules that generally have more conserved amino acid sequences compared to immunoglobulin variable domains.

[0060] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (e.g., a native sequence Fc region or an 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0061] As used herein, the term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of about 50 to 70 kDa, the amino-terminal portion of which contains a variable region of about 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains one or more constant regions. "Heavy chain" can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, including the IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4.

[0062] As used herein, the term "light chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art.

[0063] The term "antigen-binding fragment" refers to a portion of an antibody that contains the amino acid residues (e.g., CDRs) that interact with an antigen and confer its specificity and affinity to the binding fragment, domain, or region for the antigen. As used herein, "antigen-binding fragment" includes "antibody fragments" that include portions of an antibody that contain one or more CDRs, such as the antigen-binding or variable region of an antibody.

[0064] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including, e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In certain embodiments, the antibody is a humanized antibody.

[0065] In certain embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain one or more antigen-binding sites that bind to α5 integrin.

[0066] An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the antibodies described herein are IgG antibodies (e.g., human IgG), or classes (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclasses thereof.

[0067] In certain embodiments, the antibody is a four-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 certain embodiments, the H chain and L chain comprise a constant region, e.g., a human constant region. In certain embodiments, the L chain constant region of such an antibody is a kappa or lambda light chain constant region, e.g., a human kappa or lambda light chain constant region. In certain embodiments, the H chain constant region of such an antibody comprises a gamma heavy chain constant region, e.g., a human gamma heavy chain constant region. In certain embodiments, such an antibody comprises an IgG constant region, e.g., a human IgG constant region (e.g., an IgG1, IgG2, IgG3, and / or IgG4 constant region).

[0068] The term "variable region" or "variable domain" refers to the portion of an antibody light or heavy chain that is generally located at the amino terminus of the light or heavy chain and that is responsible for the binding and specificity of each particular antibody for a particular antigen. The variable region of a heavy chain is referred to as the "V H The variable region of the light chain may be represented as "V LThe term "variable" refers to the fact that the sequences of certain segments of the variable regions differ significantly among antibodies. The V regions mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, variability is not evenly distributed throughout the variable regions. Rather, the V regions consist of stretches of less variable (e.g., relatively invariant) regions called framework regions (FRs), separated by shorter regions of more variable (e.g., extreme variability) called "hypervariable regions" or "complementarity-determining regions" ("CDRs"). The heavy and light chain variable regions each contain four FRs (FR1, FR2, FR3, and FR4), which largely adopt a β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases, form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th (See, Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant region is not directly involved in binding of the antibody to an antigen, but can exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen.

[0069] Generally, antibodies contain six hypervariable regions: V H three (HCDR1, HCDR2, and HCDR3), and V LCDRs include three (LCDR1, LCDR2, and LCDR3). CDRs can be identified according to several known numbering systems. In certain embodiments, CDRs are identified according to the Kabat numbering system. Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In certain embodiments, CDRs are identified according to the Chothia numbering system. Chothia refers to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). In certain embodiments, CDRs are identified according to the AbM numbering system. AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). In certain embodiments, CDRs are identified according to the Contact numbering 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. In certain embodiments, CDRs are identified according to the ImMunoGeneTics (IMGT) Information System®. A universal numbering system has been developed and widely adopted (ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system dedicated to immunoglobulins (IGs), T-cell receptors (TRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates.

[0070] As used herein, CDRs are referred to both in terms of amino acid sequence and location within the light or heavy chain. Because the "location" of CDRs within the structure of immunoglobulin variable domains is conserved across species and occurs 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 to graft and replace CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. An additional numbering system (Ahon) was developed by Honegger and Pluckthun, J. Mol. Biol. 309:657-670 (2001). Correspondence between numbering systems, e.g., the Kabat numbering system and the IMGT unique numbering system, is 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 exemplified below. The exemplary system presented herein identifies the CDRs as Kabat and AbM long sequences. An exemplary demonstration of various numbering systems is shown in Table 1. [Table 1]

[0071] The hypervariable region may include an "extended hypervariable region" such as: L 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and V H 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). As used herein, the terms "hypervariable region," "HVR," "HV," "complementarity-determining region," or "CDR" are used interchangeably.

[0072] The term "vector" refers to a substance used to carry or contain a nucleic acid, e.g., to introduce the nucleic acid into a host cell. Usable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, and can contain a selection sequence or selection marker that can be used for stable integration into a host cell chromosome. In addition, a vector can contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be incorporated, for example, confer resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply important nutrients not present in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. Two or more nucleic acid molecules (e.g., antibody heavy and light chains or antibody V) can be expressed in a single vector. H and V L When both nucleic acid molecules (e.g., the α5 integrin antibody or the α5 integrin antibody described herein) are co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single vector expression, the encoding nucleic acids can be operably linked to a common expression control sequence or can be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Introduction of the nucleic acid into the host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblot for expression of the gene product, or other analytical methods suitable for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the nucleic acid will be expressed in an amount sufficient to produce the desired product (e.g., the anti-α5 integrin antibody or antigen-binding fragment thereof described herein), and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0073] 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 underlying causes, prevent the occurrence of one or more symptoms and / or their underlying causes, and / or ameliorate or repair damage caused by or associated with a disease, disorder, or condition. In certain embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0074] 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, shorten, and / or ameliorate the severity and / or duration of a given disease, disorder, or condition, and / or its associated symptoms. A therapeutically effective amount of an agent, including a therapeutic agent, can be the amount necessary to (i) inhibit or ameliorate the progression or worsening of a given disease, disorder, or condition, (ii) inhibit or ameliorate the recurrence, progression, or onset of a given disease, disorder, or condition, and / or (iii) improve or enhance the prophylactic or therapeutic effects of another therapy (e.g., a therapy other than administration of an anti-α5 integrin antibody described herein). A "therapeutically effective amount" of a substance / molecule / agent (e.g., an anti-α5 integrin antibody) of the present disclosure can vary based 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 includes an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term "therapeutically effective amount" refers to an amount of an antibody or other agent (e.g., a drug) effective in "treating" a disease, disorder, or condition in a subject or mammal.

[0075] A "prophylactically effective amount" is the amount of a pharmaceutical composition that, when administered to a subject, has the intended prophylactic effect, such as 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 symptom(s). A complete therapeutic or prophylactic effect does not necessarily occur with the administration of a single dose, but may occur only after the administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0076] As used herein, a "carrier" includes a carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. In most cases, the carrier is a pH-buffered aqueous solution. Examples of carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable carriers (e.g., pharmaceutical carriers) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The compositions can be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation or the like.Oral compositions containing formulations can contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions containing pharmaceutical compounds can contain, for example, a prophylactically or therapeutically effective amount of an anti-α5 integrin antibody or antigen-binding fragment thereof in isolated or purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject (e.g., a patient). The formulation should be suitable for the method of administration.

[0077] As used herein, "individual" or "subject" refers to a vertebrate, such as a human or a non-human animal (e.g., a mammal). Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Examples of non-human animal subjects include, but are not limited to, rodents, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, and horses, and non-human primates, such as apes and monkeys. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0078] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, and some of this is related to the limitations of the method of measuring or determining the value, i.e., the measurement system. For example, "about" can mean within 3 or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, for example, up to 15%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean, for example, within 5 or 2 orders of magnitude of a value.

[0079] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0080] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0081] The term "between" as used in the phrase "between A and B" or "between AB" refers to a range that includes both A and B.

[0082] As used herein, the term "and / or" in the phrase "A and / or B" is intended to include both A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in the phrase "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0083] Integrin Integrins are heterodimeric, ubiquitous transmembrane glycoprotein receptors that function primarily as signaling proteins in mammals. Each integrin is composed of an α subunit and a β subunit. There are 18 variants of the α subunit and 8 variants of the β subunit, resulting in 24 unique heterodimeric integrins. The α and β subunits combine to form a noncovalent complex with a ligand-binding site at the interface. These heterodimers regulate cell behavior through mechanisms known as "inside-out" and "outside-in" signaling. In the former, intracellular proteins bind to the cytoplasmic domain of the integrin, stabilizing a conformation that binds extracellular ligands with high affinity. In the latter, the ligand-bound integrin then stimulates intracellular signaling cascades that regulate cell behavior.

[0084] Alpha 5 is also referred to as A5, integrin alpha-5, ITGA5 protein, CD49e antigen, glycoprotein Ic (GPIc), VLA5A, FNRA, and fibronectin receptor subunit alpha. The term "alpha 5 integrin" refers to any native alpha 5 integrin polypeptide or protein from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs, and rodents (e.g., mice and rats). The term "alpha 5 integrin" includes "full-length," unprocessed alpha 5 integrin, as well as any form of alpha 5 integrin or any fragment thereof resulting from intracellular processing. The term "alpha 5 integrin" also includes naturally occurring variants of alpha 5 integrin, such as SNP variants, splice variants, and allelic variants. It is known in the art that α5 integrin, associated with β1 integrin as a heterodimer, interacts with numerous ligands (e.g., fibronectin). This interaction leads to conformational changes in the protein and signal transduction, resulting in changes in cellular activities such as cell adhesion, proliferation, apoptosis, migration, and phagocytosis. α5β1 integrin is known as a fibronectin receptor due to its high affinity for fibronectin in the extracellular matrix (ECM). This binding is mediated by a ligand-binding site at the interface between the α and β subunits within the head of α5β1 and the arginine-glycine-aspartic acid (RGD) peptide motif of the type III repeats of fibronectin. In addition to fibronectin, α5β1 integrin also binds to other RGD-containing proteins, such as osteopontin and fibrillin, as well as proteins lacking the RGD motif, such as CD40L, IL-1b, and the TNF-α converting enzyme ADAM-17. Consistent with the tissue distribution of the ligand, α5β1 is expressed by a variety of cell types, including endothelial cells of peripheral tissues and the central nervous system (CNS) (e.g., microglia and perivascular macrophages), mast cells, and macrophage lineages.

[0085] In certain embodiments, the anti-α5 integrin antibody disclosed herein, or an antigen-binding fragment thereof, binds to human α5 integrin. In certain embodiments, the human α5 integrin is wild-type human α5 integrin or a fragment thereof. In certain embodiments, the wild-type human α5 integrin comprises the amino acid sequence of Uniprot reference number P08648 (SEQ ID NO: 1). SEQ ID NO: 1 is shown below.

[0086] In certain embodiments, the human α5 integrin comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In certain embodiments, the extracellular domain of the human α5 integrin comprises amino acids 42-995 of SEQ ID NO:1. In certain embodiments, the transmembrane domain of the human α5 integrin comprises amino acids 996-1021 of SEQ ID NO:1. In certain embodiments, the cytoplasmic domain of the human α5 integrin comprises amino acids 1022-1049 of SEQ ID NO:1. In certain embodiments, the human α5 integrin comprises a signal peptide. In certain embodiments, the signal peptide of the human α5 integrin comprises amino acids 1-41 of SEQ ID NO:1.

[0087] In certain embodiments, the alpha5 integrin comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof, and / or may contain at most one, at most two, or at most three conservative amino acid substitutions.

[0088] For example, in certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein bind to the extracellular domain of α5 integrin (e.g., human α5 integrin). In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof bind to a fragment of SEQ ID NO: 1. In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein bind to amino acids 42-995 of SEQ ID NO: 1.

[0089] In certain embodiments, the anti-α5 integrin antibodies, or antigen-binding fragments thereof, disclosed herein bind to mouse α5 integrin. In certain embodiments, the anti-α5 integrin antibodies, or antigen-binding fragments thereof, disclosed herein do not bind to mouse α5 integrin.

[0090] In certain embodiments, the mouse alpha5 integrin comprises a wild-type mouse alpha5 integrin or a fragment thereof. In certain embodiments, the wild-type mouse alpha5 integrin comprises the amino acid sequence of Uniprot reference number P11688 (SEQ ID NO: 2). SEQ ID NO: 2 is shown below.

