Compositions and methods containing integrin alpha-3beta-1

Abstract

A composition and method comprising an antibody that binds to integrin α3β1 are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 420,964, filed on 31 October 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Integrin α3β1 is a crucial integrin on the surface of cells such as podocytes, which are cells in Bowman's capsule of the kidney that surround the glomerular capillaries. Integrin α3β1 is essential for podocytes to adhere to the outside of blood vessels in order to form healthy glomeruli in the kidney. Allosteric agonist antibodies against integrin α3β1 can enhance integrin-dependent ligand binding and cell adhesion. [Overview of the project]

[0003] In one embodiment, the present disclosure relates to an isolated antibody that binds to integrin α3β1 or a portion thereof, (1) A heavy chain complementarity determination region 1 (CDR H1) containing the sequence X1X2SGX3TFX4X5YX6X7X8 (Sequence ID 38), wherein X1 is A or K, X2 is A or T, X3 is F, G, or F, X4 is S or T, X5 is S or N, X6 is G, S, or A, X7 is M or I, and X8 is H, N, or S; CDR H1; (2) CDR H2 having a sequence that includes up to two amino acid substitutions to the sequence GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), or the sequence WISAX1NGNX2N (SEQ ID NO: 39), where X1 is Y or N and X2 is T or S; (3) CDR H3 containing a sequence having up to two amino acid substitutions to the sequence of VRDDIQLRD (SEQ ID NO: 11) or AREFPGWYFDY (SEQ ID NO: 13), or a sequence having up to four amino acid substitutions to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), or ARGVPSGSGYYLGLDY (SEQ ID NO: 15); (4) A light chain complementarity determination region 1 (CDR L1) comprising the sequence X1ASQX2ISX3YLN (Sequence ID 40), wherein X1 is Q or A, X2 is D or Y, and X3 is N or S, or a sequence having up to three amino acid substitutions relative to the sequence QGDSLRSYYAS (Sequence ID 23) or SGSSSNIGSNYVY (Sequence ID 24); (5) CDR L2 having a sequence that includes at most one amino acid substitution to the sequence of YDASNLET (SEQ ID NO: 25) or the sequence of YX1X2NX3RPS (SEQ ID NO: 41), wherein X1 is G or R, X2 is K or N, and X3 is N or Q; and (6) CDR L3 having the sequence X1QX2YX3X4PX5T (SEQ ID NO: 42), where X1 is L or Q, X2 is D or S, X3 is N, S or R, X4 is Y or T, and X5 is L or P, or a sequence having up to two amino acid substitutions to the sequence NSRDSSGNHWV (SEQ ID NO: 31) or AAWDDSLSGPV (SEQ ID NO: 32), It is characterized by isolated antibodies containing [specific component].

[0004] In some embodiments of this aspect, (1) CDR H1 comprises any one of the sequences AASGFTFSSYGMH (SEQ ID NO: 1), KASGYTFTSYGIS (SEQ ID NO: 2), KTSGFTFTNYGIS (SEQ ID NO: 3), AASGFTFSSYSMN (SEQ ID NO: 4), and KASGGTFSSYAIN (SEQ ID NO: 5); (2) CDR H2 comprises any one of the sequences GISGSADTTY (SEQ ID NO: 6), WISAYNGNTN (SEQ ID NO: 7), WISANNGNSN (SEQ ID NO: 8), SISSSSSYIY (SEQ ID NO: 9), and GIIPIFGTAN (SEQ ID NO: 10); (3) CDR H3 comprises any one of the sequences VRDDIQLRD (SEQ ID NO: 11), ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREFPGWYFDY (SEQ ID NO: 13), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), and ARGVPSGSGYYLGLDY (SEQ ID NO: 15); (4) CDR L1 comprises any one of the sequences QASQDISNYLN (SEQ ID NO: 21), RASQYISSYLN (SEQ ID NO: 22), QGDSLRSYYAS (SEQ ID NO: 23), and SGSSSNIGSNYVY (SEQ ID NO: 24); (5) CDR L2 comprises any one of the sequences YDASNLET (SEQ ID NO: 25), YGKNNRPS (SEQ ID NO: 26), and YRNNQRPS (SEQ ID NO: 27); (6) CDR L3 comprises any one of the sequences LQDYNYPLT (SEQ ID NO: 28), LQDYSYPLT (SEQ ID NO: 29), QQSYRTPPT (SEQ ID NO: 30), NSRDSSGNHWV (SEQ ID NO: 31), and AAWDDSLSGPV (SEQ ID NO: 32).