[0091] Other related α5 integrin polypeptides also encompassed within the term α5 integrin include fragments, derivatives (e.g., substitution, deletion, truncation, and insertion variants), fusion polypeptides, and interspecies homologs that retain the activity of α5 integrin and / or are sufficient to generate an anti-α5 integrin immune response. Those skilled in the art will understand that the anti-α5 integrin antibodies or antigen-binding fragments thereof described herein can bind to an α5 integrin polypeptide, an α5 integrin polypeptide fragment, an α5 integrin antigen, and / or an α5 integrin epitope. An epitope may be part of a larger α5 integrin antigen, which may be part of a larger α5 integrin polypeptide fragment, which may be part of a larger α5 integrin polypeptide. α5 integrin may exist in native or denatured form. The α5 integrin polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or another source, or may be prepared by recombinant or synthetic methods. An α5 integrin polypeptide can include a polypeptide having the same amino acid sequence as a corresponding α5 integrin polypeptide derived from nature. Orthologs of α5 integrin polypeptides are also well known in the art.

[0092] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein do not bind to β1 integrin (e.g., the β1 integrin subunit of α1β1 integrin). β1 integrin is also referred to as B1 integrin, integrin beta1, ITGB1 protein, CD29 antigen, fibronectin receptor subunit beta, and glycoprotein Iia. The term "β1 integrin" refers to any native β1 integrin polypeptide or protein from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno), dogs, and rodents (e.g., mice and rats). The term "β1 integrin" includes all forms of "full-length," unprocessed β1 integrin as well as any fragment thereof resulting from processing within cells. The term β1 integrin also includes naturally occurring variants of β1 integrin, such as SNP variants, splice variants, and allelic variants.

[0093] In certain embodiments, the anti-α5 integrin antibody, or antigen-binding fragment thereof, disclosed herein does not bind to human β1 integrin. In certain embodiments, the human β1 subunit comprises wild-type human β1 integrin or a fragment thereof. In certain embodiments, the wild-type human β1 integrin comprises the amino acid sequence of Uniprot reference number P05556-1 (SEQ ID NO: 3). SEQ ID NO: 3 is shown below. (SEQ ID NO: 3)

[0094] In certain embodiments, the anti-α5 integrin antibody, or antigen-binding fragment thereof, disclosed herein does not bind to mouse β1 integrin. In certain embodiments, the mouse β1 subunit comprises wild-type mouse β1 integrin or a fragment thereof. In certain embodiments, the mouse β1 integrin comprises the amino acid sequence of Uniprot reference number P09055-1 (SEQ ID NO: 4). SEQ ID NO: 4 is shown below. (SEQ ID NO: 4)

[0095] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein can block or inhibit binding between α5β1 (e.g., human α5β1) and fibronectin (e.g., human fibronectin). The term "fibronectin" refers to any native fibronectin polypeptide or protein from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cynos)), dogs, and rodents (e.g., mice and rats). Fibronectin (FN) exists as a dimer or multimer linked via disulfide bonds and has a multimodular structure composed primarily of three distinct repeats designated FN-I, FN-II, and FN-III. In the dimeric form, two fibronectin subunits each consist of 12 FN-I, 2 FN-II, and 15–17 FN-III modules. The term "fibronectin" also includes variants present in native fibronectin, such as SNP variants, splice variants, and allelic variants. Fibronectin is an essential component of the extracellular matrix and has multiple protein-binding domains, including domains for fibrin binding, collagen binding, fibulin-1 binding, heparin binding, and syndecan binding. It is known in the art that fibronectin interacts with integrins (e.g., via RGD) and is a ligand for α5β1 integrin, α8β1 integrin, and αvβ3 integrin.

[0096] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein can block or inhibit the binding of human α5β1 to human fibronectin. In certain embodiments, the human fibronectin comprises wild-type human fibronectin or a fragment thereof. In certain embodiments, the wild-type human fibronectin comprises the amino acid sequence of Uniprot reference number P02751-15 (SEQ ID NO: 5). SEQ ID NO: 5 is shown below.

[0097] 4.3. Anti-α5 integrin antibodies and antigen-binding fragments thereof The present disclosure provides anti-α5 integrin antibodies and antigen-binding fragments thereof. The antibodies can be polyclonal, monoclonal, humanized, human, multispecific, bispecific, or heteroconjugate antibodies, as well as variants thereof with improved or decreased affinity or other properties. In certain embodiments, the antibodies disclosed herein are humanized antibodies.

[0098] In certain embodiments, the anti-α5 integrin antibodies and antigen-binding fragments thereof disclosed herein bind to human α5 integrin. In certain embodiments, the anti-α5 integrin antibodies and antigen-binding fragments thereof disclosed herein bind to the extracellular domain of human α5 integrin. In certain embodiments, the α5β1 integrin is human α5β1 integrin. In certain embodiments, the anti-α5 integrin antibodies and antigen-binding fragments thereof disclosed herein bind to human α5 integrin comprising the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof (e.g., amino acids 42-995 of SEQ ID NO: 1 or a fragment thereof). In certain embodiments, the anti-α5 integrin antibodies and antigen-binding fragments thereof disclosed herein bind to α5 integrin expressed on the surface of mammalian (e.g., human) cells, for example, α5 integrin-expressing tumor cells. In certain embodiments, the anti-α5 integrin antibodies and antigen-binding fragments thereof disclosed herein bind to an α5 integrin extracellular epitope (e.g., an α5 integrin epitope) exposed on cells, for example, tumor cells.

[0099] 4.3.1. Monospecific Antibodies and Antigen-Binding Fragments Thereof In certain embodiments, the anti-α5 integrin antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (V H ) and the light chain variable region (V L In certain embodiments, V Hcomprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24, or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:26, or a modified version thereof. SEQ ID NOs:6, 7, 9, 13-15, 18-21, 24-26, and 30 are provided in Table 2.

[0100] In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24, or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:30, or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:26, or a modified version thereof. SEQ ID NOs:6, 8, 9, 13-15, 18-21, 24-26, and 30 are provided in Table 3.

[0101] In certain embodiments, V L comprises CDR1 containing the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 27, or a modified version thereof, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 28, or a modified version thereof, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 23, SEQ ID NO: 29, or a modified version thereof. SEQ ID NOs: 10 to 12, 16, 17, 22, 23, and 27 to 29 are disclosed in Table 4.

[0102] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 13 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 14 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 15 or a modified version thereof. HIn certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a modified version thereof. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the CDRs are identified according to the IMGT numbering system, as described, for example, in Lefranc, M.-P., 1999, The Immunologist, 7:132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212.

[0103] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 18 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 7 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 9 or a modified version thereof. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a modified version thereof. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. LIn certain embodiments, the CDRs are recognized by the Kabat numbering system, e.g., as described in Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242.

[0104] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 18 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 8 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 9 or a modified version thereof. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a modified version thereof. L In certain embodiments, the anti-α5 integrin antibody, or antigen-binding fragment thereof, disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. LIn certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9. H and V comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0105] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 19 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 20 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 21 or a modified version thereof. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23 or a modified version thereof. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23. LIn certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21. H and V comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, CDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 23. L In certain embodiments, the CDRs are identified according to the Chothia system, e.g., as described in 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.

[0106] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 24 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 25 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 26 or a modified version thereof. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29 or a modified version thereof. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26. H , a V comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 28, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 29 L In certain embodiments, the CDRs are identified according to the Contact system, e.g., as described in MacCallum RM et al., 1996, J Mol Biol 5:732-745. Contact CDRs are based on an analysis of available complex crystal structures.

[0107] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein has a V domain comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:6 or a modified version thereof, a CDR2 having the amino acid sequence set forth in SEQ ID NO:30 or a modified version thereof, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:9 or a modified version thereof. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a modified version thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a modified version thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12 or a modified version thereof. LIn certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a VCR comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9. H and V comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 12. L In certain embodiments, the CDRs are identified 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 Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). [Table 2] [Table 3] [Table 4] [Table 5]

[0108] In certain embodiments, conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in certain embodiments, a predicted non-essential amino acid residue in an anti-α5 integrin antibody or antigen-binding fragment thereof is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In certain embodiments, amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions for a CDR, as set forth in Table 2. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, e.g., up to 4 (or fewer) conservative amino acid substitutions, e.g., up to 3 (or fewer) conservative amino acid substitutions, e.g., up to 2 (or fewer) conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In certain embodiments, the conservative modifications are outside of the CDR sequences.

[0109] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein comprise a V antibody or V CDR comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:31. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:33. L SEQ ID NOs: 31 and 33 are disclosed in Tables 2 and 4. In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 33. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 L Includes.

[0110] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein comprise a V antibody or V CDR comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:31. HIn certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:34. L SEQ ID NO: 34 is disclosed in Table 5. In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 34. L In certain embodiments, the anti-α5 integrin antibody, or antigen-binding fragment thereof, disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO:31. H and V comprising the amino acid sequence set forth in SEQ ID NO: 34. L Includes.

[0111] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein comprise a V antibody comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:32. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:33. LSEQ ID NO: 32 is disclosed in Table 3. In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 33. L In certain embodiments, the anti-α5 antibodies or antigen-binding fragments thereof disclosed herein comprise a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 L Includes.

[0112] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein comprise a V antibody comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:32. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:34. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 34. L In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. Hand V comprising the amino acid sequence set forth in SEQ ID NO: 34. L Includes.

[0113] The antibody designated "A2-M5-Low(H) / Low(L)" is a V-type antibody disclosed in Table 2. H CDR1, V H CDR2 and V H CDR3 sequences and V sequences disclosed in Table 4 L CDR1, V L CDR2 and V L In a specific embodiment, the A2-M5-Low(H) / Low(L) antibody comprises a V CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 31 (disclosed in Table 2). H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 (disclosed in Table 4). L Includes.

[0114] The antibody designated "A2-M5-Low(H) / Low+Mod(L)" is a V-type antibody disclosed in Table 2. H CDR1, V H CDR2 and V H CDR3 sequences, and V sequences disclosed in Table 5 L CDR1, V L CDR2 and V L In a specific embodiment, the A2-M5-Low(H) / Low+Mod(L) antibody comprises a V CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 31 (disclosed in Table 2). H and V comprising the amino acid sequence set forth in SEQ ID NO: 34 (disclosed in Table 5). L Includes.

[0115] The antibody designated "A2-M5-Mod(H) / Low(L)" is a V-type antibody disclosed in Table 3. H CDR1, V H CDR2 and V H CDR3 sequences and V sequences disclosed in Table 4 L CDR1, V L CDR2 and V LIn a specific embodiment, the A2-M5-Low+Mod(H) / Low(L) antibody comprises a V CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 32 (disclosed in Table 3). H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 (disclosed in Table 4). L Includes.

[0116] The antibody designated "A2-M5-Low+Mod(H) / Low+Mod(L)" is a V-type antibody disclosed in Table 3. H CDR1, V H CDR2 and V H CDR3 sequences, and V sequences disclosed in Table 5 L CDR1, V L CDR2 and V L In a specific embodiment, the A2-M5-Low+Mod(H) / Low+Mod(L) antibody comprises a V CDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO: 32 (disclosed in Table 3). H and V comprising the amino acid sequence set forth in SEQ ID NO: 34 (disclosed in Table 5). L Includes.

[0117] In certain embodiments, the anti-α5 integrin antibody disclosed herein comprises a fragment crystallizable region (Fc region). The Fc region is the C-terminal region of an immunoglobulin heavy chain. The Fc region can be a functional Fc region, a native Fc region, a recombinant Fc region, or a mutated Fc region.

[0118] A "functional Fc region" possesses an "effector function" 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, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), etc. These effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain), and can be assessed using a variety of assays, such as those disclosed.

[0119] A "native Fc region" includes an Fc region found in nature and has not been manipulated, modified, and / or altered (e.g., isolated, purified, selected, or combined with other sequences such as variable region sequences) by humans. Examples of native human Fc regions include, but are not limited to, native human IgG1 Fc regions (non-A and A allotypes), native human IgG2 Fc regions, native human IgG3 Fc regions, and native human IgG4 Fc regions, as well as native variants thereof.

[0120] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitution(s). In certain embodiments, a variant Fc region comprises at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, or about one to about five amino acid substitutions, relative to a native Fc region or the Fc region of a parent polypeptide. The variant Fc regions described herein can have at least about 80%, or at least about 90%, at least about 95%, or at least 99% sequence identity with a native Fc region and / or the Fc region of a parent polypeptide. A variant Fc region can also comprise a loss of effector function (e.g., a silent Fc). Examples of variant Fc regions include, but are not limited to, 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).

[0121] In certain embodiments, the Fc region comprises H, and one or more heavy chain constant domains (e.g., CH1, hinge, CH2, and CH3). In certain embodiments, the heavy chain constant region is selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In certain embodiments, the heavy chain constant region is selected from IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the immunoglobulin isotype is IgG4. In certain embodiments, the immunoglobulin isotype is human IgG4. In certain embodiments, the immunoglobulin isotype is IgG1. In certain embodiments, the immunoglobulin isotype is human IgG1.

[0122] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a human IgG1 Fc region. In certain embodiments, the human IgG1 Fc region is a native human IgG1 Fc region. In certain embodiments, the native human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 35 below. Amino acids 6 to 115 of SEQ ID NO: 35 are the CH2 domain, and amino acids 116 to 222 of SEQ ID NO: 35 are the CH3 domain. CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35)

[0123] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a human IgG1 heavy chain constant domain. In certain embodiments, the human IgG1 heavy chain constant domain comprises a native human IgG1 Fc region. In certain embodiments, the human IgG1 heavy chain constant domain comprises a native human IgG1 Fc region consisting of the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the human IgG1 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 36 below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36)

[0124] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a human IgG4 Fc region. In certain embodiments, the human IgG4 Fc region is a native human IgG4 Fc region. In certain embodiments, the native human IgG4 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 37 below. CPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 37)

[0125] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG1 Fc region. In certain embodiments, the variant human IgG1 Fc region is a silent Fc region. In certain embodiments, the variant human IgG1 Fc region comprises two L→A substitutions in the CH2 domain. In certain embodiments, the variant human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 38. SEQ ID NO: 38 is shown below. In certain embodiments, the CH2 domain comprises amino acids 6 to 115 of SEQ ID NO: 38. In certain embodiments, the CH3 domain comprises amino acids 116 to 222 of SEQ ID NO: 38. CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38)

[0126] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a human IgG1 heavy chain constant domain. In certain embodiments, the human IgG1 heavy chain constant domain comprises a variant human IgG1 Fc region. In certain embodiments, the human IgG1 heavy chain constant domain comprises a variant human IgG1 Fc region consisting of the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the human IgG1 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 39 below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39)

[0127] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG1 Fc region. In certain embodiments, the variant human IgG1 Fc region comprises one N→Q substitution in the CH2 domain. In certain embodiments, the variant human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 40 below. CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0128] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG1 Fc region. In certain embodiments, the variant human IgG1 Fc region comprises two L→A substitutions and one P→S substitution in the CH2 domain. In certain embodiments, the variant human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 41 below. CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41)

[0129] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG1 Fc region. In certain embodiments, the variant human IgG1 Fc region comprises two L→A substitutions and one P→G substitution in the CH2 domain. In certain embodiments, the variant human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 42 below. CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42)

[0130] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG1 Fc region. In certain embodiments, the variant human IgG1 Fc region comprises one L→F substitution, one L→E substitution, and one P→S substitution in the CH2 domain. In certain embodiments, the variant human IgG1 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 43 below. CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 43)

[0131] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a variant human IgG4 Fc region. In certain embodiments, the variant human IgG4 Fc region comprises one P→S substitution, one A→F substitution, and one A→L substitution in the CH2 domain. In certain embodiments, the variant human IgG4 Fc region comprises the amino acid sequence set forth in SEQ ID NO: 44 below. CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 44)

[0132] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein comprises a human IgG4 heavy chain constant domain. In certain embodiments, the human IgG4 heavy chain constant domain comprises a variant human IgG4 Fc region. In certain embodiments, the human IgG4 heavy chain constant domain comprises a variant human IgG4 Fc region consisting of the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the human IgG4 heavy chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 45 below. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 45)

[0133] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein comprise a light chain constant domain. In certain embodiments, the light chain constant region is selected from kappa and lambda. In certain embodiments, the light chain constant region is kappa. In certain embodiments, the light chain constant domain comprises the amino acid sequence set forth in SEQ ID NO: 46 below. RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 46)

[0134] In certain embodiments, the anti-α5 integrin antibody disclosed herein is a humanized antibody. In certain embodiments, the anti-α5 integrin antibody disclosed herein is a monoclonal antibody. In certain embodiments, the anti-α5 integrin antibody disclosed herein is a humanized monoclonal antibody. In certain embodiments, the antigen-binding fragment disclosed herein is a Fab, Fab', F(ab')2, Fv, or single-chain variable fragment (scFv). In certain embodiments, the antigen-binding fragment disclosed herein is an scFv.

[0135] 4.3.2. Multispecific molecules The present disclosure provides a multispecific molecule comprising the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the multispecific molecule is a multispecific antibody. In certain embodiments, the multispecific molecule (e.g., a multispecific antibody) is a bispecific molecule (e.g., a bispecific antibody).

[0136] In certain embodiments, a multispecific molecule binds to at least two different binding sites or target molecules. In certain embodiments, a multispecific molecule comprises at least a first binding specificity for α5 integrin or α5β1 integrin and a second binding specificity for a second target epitope. The second target epitope can be an α5 integrin epitope or a non-α5 integrin epitope, e.g., a different second target antigen. Examples of second target antigens include, but are not limited to, αv integrin, β3 integrin, α4 ​​integrin, β1 integrin, α4 ​​integrin, β7 integrin, TREM2, TNFα, IL-6, IL-1β, CSF1, CSF-1R, C1Q, CD40L, FGFR, IL-12, and type I interferon.

[0137] Methods for creating bispecific antibodies are known in the art, including by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities. Exemplary structures of multispecific molecules are known in the art and are further described in Weidle et al., 2013, Cancer Genomics & Proteomics 10:1-18; Brinkman et al., 2017, MABS 9:2,182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents 28:3,251-276; and Spiess et al., 2015, Mol. Immunol. 67 95-106.

[0138] For example, bispecific molecules can be classified into different structural groups: (i) bispecific immunoglobulin G (BsIgG), (ii) IgG with additional antigen-binding moieties appended, (iii) bispecific antibody fragments, (iv) bispecific fusion proteins, and (v) bispecific antibody conjugates. By way of example and not limitation, BsIgG formats can include crossMab, DAF (2in1), DAF (4in1), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab arm exchange, SEED body, triomab, LUZ-Y, Fcab, κλ-body, and orthogonal Fab.

[0139] In certain embodiments, the BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using the "knobs-into-hole" strategy, the SEED platform, a common heavy chain (e.g., in κλ-bodies), and heterodimeric Fc regions. Strategies for avoiding homodimeric heavy chain pairing in BsIgG are known in the art, including knobs-into-holes, duobodies, azimetric, charge-pair, HA-TF, SEED bodies, and differential Protein A affinity.

[0140] Another bispecific molecule format is an IgG with an additional antigen-binding moiety attached. For example, a monospecific IgG can be engineered to have bispecificity by adding an additional antigen-binding unit, for example, to the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single-chain variable fragments or variable fragments). Examples of added IgG formats include, but are not limited to, 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 (4in1). See Spiess et al. Mol. Immunol. 67(2015):95-106. In certain embodiments, an exemplary antibody format is the B-Body format for monospecific or multispecific (e.g., bispecific) antibodies, as described, for example, in International Patent Application Publication No. WO2018 / 075692 and U.S. Patent Application Publication No. 2018 / 0118811.

[0141] Bispecific antibody fragments (BsAbs) are a format of bispecific molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In certain embodiments, bispecific antibody fragments have heavy and light chain regions connected by a peptide linker, which allows for efficient expression of the BsAb in a single host cell. Examples of bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scDiabodies, scDiabody-CH3, diabody-CH3, triplebodies, miniantibodies, minibodies, TriBiminibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAbs, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies.

[0142] Bispecific fusion proteins include antibody fragments conjugated to other proteins. For example, bispecific fusion proteins can be conjugated to other proteins to add additional specificity and / or functionality. In certain embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with even greater valency. For example, fusion of bispecific antibodies to albumin-binding proteins or human serum albumin can extend the serum half-life of antibody fragments. In certain embodiments, chemical conjugation, e.g., of antibodies and / or antibody fragments, can be used to create BsAb molecules. An exemplary bispecific antibody conjugate is the CovX-body format, in which a low-molecular-weight drug is site-specifically conjugated to a single reactive lysine within each Fab arm or antibody or fragment thereof. In certain embodiments, the conjugation improves serum half-life.

[0143] Methods for producing multispecific molecules, including bispecific molecules, are known in the art. For example, multispecific molecules, including bispecific molecules, can be produced by separate expression of antibody components in different host cells followed by purification / assembly, or by expression of antibody components in a single host cell. Purification of multispecific (e.g., bispecific) molecules can be achieved by various methods known in the art, such as affinity chromatography.

[0144] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein can be provided in any antibody format disclosed herein or known in the art. In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof can be Fabs-in-tandem-Ig (FIT-Ig), DVD-Ig, hybrid hybridomas (quadromas or tetradomas), anticalin platforms (Pieris), diabodies, single-chain diabodies, tandem single-chain Fv fragments, TandAbs, Trispecific Abs (Affimed), Darts Dual Affinity Retargeting (Macrogenics), Bispecific The antibody may be selected from Xmabs (Xencor), bispecific T cell engager (BiTE, Amgen, 55 kDa), triple body, tribody = Fab-scFv fusion protein multifunctional recombinant antibody derivative (CreativeBiolabs), duobody platform (Genmab), dock-and-lock platform, knob-into-hole (KIH) platform, humanized bispecific IgG antibody (REGN1979) (Regeneron), Mab2 bispecific antibody (F-Star), DVD-Ig = dual variable domain immunoglobulin (AbbVie), kappa-lambda body, TBTI = tetravalent bispecific tandem Ig, and CrossMab (Roche).

[0145] In certain embodiments, the multispecific (e.g., bispecific) molecule comprises a V HIn certain embodiments, the α5 integrin binding domain comprises a V integrin binding domain comprising a sequence (e.g., those disclosed in Section 4.3.1, e.g., those disclosed in Tables 2 and 3). In certain embodiments, the α5 integrin binding domain comprises a V integrin binding domain comprising a V integrin binding domain as disclosed herein. L In certain embodiments, the α5 integrin binding domain comprises a V sequence (e.g., those disclosed in Section 4.3.1, e.g., those disclosed in Tables 4 and 5). In certain embodiments, the α5 integrin binding domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:31. H In certain embodiments, the α5 integrin binding domain comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:33. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 33. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 L Includes.

[0146] In certain embodiments, the alpha5 integrin binding domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:31. HIn certain embodiments, the α5 integrin binding domain comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:34. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 34. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 31. H and V comprising the amino acid sequence set forth in SEQ ID NO: 34. L Includes.

[0147] In certain embodiments, the alpha5 integrin binding domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:32. H In certain embodiments, the α5 integrin binding domain comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:33. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 33. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H and V comprising the amino acid sequence set forth in SEQ ID NO: 33 L Includes.

[0148] In certain embodiments, the alpha5 integrin binding domain comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:32. H In certain embodiments, the α5 integrin binding domain comprises a V that comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:34. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 34. L In certain embodiments, the α5 integrin binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 32. H and V comprising the amino acid sequence set forth in SEQ ID NO: 34. L Includes.