[0005] In some embodiments, CDR H1 comprises the sequence of SEQ ID NO: 1, CDR H2 comprises the sequence of SEQ ID NO: 6, and CDR H3 comprises the sequence of SEQ ID NO: 11.

[0006] In some embodiments, CDR H1 comprises the sequence of SEQ ID NO: 2, CDR H2 comprises the sequence of SEQ ID NO: 7, and CDR H3 comprises the sequence of SEQ ID NO: 12.

[0007] In some embodiments, CDR H1 comprises the sequence of SEQ ID NO: 3, CDR H2 comprises the sequence of SEQ ID NO: 8, and CDR H3 comprises the sequence of SEQ ID NO: 13.

[0008] In some embodiments, CDR H1 comprises the sequence of SEQ ID NO: 4, CDR H2 comprises the sequence of SEQ ID NO: 9, and CDR H3 comprises the sequence of SEQ ID NO: 14.

[0009] In some embodiments, CDR H1 comprises the sequence of SEQ ID NO: 5, CDR H2 comprises the sequence of SEQ ID NO: 10, and CDR H3 comprises the sequence of SEQ ID NO: 15.

[0010] In some embodiments, CDR L1 comprises the sequence of SEQ ID NO: 21, CDR L2 comprises the sequence of SEQ ID NO: 25, and CDR L3 comprises the sequence of SEQ ID NO: 28.

[0011] In some embodiments, CDR L1 comprises the sequence of SEQ ID NO: 22, CDR L2 comprises the sequence of SEQ ID NO: 25, and CDR L3 comprises the sequence of SEQ ID NO: 29.

[0012] In some embodiments, CDR L1 comprises the sequence of SEQ ID NO: 21, CDR L2 comprises the sequence of SEQ ID NO: 25, and CDR L3 comprises the sequence of SEQ ID NO: 30.

[0013] In some embodiments, CDR L1 comprises the sequence of SEQ ID NO: 23, CDR L2 comprises the sequence of SEQ ID NO: 26, and CDR L3 comprises the sequence of SEQ ID NO: 31.

[0014] In some embodiments, CDR L1 comprises the sequence of SEQ ID NO: 24, CDR L2 comprises the sequence of SEQ ID NO: 27, and CDR L3 comprises the sequence of SEQ ID NO: 32.

[0015] In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity to any one of the sequences of SEQ ID NOs: 16-20. In some embodiments, the antibody includes a light chain variable region having at least 90% identity to any one of the sequences of SEQ ID NOs: 33-37.

[0016] In some embodiments, the antibody includes HCDR1 having the sequence of SEQ ID NO: 1, HCDR2 having the sequence of SEQ ID NO: 6, HCDR3 having the sequence of SEQ ID NO: 11, LCDR1 having the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 28. In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO: 16. In some embodiments, the antibody includes a light chain variable region having at least 90% identity with the sequence of SEQ ID NO: 33.

[0017] In some embodiments, the antibody includes HCDR1 having the sequence of SEQ ID NO: 2, HCDR2 having the sequence of SEQ ID NO: 7, HCDR3 having the sequence of SEQ ID NO: 12, LCDR1 having the sequence of SEQ ID NO: 22, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 29. In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO: 17. In some embodiments, the antibody includes a light chain variable region having at least 90% identity with the sequence of SEQ ID NO: 34.