[0149] In certain embodiments, the α5 integrin binding domain comprises one or more CDRs, e.g., the V H CDR1, V H CDR2 and / or V H Three Vs in CDR3 H V containing CDRs H In certain embodiments, the α5 integrin binding domain comprises one or more CDRs, e.g., the V L CDR1, V L CDR2 and / or V L Three Vs in CDR3 L V containing CDRs L Includes.

[0150] 4.3.3. Cross-competing antibodies The present disclosure further provides antibodies or antigen-binding fragments thereof that cross-compete for binding to alpha5 integrin (e.g., alpha5 integrin) with any of the anti-alpha5 integrin antibodies or antigen-binding fragments thereof disclosed herein (e.g., those disclosed in Section 4.3.1, e.g., A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L)). For example, a cross-competing antibody binds to the same epitope region, e.g., the same epitope, an adjacent epitope, or an overlapping epitope, as any of the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein (e.g., those disclosed in Section 4.3.1, e.g., A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L)).

[0151] Competition can be determined by an assay in which the test antibody under investigation prevents or inhibits specific binding of a reference antibody to a common epitope or antigen (e.g., alpha5 integrin). Many types of competitive binding assays can be used to determine whether a test antibody competes with a reference antibody for binding to alpha5 integrin (e.g., human alpha5 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., Stahl 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 label assays, solid-phase direct label sandwich assays (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using I-125 labels (see, e.g., Morel et al., (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and the like. 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 cells bearing either purified antigen (e.g., α5 integrin, such as human α5 integrin) bound to a solid surface, or an unlabeled test antigen-binding protein (e.g., a test α5 integrin antibody), or a labeled reference antigen-binding protein (e.g., a reference anti-α5 integrin antibody). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells 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 an adjacent epitope sufficiently close to the epitope bound by the reference antibody (e.g., a similar or overlapping epitope) to cause steric hindrance of the antibody. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common epitope or common antigen by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, or at least about 97%, at least about 98%, or at least about 99% or more.

[0152] 4.3.4. Conjugates The present disclosure further provides a conjugate comprising the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the anti-α5 antibody or antigen-binding fragment thereof disclosed herein is linked or conjugated (directly or indirectly) to a moiety having an effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or immunomobilizing activity. In certain embodiments, the conjugate is an immunoconjugate. In certain embodiments, the conjugate is an antibody-drug conjugate (ADC). The moiety bound or conjugated (directly or indirectly) comprises a cytotoxic or non-cytotoxic drug. In certain embodiments, the anti-α5 integrin or antigen-binding fragment thereof disclosed herein is linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety having reporter activity (e.g., a detectable label or reporter protein).

[0153] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent). In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are conjugated or recombinantly linked (directly or indirectly) to a detectable agent (e.g., a labeled agent, including a labeled antibody). In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are conjugated or recombinantly linked (directly or indirectly) to a diagnostic agent. Conjugated or recombinantly linked antibodies can be useful for the diagnosis or treatment of α5 integrin-mediated diseases, disorders, and conditions, including, for example, cancer (e.g., cancers associated with or characterized by tumor cells that express or overexpress α5 integrin), angiogenesis-related diseases (e.g., diseases associated with or characterized by aberrant angiogenesis), and inflammatory diseases (e.g., neuroinflammatory diseases such as MS and ALS).

[0154] Such diagnosis and detection can include using a diagnostic and / or detectable agent, for example, by combining an anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein with a detectable substance (e.g., a labeled agent such as a labeled antibody), e.g., Enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent substances, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent substances, such as, but not limited to, luciferase, luciferin, or aequorin; Chemiluminescent materials, such as, but not limited to, acridinium-based compounds or HALOTAG, radioactive materials, such as, but not limited to, iodine (I, I, I, and I), carbon (C), sulfur (S), tritium (H), indium (In, In, In, and In), technetium (Tc), thallium (Ti), gallium (Ga), and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 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 This can be done by combining

[0155] A labeled agent (e.g., a labeled antibody) that specifically binds to an anti-α5 integrin antibody or antigen-binding fragment thereof can be used for diagnostic purposes to detect, diagnose, or monitor an α5β1 integrin-mediated disease, disorder, or condition.

[0156] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are recombinantly linked or conjugated (directly or indirectly, covalently or non-covalently) to a heterologous protein or polypeptide (or fragment thereof), a polypeptide (e.g., of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate a fusion protein. The heterologous protein, polypeptide, or peptide to which the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are linked is useful for targeting the anti-α5 integrin antibodies or antigen-binding fragments thereof to specific cells (e.g., α5β1 integrin-expressing cells such as tumor cells). In certain embodiments, the fusion protein retains the biological activity of the anti-α5 integrin antibody or antigen-binding fragment thereof. Fusion proteins can be generated, for example, through techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of an anti-α5 integrin antibody or antigen-binding fragment thereof, e.g., an anti-α5 integrin antibody or antigen-binding fragment thereof with higher affinity and lower dissociation rate. In certain embodiments, an anti-α5 integrin antibody or antigen-binding fragment thereof can be modified prior to recombination by error-prone PCR, random nucleotide insertion, or other random mutagenesis methods. Nucleic acids encoding the anti-α5 integrin antibodies or antigen-binding fragments thereof described herein can be recombined with one or more components, motifs, sections, portions, domains, fragments, etc., of one or more heterologous molecules.

[0157] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are conjugated (directly or indirectly) to a marker or "tag" sequence, such as a peptide, to facilitate purification. In certain embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the "FLAG" tag.

[0158] Methods for attaching or conjugating (directly or indirectly) a moiety (such as a polypeptide) to an antibody are well known in the art, any of which can be used to create the antibody-drug conjugates or fusion proteins described herein.

[0159] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof (e.g., antibodies, e.g., humanized antibodies) disclosed herein are bound to a solid support and can be used in immunoassays or purification of target antigens (e.g., α5 integrin or α5β1 integrin). Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0160] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are bound or conjugated (directly or indirectly) to a second antibody to form an antibody heteroconjugate.

[0161] The linker can be a "cleavable moiety" that facilitates intracellular release of the attached or conjugated drug, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates (e.g., antibody-drug conjugates) of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transporter-mediated resistance.

[0162] Conjugates of antibodies and drugs, such as when the drug is a drug for the preparation of an ADC, can be made using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further provides conjugates of antibodies and drugs, such as when the drug is a drug for the preparation of an ADC, that can be prepared using any suitable method disclosed in the art (e.g., Bioconjugate Techniques (Hermanson ed., 2002)). nd ed. 2008).

[0163] Traditional antibody-drug conjugation strategies, such as those for preparing ADCs, based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues yield heterogeneous conjugates. Recently developed technologies allow site-specific conjugation to antibodies, resulting in uniform loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. These techniques include engineering "thiomab" antibodies containing cysteine ​​substitutions at positions in the heavy and light chains that provide reactive thiol groups without perturbing immunoglobulin folding and assembly or altering the antigen. Another method involves cotranslationally inserting selenocysteine ​​into the antibody sequence by recoding the stop codon UGA from the terminus to a selenocysteine ​​insertion, allowing site-specific covalent conjugation with the nucleophilic selenol group of selenocysteine ​​in the presence of other native amino acids.

[0164] In certain embodiments, the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein are conjugated to one or more cytotoxic agent(s) disclosed herein or known in the art to generate ADCs. In certain 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 certain embodiments, the cytotoxic agent is an enzymatically active toxin, such as, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Papaya amago proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. In certain embodiments, the cytotoxic agent is a radioconjugate or a radioisotope that generates a radioconjugated agent. A variety of radionuclides are available for preparing radioconjugate agents, including, but not limited to, 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re, and 212Bi. Conjugates of a polypeptide or molecule with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothenes, and CC1065, and derivatives of these toxins that retain toxin activity, can also be used.Conjugates of polypeptides or molecules with cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridiidithiol)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).

[0165] In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein is conjugated to a drug, such as a signal transduction modulator, a proapoptotic agent, a mitotic inhibitor, an antitumor antibiotic, an immunomodulator, a nucleic acid for gene therapy, an alkylating agent, an antiangiogenic agent, an antimetabolite, a boron-containing agent, a chemoprotectant, a hormone, an antihormonal agent, a corticosteroid, a photoactive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor. In certain embodiments, the mitotic inhibitor is a dolastatin, an auristatin, a maytansinoid, or a plant alkaloid. In certain embodiments, the drug is a dolastatin, an auristatin, a maytansinoid, or a plant alkaloid. Examples of auristatins include monomethylauristatin F (MMAF) or monomethylauristatin E (MMAE). Examples of maytansinoids include DM1, DM2, DM3, and DM4. In certain embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracycline, calicheamicin, and duocarmycin, hi certain embodiments, the actinomycin is a pyrrolobenzodiazepine (PBD).

[0166] 4.3.5. Antibody generation The anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein can be obtained by any suitable method, including (but not limited to) immunization with whole tumor cells containing α5 integrin and collecting antibodies, recombinant techniques, or screening libraries of antibodies or antibody fragments using α5 integrin extracellular domain epitopes. 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 Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)). For example, an exemplary technique for producing monoclonal antibodies involves immunizing an animal with a human α5 integrin antigen and producing hybridomas from spleen cells harvested from the animal, which hybridomas are capable of producing monoclonal antibodies or antibody fragments that bind to the α5 integrin antigen.

[0167] In certain embodiments, antibodies or antigen-binding fragments thereof can be isolated from antibody phage libraries, such as those described herein. In certain embodiments, antibody phage libraries can be generated using techniques described, for example, in *Antibody Phage Display: Methods and Protocols*, P.M.O. Brien and R. Aitken, eds., Humana Press, Totawa NJ, 2002. In certain embodiments, antibody clones can be selected by screening phage libraries. Phage libraries can contain phages displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins (e.g., Fab, scFv). Such phage libraries are screened for antibodies against a desired antigen. Clones expressing Fv fragments (e.g., Fab, scFv) capable of binding to the desired antigen adsorb to the antigen and are therefore separated from non-binding clones in the library. Binding clones can then be eluted from the antigen and further enriched by additional cycles of antigen adsorption / elution.

[0168] The variable domains may be V or VL as described, for example, in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). H and V L can be functionally displayed on phage either as single-chain Fv (scFv) fragments covalently linked via a short, flexible peptide, or as Fab fragments, each fused to a constant domain and interacting non-covalently.

[0169] V H and V LGene repertoires can be cloned separately by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding clones, for example, as described in Winter et al., supra. Libraries derived from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned, for example, as described in Griffiths et al., EMBO J, 12:725-734 (1993), to provide a single source of human antibodies to a wide range of non-self and self antigens without any immunization. Finally, naive libraries can be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding the highly variable CDR3 regions to achieve in vitro rearrangement, for example, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992).

[0170] Screening of libraries can be accomplished by various techniques known in the art. For example, α5 integrin (e.g., α5 integrin polypeptide, fragment, or epitope) or α5β1 integrin (e.g., α5β1 integrin polypeptide, fragment, or epitope) can be used to coat the wells of an adsorption plate, expressed in host cells immobilized on an adsorption plate, used in cell sorting, conjugated to biotin for capture on streptavidin-coated beads, or used in any other method of panning a display library. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be facilitated by using extended washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by using a low antigen coating density, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0171] The anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein can be used to design an antigen screening procedure suitable for selecting a desired phage clone, followed by isolation of V from the desired phage clone. H and / or V L sequence (e.g., Fv sequence), or V H and V L A full-length anti-α5 integrin antibody clone can be obtained by constructing a variety of CDR sequences derived from the sequence and suitable constant region (e.g., Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3.

[0172] The present disclosure provides humanized antibodies that bind to alpha5 integrin (e.g., human alpha5 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 from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically from an "import" variable domain. Humanized antibodies that bind to α5 integrin and / or α5β1 integrin can be produced by 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).

[0173] In certain embodiments, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of six complementarity-determining regions (CDRs) of a parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J.9:133-139,1995) determined that only about one-third of the residues of CDRs actually contact antigen, and named them "specificity-determining residues" or SDRs. In the technique of SDR grafting, only SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34,2005).