[0018] In some embodiments, the antibody includes HCDR1 having the sequence of SEQ ID NO: 3, HCDR2 having the sequence of SEQ ID NO: 8, HCDR3 having the sequence of SEQ ID NO: 13, LCDR1 having the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 30. In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO: 18. In some embodiments, the antibody includes a light chain variable region having at least 90% identity with the sequence of SEQ ID NO: 35.

[0019] In some embodiments, the antibody includes HCDR1 having the sequence of SEQ ID NO: 4, HCDR2 having the sequence of SEQ ID NO: 9, HCDR3 having the sequence of SEQ ID NO: 14, LCDR1 having the sequence of SEQ ID NO: 23, LCDR2 having the sequence of SEQ ID NO: 26, and LCDR3 having the sequence of SEQ ID NO: 31. In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO: 19. In some embodiments, the antibody includes a light chain variable region having at least 90% identity with the sequence of SEQ ID NO: 36.

[0020] In some embodiments, the antibody includes HCDR1 having the sequence of SEQ ID NO: 5, HCDR2 having the sequence of SEQ ID NO: 10, HCDR3 having the sequence of SEQ ID NO: 15, LCDR1 having the sequence of SEQ ID NO: 24, LCDR2 having the sequence of SEQ ID NO: 27, and LCDR3 having the sequence of SEQ ID NO: 32. In some embodiments, the antibody includes a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO: 20. In some embodiments, the antibody includes a light chain variable region having at least 90% identity with the sequence of SEQ ID NO: 37.

[0021] In some embodiments, the antibody comprises an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO: 43.

[0022] In some embodiments of the antibodies described herein, the antibody binds to cells expressing integrin α3β1 or a portion thereof. In certain embodiments, the cells are podocytes, T cells, cancer cells, or neutrophils.

[0023] In some embodiments, the antibody binds to the α3 portion of integrin α3β1. In some embodiments, the antibody binds to a sequence within the thigh-genu region of the α3 portion. In certain embodiments, the antibody binds to the sequence of SEQ ID NO: 44 or a sequence within the sequence of SEQ ID NO: 44. In some embodiments, the antibody binds to a specific three-dimensional structure of α3. In some embodiments, the antibody binds to α3 and stabilizes it to a specific three-dimensional structure.

[0024] In certain embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, or single-chain Fv(scFv) antibody. In some embodiments, the antibody is a bispecific antibody.

[0025] In another aspect, the disclosure also provides isolated nucleic acids encoding the isolated antibodies described herein.

[0026] In another aspect, the disclosure provides an expression vector comprising a nucleic acid encoding an isolated antibody described herein.

[0027] In another aspect, the disclosure provides isolated host cells containing the above-mentioned vector.

[0028] In another aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as described herein and a pharmaceutically acceptable carrier.

[0029] In another embodiment, the Disclosure provides a method for treating a disease or condition associated with podocyte loss in a subject requiring treatment of such disease or condition, comprising administering to the subject an isolated antibody described herein. In some embodiments, the disease or condition is a kidney disease, an autoimmune disease, cancer, or inflammation. In some embodiments, the disease or condition is a transplant surgery.

[0030] In some embodiments of this method, the kidney disease is a glomerular disease, such as nephritis, nephrotic syndrome, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

[0031] In another aspect, the present disclosure relates to a method for identifying an antibody that binds to integrin α3β1 or a portion thereof, 1) Removing an antibody that binds to the β1 chain of integrin α3β1 in the presence or absence of a ligand-mimicking peptide and / or the antibody; 2) Select an antibody from the remaining antibodies in step 1) that binds to integrin α3β1 in the presence or absence of the β1 agonist antibody; 3) Counter-selection of an antibody that binds to integrin α3β1 against an immobilized β1 agonist antibody or ligand-mimicking peptide alone; and 4) Repeat steps 1), 2), and 3) above in the presence of cell surface-expressed integrin α3β1 to enrich the antibody which is an integrin α3 allosteric agonist. The method is characterized by including the following:

[0032] In some embodiments of this method, the ligand-mimicking peptide is LXY2. In some embodiments of this method, steps 1) and / or 3) are performed using β1-containing integrin dimers other than α3β1, such as α4β1 and α5β1. In some embodiments of this method, steps 1) and / or 2) are performed using human K562 cells that primarily express human α5β1 integrin and do not overexpress α3β1.

[0033] In some embodiments of this method, steps 2) and / or 3) are carried out using human K562 cells overexpressing α3β1. In some embodiments, steps 1) and / or 2) and / or 3) are carried out in the presence of an agent that blocks the ligand-binding site or domain of the integrin, such as an antibody and a ligand.

[0034] In some embodiments, integrin α3β1 is stabilized to a specific conformation by pre-complexing with an activator or inhibitor, such as the activating antibody 9EG7 or TS2 / 16. In some other embodiments, integrin α3β1 is stabilized to a specific conformation by pre-complexing with an active agent that selectively binds to the β-chain of the integrin dimer. [Brief explanation of the drawing]

[0035] [Figure 1A] Evaluation of integrin agonist antibody binding characteristics by direct integrin ELISA. Bovine serum albumin (BSA), recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD were coated onto plates and incubated with human anti-α3 antibody or isotype (n=8 for each coated protein, n=4 for BSA). Binding of (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 was detected by incubation with anti-hIgG1 antibody HRP conjugate, development of a fluorescent substrate, and then reading the average fluorescence intensity with a plate reader. [Figure 1B] Evaluation of integrin agonist antibody binding characteristics by direct integrin ELISA. Bovine serum albumin (BSA), recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD were coated onto plates and incubated with human anti-α3 antibody or isotype (n=8 for each coated protein, n=4 for BSA). Binding of (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 was detected by incubation with anti-hIgG1 antibody HRP conjugate, development of a fluorescent substrate, and then reading the average fluorescence intensity with a plate reader. [Figure 1C]Evaluation of integrin agonist antibody binding characteristics by direct integrin ELISA. Bovine serum albumin (BSA), recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD were coated onto plates and incubated with human anti-α3 antibody or isotype (n=8 for each coated protein, n=4 for BSA). Binding of (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 was detected by incubation with anti-hIgG1 antibody HRP conjugate, development of a fluorescent substrate, and then reading the average fluorescence intensity with a plate reader. [Figure 1D] Evaluation of integrin agonist antibody binding characteristics by direct integrin ELISA. Bovine serum albumin (BSA), recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD were coated onto plates and incubated with human anti-α3 antibody or isotype (n=8 for each coated protein, n=4 for BSA). Binding of (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 was detected by incubation with anti-hIgG1 antibody HRP conjugate, development of a fluorescent substrate, and then reading the average fluorescence intensity with a plate reader.

[0036] [Figure 2] Epitope mapping of integrin agonist antibodies by direct integrin ELISA. Recombinant integrin α3β1 domain or bovine serum albumin (BSA) was coated onto plates and incubated with human anti-α3 antibody (red) or isotype (blue) (n = 4). Binding of Ab74 A101 was detected by incubation with anti-hIgG1 antibody HRP conjugate, development of a fluorescent substrate, and then reading the average fluorescence intensity with a plate reader.