[0174] The selection of human variable domains, both light and heavy, used to create a humanized antibody can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a nonhuman (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that most closely resembles the rodent sequence 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 method uses a specific framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. 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 based on the most abundant human subclass, V. L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) are derived from the consensus sequence. Alternatively, human germline genes are used as the source of the framework regions.

[0175] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. This method involves comparing non-human sequences with functional human germline gene repertoires. Then, genes encoding standard structures identical to or closely related to mouse sequences are selected. Next, among the genes that share standard structures with non-human antibodies, the gene with the highest CDR homology is selected as a FR donor. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169:1119-1125, 2002).

[0176] Antibodies are humanized while retaining their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are known to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0177] Another method for antibody humanization is based on a measure of antibody humanization called Human String Content (HSC). This method compares mouse sequences with the repertoire of human germline genes, and scores differences as HSC. Then, instead of using overall identity index, the target sequence is humanized to maximize its HSC, resulting in the generation of multiple diverse humanized variants (see, for example, Lazar et al., Mol. Immunol. 44:1986-1998, 2007).

[0178] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries, as well as by bacterial 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).

[0179] In the FR library approach, a collection of residue variants is introduced into a specific position of the FR, and then the library is selected to select the FR that best supports the grafted CDR. The substituted residues can include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, for example, Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or those from a more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

[0180] Instead of generating a combinatorial library of selected residue variants, FR shuffling combines entire FRs with non-human CDRs (see, e.g., Dall'Acqua et al., Methods 36:43-60, 2005). L , followed by V HAntibodies can be screened for binding in a two-step selection process that humanizes the target antibody. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than two-step screening, as the resulting antibodies have shown improved biochemical and physicochemical properties, such as enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60, 2007).

[0181] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and the evaluation of sequential substitution and binding of non-human fragments to a library of human FRs. H and V L Starting from the CDR3 region of the V H and V L Other regions of the non-human antibody, including both CDR1 and CDR2, are gradually replaced with human FRs. This methodology typically identifies multiple subclasses of antibodies that retain the epitope but differ in the human V-segment CDRs. Humaneering allows for 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).

[0182] The "human engineering" method involves modifying non-human antibodies or antibody fragments, such as murine or chimeric antibodies or antibody fragments, by introducing specific changes into the antibody's amino acid sequence to produce modified antibodies that have reduced immunogenicity in humans but nonetheless retain the desired binding characteristics of the original non-human antibody. Generally, this technique involves classifying amino acid residues in non-human (e.g., murine) antibodies as "low risk," "medium risk," or "high risk" residues. Classification is performed using an overall risk / reward calculation that evaluates the predicted benefit of making a particular substitution (e.g., immunogenicity in humans) against the risk that the substitution will affect the folding of the resulting antibody and / or substitutions using human residues. Specific human amino acid residues to be substituted at a given position (e.g., low or medium risk) in a non-human (e.g., murine) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific human antibody sequence or a consensus human antibody sequence. Amino acid residues at low ("Low") and / or moderate ("Mod") risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for producing engineered human proteins are described in further detail in Studnicka et al., Protein Engineering 7:805-814 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication No. WO 93 / 11794.

[0183] 4.4. Antibody-Encoding Nucleic Acids and Vectors The present disclosure provides nucleic acids encoding the anti-α5 integrin antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the nucleic acids encode the anti-α5 integrin V antibodies or antigen-binding fragments thereof disclosed herein. HIn certain embodiments, the first polynucleotide comprises an anti-α5 integrin V polypeptide (e.g., as disclosed in Section 4.3.1, e.g., Tables 2 and 3) encoding an anti-α5 integrin V polypeptide comprising the amino acid sequence set forth in SEQ ID NO:31. H In certain embodiments, the first polynucleotide encodes an anti-α5 integrin V comprising the amino acid sequence set forth in SEQ ID NO: 32. H In certain embodiments, the first polynucleotide encodes an anti-α5 integrin V comprising CDR1, CDR2, and CDR3 disclosed in Tables 2 and 3. H Code the following.

[0184] In certain embodiments, the nucleic acid is an anti-α5 integrin V antibody disclosed herein. L In certain embodiments, the second polynucleotide comprises an anti-α5 integrin V polypeptide (e.g., as disclosed in Section 4.3.1, e.g., Tables 4 and 5) encoding an anti-α5 integrin V polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33. L In certain embodiments, the second polynucleotide encodes an anti-α5 integrin V comprising the amino acid sequence set forth in SEQ ID NO: 34. L In certain embodiments, the second polynucleotide encodes an anti-α5 integrin V comprising CDR1, CDR2, and CDR3 disclosed in Table 4. L Code the following.

[0185] Also provided are vectors comprising the nucleic acids disclosed herein. In certain embodiments, the vector is an expression vector.

[0186] In certain embodiments, the nucleic acids disclosed herein are operably linked to one or more polynucleotides comprising expression control sequences. Expression control sequences include promoters, enhancers, and operators and are generally selected based on the expression system in which the expression construct (e.g., expression vector) will be utilized. Promoter and enhancer sequences are generally selected for their ability to enhance gene expression, while operator sequences are generally selected for their ability to regulate gene expression. In certain embodiments, the expression vector further comprises sequences encoding one or more selectable markers that allow identification of host cells harboring the construct. In certain embodiments, the expression vector further comprises sequences that facilitate, and preferably promote, homologous recombination within the host cell. In certain embodiments, the expression vector further comprises sequences necessary for replication in the host cell.

[0187] Exemplary expression control sequences include promoter / enhancer sequences, such as the cytomegalovirus promoter / enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985), the Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4:151, 1993), the Thai promoter (Korhonen et al., Blood, 86(5):1828-1835, 1995), the simian virus 40 promoter, DRA (downregulated in adenomas; Alrefai et al., Am. J. Physiol. Gastrointest. Liver Physiol., 293:G923-G934, 2007), MCT1 (monocarboxylate transporter 1; Cuff et al. For expression in mammalian cells, a promoter is operably linked upstream (e.g., 5') of the polypeptide coding sequence. In another embodiment, the promoter is an epithelial-specific promoter or an endothelial-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.

[0188] In certain embodiments, the one or more polynucleotides 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 by the cell from the secreted polypeptide. In certain embodiments, the one or more polynucleotides comprise a sequence whose sole function is to facilitate large-scale production of the vector. Polynucleotides for gene therapy can be produced and administered using procedures described in the literature for various transgenes. See, e.g., Isner et al., Circulation, 91:2687-2692, 1995, and Isner et al., Human Gene Therapy, 7:989-1011, 1996.

[0189] Any suitable vector can be used to introduce the nucleic acids disclosed herein into a host cell. Exemplary vectors described include, but are not limited to, replication-deficient retroviral vectors, including 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 viral (AAV) vectors (see, e.g., U.S. Pat. Nos. 5,474,935l; 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. al., J. Invest. Med., 45:87-98, 1997), adenovirus (AV) vectors (see, e.g., U.S. Patent Nos. 5,792,453, 5,824,544, 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. al., Cell, 68:143-155, 1992), adenovirus-adeno-associated virus chimeras (U.S. Pat. No. 5,856,152) or vaccinia virus or herpes virus vectors (U.S. Pat. Nos. 5,879,934, 5,849,571, 5,830,727, 5,661,033, 5,328,688), lipofectin-mediated gene transfer (BRL), liposomal vectors (U.S. Pat. No. 5,631,237), and combinations thereof.All 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 edition, 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 made replication-deficient, for example, by deleting or disrupting a selected gene required for viral replication.

[0190] Other contemplated non-viral delivery mechanisms include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990), DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), and direct microinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979; Felgner, Sci Am., 276(6):102-6, 1997; Felgner, Hum Gene Ther., 7(15):1791-3, 1996), cell sonication (Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene bombardment using high-velocity microprojectiles (Yang et al. al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990), and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993).

[0191] Expression vectors can be encapsulated in liposomes. See, for example, Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104 (1991); Radler et al., Science, 275 (5301): 810-814, 1997). Various commercial approaches involving "lipofection" technology are also contemplated. In certain embodiments, liposomes can be complexed with hemagglutinating virus (HVJ). This has been shown to promote fusion with the cell membrane and facilitate the transfer of liposome-encapsulated DNA into cells (see, for example, Kaneda et al., Science, 243: 375-378, 1989). In certain embodiments, liposomes are complexed or used in combination with nuclear non-histone chromosomal proteins (HMG-1) (see, e.g., Kato et al., J. Biol. Chem., 266:3361-3364, 1991). In certain embodiments, liposomes are complexed or used in combination with both HVJ and HMG-1. Such expression constructs have been effectively used for the transfer and expression of nucleic acids in vitro and in vivo. In certain embodiments, the anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein is included in the liposome to target the liposome to cells expressing α5 integrin on their surface (e.g., tumor cells).

[0192] The present disclosure further provides host cells comprising the vectors disclosed herein. Host cells can be prokaryotic cells, such as Escherichia coli (see, e.g., Pluckthun et al., Methods Enzymol., 178:497-515, 1989), or eukaryotic cells, such as animal cells (e.g., myeloma cells, Chinese hamster ovary (CHO) cells, or hybridoma cells), yeast (e.g., Saccharomyces cerevisiae), or plant cells (e.g., tobacco, corn, soybean, or rice cells). The use of mammalian host cells can provide for translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) desired to confer optimal biological activity to the recombinant expression product. Similarly, a polypeptide (e.g., an anti-α5 integrin antibody or antigen-binding fragment thereof) may be glycosylated or non-glycosylated and / or may be covalently modified to include one or more water-soluble polymer attachments, such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0193] Methods for introducing DNA or RNA into host cells are well known, including transformation, transfection, electroporation, intranuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Such host cells are also useful for amplifying polynucleotides and expressing polypeptides encoded by the polynucleotides. In this regard, a process for producing an anti-α5 integrin antibody (e.g., a humanized antibody) can include culturing host cells and isolating the anti-α5 integrin antibody. Particle bombardment can be used to introduce naked DNA expression constructs into cells, but this method relies on the ability to accelerate DNA-coated microparticles to high speeds to penetrate cell membranes and enter the cells without killing them (see, e.g., Klein et al., Nature, 327:70-73, 1987). Several instruments for accelerating small particles have been developed. One such device utilizes a high-voltage discharge to generate an electric current, which in turn provides the driving force (see, e.g., Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). The microparticles used are composed of biologically inert materials such as tungsten or gold beads. The host cells may be isolated and / or purified. The host cells may also be cells transformed in vitro to cause transient or permanent expression of a polypeptide in vivo. The host cells may also be isolated cells that have been transformed ex vivo and transduced after transformation, e.g., to produce a polypeptide in vivo for therapeutic purposes. The definition of a host cell specifically excludes genetically modified humans.

[0194] Various methods for producing antibodies from polynucleotides are generally known. For example, basic molecular biology procedures are described by Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, 2001). Furthermore, numerous publications describe techniques suitable for manipulating DNA, creating expression vectors, and transforming and culturing appropriate cells to prepare antibodies (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).

[0195] In certain embodiments, anti-α5 integrin antibodies (e.g., humanized antibodies) are produced by any suitable method, such as by isolation from an immunized animal, recombinant or synthetic production, or genetic engineering, including those described above. Antibody fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. For example, digestion of whole antibodies with papain or pepsin produces a 5S fragment designated F(ab')2, or two monovalent Fab fragments and an Fc fragment, respectively. F(ab)2 can be further cleaved using a thiol reducing agent to produce a 3.5S Fab monovalent fragment. Methods for producing antibody fragments are further described, for example, in Edelman et al., Methods in Enzymology, 1:422 Academic Press (1967), Nisonoff et al., Arch. Biochem. Biophys., 89:230-244, 1960, Porter, Biochem. J., 73:119-127, 1959, U.S. Patent 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), pages 2.8.1 2.8.10 and 2.10A.1 2.10A.5.

[0196] Anti-α5 integrin antibodies (e.g., humanized antibodies) can be genetically engineered. For example, anti-α5 integrin antibodies (e.g., humanized antibodies) contain variable regions or domains produced, for example, by recombinant DNA engineering techniques. In this regard, the variable regions are optionally modified by insertion, deletion, or alteration of the amino acid sequence of the antibody to produce the desired antibody, including those described above. Polynucleotides encoding the CDRs of interest, such as those listed in Table 2, are prepared, and the variable regions are synthesized using the polymerase chain reaction, e.g., using mRNA from antibody-producing cells as a template (see, e.g., Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995); and Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106-110, 1991). Current antibody engineering techniques allow for 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 remainder of the variable region domain from a second antibody. Such techniques are used, for example, to humanize an antibody or to increase its affinity for a binding target.

[0197] A "humanized antibody" is an antibody in which the CDRs of the heavy and light variable chains of a non-human immunoglobulin have been transferred into human variable domains. Constant regions need not be present, but if present, they are optionally substantially identical to human immunoglobulin constant regions, e.g., in certain embodiments, at least about 85-90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more identical. Thus, in some cases, all of a humanized immunoglobulin, except possibly the CDRs, is substantially identical to corresponding portions of native human immunoglobulin sequences. For example, a humanized antibody is a human immunoglobulin (e.g., host antibody) in which residues from a hypervariable region of the host antibody are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.

[0198] 4.5.Detection Methods The present disclosure provides methods for detecting α5 integrin in whole cells or whole tissues. In certain embodiments, the methods include: a) contacting cells or tissues with an anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein, where the anti-α5 integrin antibody or antigen-binding fragment thereof comprises a detectable label; and b) determining the amount of labeled anti-α5 integrin antibody or antigen-binding fragment thereof bound to the cells or tissues. In certain embodiments, the methods are for detecting human α5 integrin.

[0199] In certain embodiments, b) determining the amount of labeled anti-α5 integrin antibody or its antigen-binding fragment bound to the cell or tissue comprises measuring the amount of detectable label associated with the cell or tissue, wherein the amount of bound antibody or its antigen-binding fragment indicates the amount of α5 integrin (e.g., human α5 integrin) in the cell or tissue.

[0200] The cell or tissue can be any cell or tissue, including any normal, healthy, abnormal, tumor, or cancerous cell or tissue.

[0201] The present disclosure also provides methods for detecting or diagnosing α5 integrin-related diseases, disorders, or conditions. A more definitive diagnosis of α5 integrin-related diseases, disorders, or conditions may enable medical professionals to implement preventative measures or proactive treatments early, thereby preventing the onset or further progression of α5 integrin-related diseases, disorders, or conditions. In certain embodiments, the method includes (a) measuring the expression level of α5 integrin in a cell or tissue sample from a subject using an anti-α5 integrin antibody or antigen-binding fragment thereof disclosed herein, and (b) comparing the expression level of α5 integrin measured in (a) with a control expression level of α5 integrin, wherein an elevated expression level of α5 integrin measured in (a) compared to the control expression level of α5 integrin indicates a disease, disorder, or condition associated with α5 integrin. In certain embodiments, the control expression level of α5 integrin is the expression level of α5 integrin in a cell or tissue sample from a subject not suffering from an α5 integrin-related disease, disorder, or condition.

[0202] 4.6. Compositions and Formulations The present disclosure provides a composition comprising an anti-α5 integrin antibody or antigen-binding fragment thereof, conjugate, or multispecific molecule disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0203] The particular carrier used may depend on physicochemical considerations, such as solubility and lack of reactivity with the anti-α5 integrin antibody or its antigen-binding fragment or combination therapy, as well as the route of administration. Pharmaceutically acceptable carriers are well known in the art, and examples are described herein. Exemplary pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable aqueous solutions or dispersions. Injectable formulations are further described, for example, in "Pharmaceutics and Pharmacy Practice," J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and "ASHP ​​Handbook on Injectable Drugs," Toissel, 4th ed., pages 622-630 (1986)." In certain embodiments, the pharmaceutical compositions described herein are placed in a container with packaging material providing instructions for use of such pharmaceutical compositions. Generally, such instructions include specific wording describing the concentrations of the reagents and, in certain embodiments, may also include the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) needed to reconstitute the pharmaceutical composition.

[0204] In certain embodiments, the composition comprises a non-antibody protein scaffold. Examples of non-antibody protein scaffolds include, but are not limited to, fibronectin scaffolds, anticalins, adnectins, affibodies, DARPins, finomers, affitins, affilins, avimers, cysteine-rich knottin peptides, or engineered Kunitz-type inhibitors. Methods for producing such non-antibody protein scaffolds are well known in the art, and any of them can be used to produce anti-α5 integrin antibodies comprising non-antibody protein scaffolds (see, for example, Simeon and Chen, Protein Cell, 9(1):3-14(2018); Yang et al., Annu Rev Anal Chem (Palo Alto Calif). 10(1):293-320(2017)).

[0205] 4.7.Treatment Methods Anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, and compositions can be used to treat α5 integrin-related diseases, disorders, or conditions, such as one or more symptoms of the disease, disorder, or condition. Accordingly, the present disclosure provides methods for treating α5 integrin-related diseases, disorders, or conditions. In certain embodiments, the methods comprise administering to a subject an anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein. In certain embodiments, the subject is suffering from or has been diagnosed with an α5 integrin-related disease, disorder, or condition. In certain embodiments, the subject is a human.

[0206] In certain embodiments, the disorder, disease, or condition is associated with α5β1. In certain embodiments, an α5β1 integrin-associated disease, disorder, or condition refers to any disease, disorder, or condition that is caused in whole or in part by or results from α5β1 integrin or the interaction of α5β1 integrin with fibronectin, and / or any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of the interaction of α5β1 integrin with fibronectin. In certain embodiments, the disorder, disease, or condition is associated with overexpression of α5β1. Examples of α5 integrin-associated diseases, disorders, and conditions include, but are not limited to, tumors, angiogenesis-associated diseases (e.g., diseases associated with or characterized by aberrant angiogenesis), and inflammatory diseases.

[0207] In certain embodiments, the α5β1 integrin-associated disease, disorder, or condition is characterized by or associated with an abnormal increase in angiogenic activity of cells (e.g., tumor cells). In certain embodiments, the α5 integrin-associated disease, disorder, or condition is a tumor. In certain embodiments, cells of the tumor express or overexpress α5 integrin or α5β1 integrin. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a cancer. In certain embodiments, the tumor or cancer is characterized by or associated with tumor cells or cancer cells that express or overexpress α5 integrin or α5β1 integrin. Examples of cancers include, but are not limited to, breast cancer, bladder cancer, melanoma, prostate cancer, mesothelioma, lung cancer, brain cancer, ovarian cancer, colon cancer, testicular cancer, thyroid cancer, squamous cell carcinoma, glioblastoma, neuroblastoma, neurofibromatosis, uterine cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

[0208] The present disclosure also provides a method for modulating (e.g., inhibiting, reducing, or preventing) tumor growth in a subject suffering from a tumor. For example, the method includes administering to the subject an anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein in an amount effective to modulate tumor growth in the subject. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals typically characterized by unregulated or abnormal cell growth, and include, but are not limited to, all malignant tumors, such as carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Examples of cancer include, but are not limited to, breast cancer (including metastatic breast cancer), cervical cancer, colon cancer, colorectal cancer (including metastatic colorectal cancer), lung cancer (including non-small cell lung cancer), fibrosarcoma, non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia, bladder cancer, pancreatic cancer, renal cell carcinoma, spleen cancer, prostate cancer including hormone-refractory prostate cancer, liver cancer, head and neck cancer, gastric cancer, bladder cancer, melanoma, ovarian cancer, mesothelioma, soft tissue cancer, gastrointestinal stromal tumor, glioblastoma multiforme, and multiple myeloma.

[0209] "Inhibiting" does not require 100% inhibition. Any inhibition that reduces tumor growth and / or metastasis is contemplated. Similarly, "modulating" tumor growth refers to reducing the size of a tumor, slowing tumor growth, or inhibiting an increase in the size of an existing tumor. Complete elimination of the tumor is not required; any reduction in tumor size or slowing of tumor growth constitutes a beneficial biological effect in the subject. In this regard, tumor cell elimination can be improved, for example, by at least about 5%, at least about 10%, or at least about 20%, compared to the level of elimination observed in the absence of the method (e.g., a biologically compatible control subject or specimen not exposed to the agent of the method). This effect is detected, for example, by a reduction in tumor size or tumor metastasis, a reduction or maintenance of tumor marker levels, or a reduction or maintenance of tumor cell population. In certain embodiments, tumor cell elimination is improved, e.g., by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%), compared to tumor cell elimination in the absence of the anti-α5 integrin combination, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition disclosed herein.

[0210] In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is associated with abnormal angiogenesis. In certain embodiments, the alpha5 integrin-associated disease, disorder, or condition is an ocular disease. In certain embodiments, the ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and uveitis.

[0211] The present disclosure also provides a method for inhibiting abnormal angiogenesis in a subject. In certain embodiments, the subject is afflicted with a tumor. In certain embodiments, the method comprises administering to the subject an anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition described herein in an amount effective to inhibit abnormal angiogenesis.

[0212] In certain embodiments, the inflammatory disease is a neuroinflammatory disease. Examples of inflammatory diseases include, but are not limited to, macrophage-mediated innate immune disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and atherosclerosis.

[0213] The particular dosing regimen for a particular subject will depend, in part, on the agent used, the amount of agent administered, the route of administration, and the cause and extent of any side effects. The amount of anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition administered to a subject (e.g., a mammal such as a human) should be sufficient to affect the desired response over a reasonable time frame. Thus, in certain embodiments, the amount of anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition described herein administered to a subject is an effective amount. In certain embodiments, the effective amount is a therapeutically effective amount.

[0214] Suitable routes of administration of the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein are well known in the art. Depending on the situation, the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein can be applied or instilled into a body cavity, absorbed through the skin or mucous membranes, ingested, inhaled, and / or introduced into the circulatory system. In certain embodiments, the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein are administered via injection by intravenous, subcutaneous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intra-arterial, intraportal, intralesional, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, ​​vaginal, or rectal means, sustained release systems, or implantable devices. In certain embodiments, the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein are administered regionally via intra-arterial or intravenous administration supplying the region of interest, e.g., via the hepatic artery for delivery to the liver. In certain embodiments, the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein are administered locally via implantation of a membrane, sponge, or another suitable material into which the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions are absorbed or encapsulated. When an implantable device is used, in one embodiment, the device is implanted into any suitable tissue or organ to deliver the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein, for example, via diffusion, sustained bolus release, or continuous administration. In certain embodiments, the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein are administered directly to tissue exposed during tumor resection or other surgical procedures.

[0215] In certain embodiments, the method further comprises administering to the subject one or more additional agents. The one or more additional agents can be administered together (e.g., in the case of combination therapy) or separately (e.g., simultaneously, one after the other, sequentially) with the anti-α5 integrin antibody, antigen-binding fragment thereof, multispecific molecule, conjugate, or composition. In certain embodiments, the one or more additional agents comprise a therapeutic agent. Examples of therapeutic agents include, but are not limited to, therapeutic antibodies, immunotherapeutic agents, cytotoxic agents, chemotherapeutic agents, and inhibitors.

[0216] Therapeutic antibodies that can be used in conjunction with the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions described herein (e.g., in 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 Examples of antibodies that bind to type I interferons (IFNs) include antibodies that bind to CSF-1R (e.g., MCS110), CSF-1R (e.g., AMG820), C1Q (ANX005), CD40L (e.g., ruplizumab), FGFR (e.g., bemarituzumab), IL-1β (e.g., canakinumab, gevokizumab), IL-6 (e.g., tocilizumab), IL-12 (e.g., ustekinumab), and antibodies that bind to type I interferons (IFNs) (e.g., sifalimumab).