[0037] [Figure 3A] Increased ligand binding by mouse integrin α3β1-expressing cells in the presence of agonist antibodies. α3β1-expressing K562 cells were incubated with an α3β1 ligand-mimicking LXY2-biotin conjugate and either an integrin agonist antibody or an isotype antibody control. The cells were then stained with streptavidin-fluorophore conjugate and measured by flow cytometry. (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 show increased LXY2 binding compared to isotype control alone. [Figure 3B] Increased ligand binding by mouse integrin α3β1-expressing cells in the presence of agonist antibodies. α3β1-expressing K562 cells were incubated with an α3β1 ligand-mimicking LXY2-biotin conjugate and either an integrin agonist antibody or an isotype antibody control. The cells were then stained with streptavidin-fluorophore conjugate and measured by flow cytometry. (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 show increased LXY2 binding compared to isotype control alone. [Figure 3C] Increased ligand binding by mouse integrin α3β1-expressing cells in the presence of agonist antibodies. α3β1-expressing K562 cells were incubated with an α3β1 ligand-mimicking LXY2-biotin conjugate and either an integrin agonist antibody or an isotype antibody control. The cells were then stained with streptavidin-fluorophore conjugate and measured by flow cytometry. (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 show increased LXY2 binding compared to isotype control alone. [Figure 3D]Increased ligand binding by mouse integrin α3β1-expressing cells in the presence of agonist antibodies. α3β1-expressing K562 cells were incubated with an α3β1 ligand-mimicking LXY2-biotin conjugate and either an integrin agonist antibody or an isotype antibody control. The cells were then stained with streptavidin-fluorophore conjugate and measured by flow cytometry. (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 show increased LXY2 binding compared to isotype control alone.

[0038] [Figure 4A] Reduced cell migration in the presence of integrin agonist antibodies as measured by a wound healing assay. α3β1-expressing SK-OV-3 cells were plated onto ligand-coated wells and adhered at 37°C for 16 hours. (A) Scratch wounds were created on the cell layer using a sterile pipette tip, and then the treatment solution was added. (B) Isotyped antibody controls and (C) blocking anti-α3 antibody did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibody and (E) anti-α3 agonist antibody Ab74 A101 suppressed wound closure after 16 hours. [Figure 4B] Reduced cell migration in the presence of integrin agonist antibodies as measured by a wound healing assay. α3β1-expressing SK-OV-3 cells were plated onto ligand-coated wells and adhered at 37°C for 16 hours. (A) Scratch wounds were created on the cell layer using a sterile pipette tip, and then the treatment solution was added. (B) Isotyped antibody controls and (C) blocking anti-α3 antibody did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibody and (E) anti-α3 agonist antibody Ab74 A101 suppressed wound closure after 16 hours. [Figure 4C]Reduced cell migration in the presence of integrin agonist antibodies as measured by a wound healing assay. α3β1-expressing SK-OV-3 cells were plated onto ligand-coated wells and adhered at 37°C for 16 hours. (A) Scratch wounds were created on the cell layer using a sterile pipette tip, and then the treatment solution was added. (B) Isotyped antibody controls and (C) blocking anti-α3 antibody did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibody and (E) anti-α3 agonist antibody Ab74 A101 suppressed wound closure after 16 hours. [Figure 4D] Reduced cell migration in the presence of integrin agonist antibodies as measured by a wound healing assay. α3β1-expressing SK-OV-3 cells were plated onto ligand-coated wells and adhered at 37°C for 16 hours. (A) Scratch wounds were created on the cell layer using a sterile pipette tip, and then the treatment solution was added. (B) Isotyped antibody controls and (C) blocking anti-α3 antibody did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibody and (E) anti-α3 agonist antibody Ab74 A101 suppressed wound closure after 16 hours. [Figure 4E] Reduced cell migration in the presence of integrin agonist antibodies as measured by a wound healing assay. α3β1-expressing SK-OV-3 cells were plated onto ligand-coated wells and adhered at 37°C for 16 hours. (A) Scratch wounds were created on the cell layer using a sterile pipette tip, and then the treatment solution was added. (B) Isotyped antibody controls and (C) blocking anti-α3 antibody did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibody and (E) anti-α3 agonist antibody Ab74 A101 suppressed wound closure after 16 hours.