[0217] Immunotherapeutic and immunotherapeutic agents that can be used in conjunction with the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions described herein (e.g., in combination therapy) include, but are not limited to, cytokines, interleukins, tumor necrosis factors, and combinations thereof. In certain embodiments, the immunotherapy includes an immunotherapeutic agent that modulates the immune response, such as a checkpoint inhibitor or checkpoint agonist. In certain embodiments, the immunotherapeutic agent is an antibody modulator, including those known in the art, targeting PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFbeta, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, among others. In certain embodiments, the immunotherapeutic agent is an agent that enhances natural killer (NK) cell activity. In certain embodiments, the immunotherapeutic agent is an agent that inhibits suppression of an immune response. In certain embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or the activity of suppressor cells. In certain embodiments, the immunotherapeutic agent is an agent or therapy that inhibits Treg activity. In certain embodiments, the immunotherapeutic agent is an agent that inhibits the activity of an inhibitory immune checkpoint receptor.

[0218] In certain embodiments, the immunotherapeutic agent comprises a T cell modulator selected from an agonist or activator of a costimulatory molecule. In one embodiment, the costimulatory molecule agonist is selected from an agonist (e.g., an agonist antibody or antigen-binding fragment thereof, or a soluble fusion) of GITR, OX40, ICOS, SLAM (e.g., 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 CD83 ligand. In other embodiments, the effector cell combination includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds CD3) and a tumor antigen (e.g., EGFR, PSCA, PSMA, EpCAM, HER2, among others).

[0219] Cytotoxic agents that can be used in conjunction with the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein (e.g., for combination therapy) include substances that inhibit or prevent cell function and / or cause cell death or perturbation. 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.

[0220] Chemotherapeutic agents that can be used with the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions 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® cyclosphosphamide; Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; Aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; Ethylenimines and methylamelamines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; Acetogenins (specifically, bullatacin and bullatacinone), camptothecin (including synthetic analogue topotecan), bryostatin, Kallistatin, CC-1065 (including its adozelesin, carzelesin, and beizelesin synthetic analogs); Cryptophycins (specifically, cryptophycin 1 and cryptophycin 8), Dolastatin, duocarmycins (such as synthetic analogs KW-2189 and CB1-TM1); Eleutherobin, Pancratistatin, Sarcodictiin, spongistatin, Nitrogen mustards, for example, chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, fenesterine, prednimustine, trophosfamide, uracil mustard, Nitrosoureas such as Camrustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, specifically calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); Dynemisin, including Dynemisin A, bisphosphonates, such as clodronate; Espera Machine, and Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (morpholino-doxorubicin, cyanomono (including pyrrolino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calisthenol, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid, Aseglaton, Aldophosphamide glycosides, aminolevulinic acid, Eniluracil, amsacrine, Best Love Sil, Bisantren, edatraxate, Defofamine, Demecolcine, Diazicon, erformitin, elliptinium acetate, Epothilones, Etoglucide, gallium nitrate, hydroxyurea, Lentinan, lonidynin, maytansinoids, such as maytansine and ansamitocins; Mitoguazone, Mitoxantrone, Mopidanmol, Nitraeline, Pentostatin, Fenamet pirarubicin, losoxantrone, Podophyllic acid, 2-ethylhydrazide, procarbazine, PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.), Razoxane, Rhizoxin, Schizophyllan, Spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, Trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine), urethane, Vindesine, dacarbazine, Mannomustine, mitobronitol, Mitolactol, Pipobroman, Gashitosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, Taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil, GEMZAR® gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogues such as cisplatin, oxaliplatin, and carboplatin; vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, Novantron, teniposide, edatrexate, daunomycin, aminopterin, Xeroda, ibandronate, irinotecan (Camptosar, CPT-11) (which includes a treatment regimen of irinotecan with 5-FU and leucovorin), topoisomerase inhibitor RFS2000, Difluoromethylornithine (DMFO), Retinoids such as retinoic acid Capecitabine, Combretastatin, Leucovorin (LV), oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); Inhibitors of PKC-alpha, Raf, H-Ras, and EGFR (e.g., erlotinib (Tarceva™)), which reduce cell proliferation; and Pharmaceutically acceptable salts, acids, or derivatives of any of the above are also included. Also included are antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogens or selective estrogen receptor modulators (SERMs), including tamoxifen (such as NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON / toremifene. Aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, holmestein, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole, and Antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and troxacitabine (1,3-dioxolane nucleoside cytosine analogue), antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozymes) and HER2 expression inhibitors; vaccines, e.g., gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® 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.

[0221] Non-limiting examples of inhibitors that can be used in conjunction with the anti-α5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions described herein (e.g., for combination therapy) include: FAK inhibitors (e.g., GSK2256098), MEK inhibitors (e.g., cobimetinib, lametinib, 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 and kinase inhibitors such as FGFR inhibitors (eg, erdafitinib).

[0222] In certain embodiments, the anti-alpha5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein may be used in combination with a PD-1 or PD-L1 inhibitor, such as, for example, an anti-PD-1 or anti-PD-L1 monoclonal antibody, such as nivolumab (Opdivo), pembrolizumab (Keytruda, MK-3475), atezolizumab, or avelumab.

[0223] In certain embodiments, the anti-alpha5 integrin antibodies, antigen-binding fragments thereof, multispecific molecules, conjugates, or compositions disclosed herein can be used in combination with a CTLA-4 inhibitor, e.g., an anti-CTLA-4 antibody, e.g., ipilimumab (Yavoy), or in combination with an antibody against a cytokine, or in combination with a bispecific antibody that binds PD-L1 and CTLA-4 or PD-1 and CTLA-4, or in combination with other anti-cancer agents.

[0224] 4.8.Kit The present disclosure provides kits comprising an anti-α5 integrin antibody, or antigen-binding fragment thereof, conjugate, or multispecific molecule disclosed herein in unit dosage form. In certain embodiments, the kit includes a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components.

[0225] In certain embodiments, the kit includes a sterile container housing the therapeutic or prophylactic vaccine, which may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container format known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In certain embodiments, the kit further includes instructions for administering the anti-α5 integrin antibody, or antigen-binding fragment thereof, conjugate, or multispecific molecule disclosed herein to a subject. The instructions may generally include information on using the anti-α5 integrin antibody, or antigen-binding fragment thereof, conjugate, or multispecific molecule disclosed herein to treat an α5-related disease, disorder, or condition. In certain embodiments, the instructions include at least one of a description of the therapeutic agent, dosing schedule and administration for treating the α5-related disease, disorder, or condition, precautions, warnings, indications, contraindications, overdose information, side effects, animal pharmacology, clinical studies, and / or references. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container.

[0226] It is understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Accordingly, the following examples are intended to illustrate the present invention, but not to limit the disclosure. [Example]

[0227] 5. Working Example The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use antibodies, bispecific antibodies, compositions comprising same, and methods of screening and treatment of the disclosed subject matter, and are not intended to limit the scope of what the inventors regard as their disclosure. It will be understood that various other embodiments may be practiced in light of the general description provided above.

[0228] Example 1: Expression of a humanized antibody derived from the A2-M5 IgG4 chimera Antibody expression plasmids were constructed encoding humanized heavy and light chain sequences with low-risk (Low) and low-moderate (Low+Mod) risk amino acid changes. Sequence accuracy was verified by Sanger sequencing, and plasmid concentrations were determined by measuring absorbance at 260 nm. The expression plasmids were V H Low and V L Low+Mod combination, V H Low+Mod and V L Low combination and V H Low+Mod and V L Heavy and light chain combinations, such as the Low+Mod combination, were combined to express the antibody. Expression clones were combined as described, transfected into suspension-adapted CHO K1 cells, and cultured in animal-component-free, serum-free medium. Supernatants were collected by centrifugation and then filtered (0.2 μm filter). Antibodies were purified using MabSelect™ SuRe™ (Cytiva, Marlborough, MA). Purity was measured by analytical size-exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8 × 300 mm, Agilent Technologies, Inc., Santa Clara, CA) at 0.8 mL / min with PBS as the running buffer. The yield from 250 mL of culture medium was measured using absorbance at 280 nm for the humanized antibodies A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L) of the present disclosure.

[0229] Antibody expression plasmids encoding humanized heavy and light chain sequences with low- and low-risk amino acid changes are constructed. Sequence accuracy is verified by Sanger sequencing, and plasmid concentration is determined by measuring absorbance at 260 nm. The expression plasmids are V H Low and V L The antibody is combined to express a combination of humanized antibodies A2-M5-Low(H) / Low(L). The expression clones are combined as described, transfected into suspension-adapted CHO K1 cells, and cultured in serum-free, animal-component-free medium. The supernatant is collected by centrifugation and then filtered (0.2 μm filter). The antibody is purified using MabSelect™ SuRe™ (Cytiva, Marlborough, MA). Purity is measured by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 μm 7.8 × 300 mm, Agilent Technologies, Inc., Santa Clara, CA) at 0.8 mL / min with PBS as the running buffer. Yields from 250 mL cultures are measured using absorbance at 280 nm for the humanized antibody A2-M5-Low(H) / Low(L) of the present disclosure.

[0230] Example 2: Binding affinity of humanized A2-M5 antibody The binding affinity of the humanized antibodies A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L) disclosed herein was measured. Surface plasmon resonance (SPR) was used to measure the binding affinity of the humanized antibodies A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L) disclosed herein to human recombinant rh-α5β1 protein using standard protocols. Briefly, rh-α5β1 protein in solution is manually applied to a gold-coated sensor chip. After incubation, the chip is washed, blocked, washed again, and dried before use. SPR measurements are performed using a high-throughput surface plasmon resonance imaging (SPRi) platform, the PlexArray HT (Plexera Bioscience, Seattle, WA). Collimated light (660 nm) is passed through a coupling prism, reflected off the SPR-active gold surface, and received by a CCD camera. Buffer and sample are injected into a flow cell attached to the coupling prism by a non-pulsatile piston pump. Each SPR measurement cycle includes the following steps: washing with running buffer at a constant rate to obtain a stable baseline, injecting the humanized antibody to bind to rh-α5β1 and reach equilibrium, and then injecting only running buffer to dissociate the humanized antibody. All measurements are performed at 25°C. The SPR binding response (a.u.) is recorded and plotted over time.

[0231] Example 3: Inhibition of fibronectin binding to α5β1 by humanized antibodies The humanized antibodies A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L) disclosed herein were tested in a quantitative FN inhibition assay in an ELISA format. Immulon4 HBX ELISA 96-well plates were coated with FN by incubating overnight at 4°C with 2.5 μg / mL human FN (R&D Systems, Minneapolis, MN 55413, catalog number 1918-FN) in 1×PBS (0.01 M phosphate buffer and 0.154 M NaCl, pH 7.4). Plates were then washed three times with wash buffer (1x Tris-buffered saline containing 0.05% Tween 20) and blocked with 2% BSA in 1x TBS for 2 hours at room temperature (RT). Antibodies were diluted in standard diluent (2% BSA, 1x TBS, 0.05% Tween 20) containing 0.1 μg / mL rh-α5β1-6xHis-tagged protein (Acro Biosystems, Newark, DE, catalog number 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 number PP102) was used to normalize data obtained between different assay runs. For the assay, after removing the blocking solution, the wells are washed three times and 100 uL of the antibody dilution series / His-tagged α5β1 mixture is added to the wells.After 1 hour at RT, wells were washed three times and incubated with biotinylated anti-6xHis tagged antibody (Invitrogen, Carlsbad, CA, Catalog No. MAI-21315-BTIN) at a standard dilution of 1:1000 for 1 hour. Washed three times, incubated with poly-HRP streptavidin (Thermo Fisher Scientific, Waltham, MA, Catalog No. N200) for 30 minutes, washed four times, and incubated with TMB substrate (Thermo Fisher Scientific, Waltham, MA, Catalog No. N301) for 2–5 minutes, followed by the addition of ELISA stop solution (Invitrogen, Carlsbad, CA, Catalog No. SS04). Absorbance at 450 nm was measured. Data points were normalized to the isotype control Ab value for each concentration and reported as % absorbance at 450 nm normalized to the control Ab. Curves (four parameters) are fitted to the data by nonlinear regression analysis using GraphPad Prism version 9.0.2 (GraphPad Software, LLC, San Diego, CA).