[0039] [Figure 5] Schematic diagram of a domain-swapped mammalian expression construct.

[0040] [Figure 6A]Antibody staining of podocytes demonstrates that a novel anti-integrin α3 antibody stains podocyte-expressed integrin α3β1. Kidney sections of C57B / L6 wild-type mice were immunofluorescently stained with various antibodies (5 μg / mL) and imaged using a confocal microscope. Representative images show staining with either Ab74_A100 (A), 9EG7 (B), or a human anti-mouse IgG1 isotype control antibody (C). [Figure 6B] Antibody staining of podocytes demonstrates that a novel anti-integrin α3 antibody stains podocyte-expressed integrin α3β1. Kidney sections of C57B / L6 wild-type mice were immunofluorescently stained with various antibodies (5 μg / mL) and imaged using a confocal microscope. Representative images show staining with either Ab74_A100 (A), 9EG7 (B), or a human anti-mouse IgG1 isotype control antibody (C). [Figure 6C] Antibody staining of podocytes demonstrates that a novel anti-integrin α3 antibody stains podocyte-expressed integrin α3β1. Kidney sections of C57B / L6 wild-type mice were immunofluorescently stained with various antibodies (5 μg / mL) and imaged using a confocal microscope. Representative images show staining with either Ab74_A100 (A), 9EG7 (B), or a human anti-mouse IgG1 isotype control antibody (C). [Modes for carrying out the invention]

[0041] I. Introduction The inventors have discovered a novel antibody that binds to integrin α3β1, specifically to a sequence within the thigh-genu region of integrin α3β1. Such an antibody acts as an agonist to integrin α3β1 and can enhance integrin-dependent functions such as ligand binding and cell adhesion. In particular, considering that integrin α3β1 is an important integrin on the surface of podocytes, this antibody may be useful in treating diseases and / or conditions associated with podocyte loss, such as nephritis, nephrotic syndrome, Alport syndrome, or renal diseases such as focal segmental glomerulosclerosis (FSGS).

[0042] The inventors also found that novel anti-integrin α3 allosteric antibodies induce intracellular signaling in the presence of external ligands. For example, when integrin α3-expressing cells were incubated with the novel anti-α3 integrin antibody in the absence of an integrin ligand, the levels of phosphorylated adhesion plaque kinase (pFAK) did not change. Co-incubation of cells with the novel antibody and ligand laminin increased the relative levels of pFAK.

[0043] II. Definition As used herein, the term “antibody” includes antibody fragments that retain binding specificity. For example, many well-characterized antibody fragments exist. For example, by digesting the antibody on the C-terminal side of the disulfide bond in the hinge region with pepsin, F(ab)'2 is produced. This is because the light chain is V by the disulfide bond. H -C H F(ab)'2 is a dimer of Fab linked to 1. Under mild conditions, F(ab)'2 can be reduced to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer to the Fab' monomer. The Fab' monomer is essentially Fab with a portion of the hinge region (for a detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). Various antibody fragments are defined in relation to the digestion of intact antibodies, but those skilled in the art will understand that fragments can be synthesized de novo, either chemically or by the use of recombinant DNA methodologies. Thus, as used herein, the term antibody includes either an antibody fragment produced by modification of the whole antibody, or an antibody fragment synthesized using recombinant DNA methodologies.

[0044] The antibodies described herein may consist of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, the antibody is IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, or IgE.

[0045] Typical immunoglobulin (antibody) structural units are known to contain tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. L ) and variable heavy chain (V H The terms ) refer to these light and heavy chains, respectively.

[0046] In antibodies, a substitutional variant involves the removal of at least one amino acid residue and the insertion of a different residue in its place. While hypervariable regions are the most important sites for mutagenesis through substitution, framework modifications are also intended. Examples of conservative substitutions are described above.