[0232] Example 4: Prospects for the development of humanized A2-M5 antibody The humanized antibodies A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L) disclosed herein are evaluated and measured for development prospects (including specificity, integrity, stability, and accelerated stress). These evaluations include mass analysis by mass spectrometry (both reduction and deglycosylation), post-translational modification evaluation by mass spectrometry, aggregation analysis by size-exclusion ultra-performance chromatography (SE-UPLC), purity analysis by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), charge variant analysis by capillary isoelectric focusing (cIEF), and thermal stability analysis. The accelerated stress test involves three cycles of freeze / thawing, followed by aggregation analysis by SE-UPLC and purity analysis by CE-SDS.

[0233] Example 5: Immunogenicity evaluation of humanized A2-M5 antibody The ProStorm® cytokine release assay, designed to aid in the prediction of first-infusion-related reactions, is used to evaluate the immunogenicity of the humanized antibodies disclosed herein: A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L). This in vitro cytokine release assay is performed on fresh human blood samples obtained from a panel of healthy donors. An appropriate amount of blood was placed in heparin Vacutainers®. Heparinized blood samples are used fresh (<3 hours after collection) in the ProStorm® assay. Each humanized antibody is analyzed at four concentrations: 0.1, 1, 10, and 100 μg / mL in undiluted whole blood, along with positive and negative control conditions. After incubation, plasma is separated and immunoassays are used to measure repeated doses of the following cytokines in pg / mL: IFNγ, TNFα, IL-2, IL-4, IL-6, IL-8, and IL-10.

[0234] Additionally, the ProScern® dendritic cell-T cell (DC-T) assay is performed against the humanized antibodies A2-M5-Low(H) / Low(L), A2-M5-Low(H) / Low+Mod(L), A2-M5-Low+Mod(H) / Low(L), and A2-M5-Low+Mod(H) / Low+Mod(L). The ProImmune Tissue Bank identifies a panel of 20 different healthy donor PBMC samples to reflect global HLA distribution. PBMC preparations are typed for HLA class II and stored in liquid nitrogen until use. Donor PBMC cultures are used to induce monocytes, which are then induced to a semi-mature DC phenotype by culturing in a defined medium. After a period of culture, dendritic cells (DCs) were incubated with humanized antibodies in multiwell plates containing untreated control wells and wells containing the positive control antigens KLH and tuberculin PPD. Treated DCs were cultured in defined media to further induce maturation. These antigen-loaded DCs were harvested and re-plated for coculture with PBMCs from the same donor sample. These DC-T cell cultures were maintained in multiwell plates for further in vitro culture, and T cell proliferation was determined using a CFSE staining assay. The percentage of stimulation above background for each sample combination was determined by subtracting the percentage of CD4+ CFSE dim cells without antigen stimulation from the percentage of CD4+ CFSE dim cells with antigen stimulation. Statistical analysis (one-way ANOVA) was applied to the entire data set.

[0235] Embodiments of the presently disclosed subject matter From the above description, it will be apparent that variations and modifications of the subject matter disclosed herein can be made to adapt it to various uses and conditions, and such embodiments also fall within the scope of the following claims.

[0236] The recitation of listed elements in any definition of a variable herein includes that definition of the variable as any single element or combination (or subcombination) of elements among the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0237] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. An anti-α5 integrin antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

31.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

31.

3. An anti-α5 integrin antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

32.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

32.

5. An anti-α5 integrin antibody, or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

33.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

33.

7. An anti-α5 integrin antibody, or an antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

34.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

34.

9. (a) a heavy chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:31 or SEQ ID NO:32; and (b) an anti-α5 integrin antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO:

34.

10. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32; (b) The antibody or antigen-binding fragment thereof according to claim 9, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO:

34.

11. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:31; (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

33.

12. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31; (b) The antibody or antigen-binding fragment thereof according to claim 11, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

33.

13. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:31; (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

34.

14. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31; (b) The antibody or antigen-binding fragment thereof according to claim 13, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

34.

15. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 32; (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

33.

16. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32; (b) The antibody or antigen-binding fragment thereof according to claim 15, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

33.

17. (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 32; (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

34.

18. (a) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32; (b) The antibody or antigen-binding fragment thereof according to claim 17, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

34.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody is a monoclonal antibody.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antibody is a humanized antibody.

21. The antigen-binding fragment may be Fab, Fab', F(ab') ２ 21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, which is a fragment variable (Fv), or a single-chain fragment variable (scFv).

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, which binds to α5β1 integrin.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, which inhibits binding between α5β1 integrin and fibronectin.

24. An antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 for binding to α5 integrin.

25. An antibody or antigen-binding fragment thereof that binds to essentially the same epitope region as the antibody or antigen-binding fragment thereof according to any one of claims 1 to 21 with respect to binding to α5 integrin.

26. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25.

27. 27. The composition of claim 26, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

28. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25.

29. 29. The conjugate of claim 28, wherein the antibody or antigen-binding fragment thereof is linked to a therapeutic, detectable, or diagnostic agent.

30. 30. The conjugate of claim 28 or 29, which is an immunoconjugate.

31. 30. The conjugate of claim 29, wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxin, or a drug.

32. A composition comprising the conjugate of any one of claims 28 to 31.

33. 33. The composition of claim 32, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

34. A multispecific molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 25 linked to a second functional moiety.

35. 35. The multispecific molecule of claim 34, wherein the second functional moiety has a different binding specificity than the antibody or antigen-binding fragment thereof.

36. 36. A composition comprising the multispecific molecule of claim 34 or 35.

37. 37. The composition of claim 36, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

38. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25.

39. V comprising the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 32 Ｈ and / or a V comprising the amino acid sequence set forth in SEQ ID NO:33 or SEQ ID NO:

34. Ｌ 39. The nucleic acid of claim 38, comprising a second polynucleotide encoding

40. The first polynucleotide and the second polynucleotide are (a) a V comprising the amino acid sequence set forth in SEQ ID NO: 31 Ｈ and a V comprising the amino acid sequence set forth in SEQ ID NO:

33. Ｌ a second polynucleotide encoding (b) a V comprising the amino acid sequence set forth in SEQ ID NO: 31; Ｈ and a V comprising the amino acid sequence set forth in SEQ ID NO:

34. Ｌ a second polynucleotide encoding (c) a V comprising the amino acid sequence set forth in SEQ ID NO: 32 Ｈ and a V comprising the amino acid sequence set forth in SEQ ID NO:

33. Ｌ a second polynucleotide encoding (d) a V comprising the amino acid sequence set forth in SEQ ID NO: 32 Ｈ and a V comprising the amino acid sequence set forth in SEQ ID NO:

34. Ｌ and a second polynucleotide encoding the nucleic acid of claim 39.

41. A vector comprising the nucleic acid of claim 40.

42. 42. The vector of claim 41, which is an expression vector.

43. A host cell comprising the vector of claim 41 or 42.

44. 44. A method for producing an anti-alpha5 integrin antibody or antigen-binding fragment thereof, comprising culturing a host cell described in claim 43 under conditions that induce expression of the antibody or antigen-binding fragment thereof from the host cell.

45. 26. A method for detecting alpha5 integrin in whole cells or tissue, comprising contacting a cell or tissue with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof comprises a detectable label, and measuring the amount of detectable label associated with the cell or tissue to determine the amount of the labeled antibody or antigen-binding fragment thereof bound to the cell or tissue, wherein the amount of bound antibody or antigen-binding fragment thereof indicates the amount of alpha5 integrin in the cell or tissue.

46. 37. A method of treating an alpha5 integrin-associated disease, disorder, or condition in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof of any one of claims 1 to 25, a multispecific molecule of claim 34 or 35, a conjugate of any one of claims 28 to 31, or a composition of any one of claims 26, 27, 32, 33, 36, and 37.

47. 47. The method of claim 46, wherein the alpha5 integrin-associated disease, disorder or condition is associated with alpha5beta1.

48. 48. The method of claim 46 or 47, wherein the alpha5 integrin-associated disease, disorder or condition is a tumor.

49. 49. The method of claim 48, wherein the tumor is a solid tumor.

50. 50. The method of claim 48 or 49, wherein the tumor is a cancer.

51. 51. The method of claim 50, wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, melanoma, prostate cancer, mesothelioma, lung cancer, brain cancer, ovarian cancer, colon cancer, testicular cancer, thyroid cancer, squamous cell carcinoma, glioblastoma, neuroblastoma, neurofibromatosis, uterine cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

52. 48. The method of claim 46 or 47, wherein the alpha5 integrin-associated disease, disorder or condition is associated with abnormal angiogenesis.

53. 53. The method of claim 52, wherein the alpha5 integrin-associated disease, disorder, or condition is an ocular disease.

54. 54. The method of claim 53, wherein the eye disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and uveitis.

55. 48. The method of claim 46 or 47, wherein the alpha5 integrin-associated disease, disorder or condition is an inflammatory disease, disorder or condition.

56. 56. The method of claim 55, wherein the inflammatory disease, disorder, or condition is a neuroinflammatory disease, disorder, or condition.

57. 56. The method of claim 55, wherein the inflammatory disease, disorder, or condition is selected from the group consisting of macrophage-mediated innate immune disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and atherosclerosis.

58. 37. A method of inhibiting abnormal angiogenesis in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, a multispecific molecule according to claim 34 or 35, a conjugate according to any one of claims 28 to 31, or a composition according to any one of claims 26, 27, 32, 33, 36 and 37.

59. 59. The method of claim 58, wherein the subject is afflicted with a tumor.

60. 60. The method of any one of claims 46 to 59, wherein the subject is a human.

61. 38. The antibody or antigen-binding fragment thereof of any one of claims 1 to 25, the multispecific molecule of claim 34 or 35, the conjugate of any one of claims 28 to 31, or the composition of any one of claims 26, 27, 32, 33, 36, and 37 for use in treating an alpha5 integrin-related disease, disorder, or condition in a subject, or in inhibiting abnormal angiogenesis in a subject.

62. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 61, wherein the α5 integrin-associated disease, disorder, or condition is associated with α5β1.

63. 63. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 61 or 62, wherein the alpha5 integrin-related disease, disorder, or condition is a tumor.

64. 64. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 63, wherein the tumor is a solid tumor.

65. 65. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 63 or 64, wherein the tumor is cancer.

66. 66. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 65, wherein the cancer is selected from the group consisting of breast cancer, bladder cancer, melanoma, prostate cancer, mesothelioma, lung cancer, brain cancer, ovarian cancer, colon cancer, testicular cancer, thyroid cancer, squamous cell carcinoma, glioblastoma, neuroblastoma, neurofibromatosis, uterine cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

67. 63. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 61 or 62, wherein the alpha5 integrin-associated disease, disorder, or condition is associated with abnormal angiogenesis.

68. 68. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 67, wherein the alpha5 integrin-associated disease, disorder, or condition is an ocular disease.

69. 69. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 68, wherein the ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, and uveitis.

70. 63. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 61 or 62, wherein the alpha5 integrin-related disease, disorder, or condition is an inflammatory disease, disorder, or condition.

71. 71. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 70, wherein the inflammatory disease, disorder, or condition is a neuroinflammatory disease, disorder, or condition.

72. 71. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to claim 70, wherein the inflammatory disease, disorder, or condition is selected from the group consisting of macrophage-mediated innate immune disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and atherosclerosis.

73. 73. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to any one of claims 61 to 72, wherein the subject is suffering from a tumor.

74. 74. The antibody or antigen-binding fragment thereof, multispecific molecule, conjugate, or composition for use according to any one of claims 61 to 73, wherein the subject is a human.

Images