[0047] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the polypeptide backbone in the substitution region, such as a β-sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain. Naturally occurring residues are grouped based on the following common side-chain properties: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Polar without charge: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His. Non-conservative substitutions can be caused by exchanging one member of these classes with another.

[0048] One type of substitution that can be caused is to change one or more cysteines in an antibody that may be chemically reactive to another residue such as, but not limited to, alanine or serine. For example, non-standard cysteine substitutions may exist. Substitutions can be made in the CDR or framework regions of the variable domain of the antibody, or in the constant region. In some embodiments, the cysteines are standard (e.g., involved in disulfide bond formation). Any cysteine residue not involved in maintaining the appropriate conformation of the antibody can also generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, particularly when the antibody is an antibody fragment such as an Fv fragment, the stability can be improved by adding cysteine bond(s) to the antibody.

[0049] Antibodies include single-chain antibodies (antibodies existing as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv), in which the variable heavy chain region and the variable light chain region are linked together (either directly or via a peptide linker) to form a continuous polypeptide. H -V L dimers are included. The single-chain Fv antibody is either directly linked or linked by a peptide coding linker, V H coding sequence and V L ​​​L (For example, Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). V H and V L Although they are connected to each other as a single polypeptide chain, V H Domain and V L The domains are associated non-covalently. Alternatively, the antibody may be another fragment. Other fragments may also be generated, for example, using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies may also include diantibodies and miniantibodies. Antibodies of this disclosure also include heavy chain dimers, such as antibodies derived from camelids. In some embodiments, the antibody is a dimer. In other embodiments, the antibody may be in monomeric form having an active isotype. In some embodiments, the antibody is in a polyvalent form, for example, trivalent or tetravalent form.

[0050] As used herein, the terms “variable region” and “variable domain” refer to portions of the light and heavy chains of an antibody that include the amino acid sequences of the complementarity-determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR3) and the framework region (FR). The variable regions of the heavy and light chains are generally V, respectively. H and V L This is referred to as [the variable region]. The variable region is contained on the Fab, F(ab')2, Fv, and scFv antibody fragments described herein and is involved in the recognition of specific antigens.

[0051] As used herein, “complementarity-determining regions (CDRs)” refers to three hypervariable regions in each chain that divide the four framework regions established by the light chain variable region and the heavy chain variable region. CDRs are primarily responsible for the binding of antigens to epitopes. The CDRs of each chain are typically numbered sequentially starting from the N-terminus and referred to as CDR1, CDR2, and CDR3, and are also typically identified by the chain on which a particular CDR is located. Therefore, V H CDR3 is located in the variable domain of the heavy chain of the antibody where it is found, whereas VL CDR1 is a CDR1 derived from the variable domain of the light chain of the antibody in which it is found.

[0052] The sequences of different light chain or heavy chain framework regions are relatively conserved within a species. The antibody framework region, that is, the framework region formed by the combination of its constituent light and heavy chains, plays a role in positioning and aligning the CDR in three-dimensional space.

[0053] The amino acid sequences of the CDR and framework regions are defined using various definitions well known in the art, e.g., Kabat, North method (e.g., North et al., J Mol Biol. 406(2):228-256, 2011), Chothia, the International ImMunoGeneTics Database (IMGT), and AbM (e.g., Johnson et al., above; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J.Mol.Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human V HThis can be determined using segments (see J.Mol.Biol.227,799-817; Al-Lazikani et al., J.Mol.Biol 1997,273(4)). The definition of antigen-binding sites is also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J.Mol.Biol., 262(5), 732-745 (1996); and Martin et al, Proc.Natl Acad.Sci.USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).

[0054] As used herein, the term "allosteric agonist" refers to a molecule (e.g., an antibody) that binds to a target (e.g., integrin α3β1 or a portion thereof, a sequence within the α3 portion of integrin α3β1, the sequence of SEQ ID NO: 44 or a portion thereof) at a site or region other than the target's active site, thereby enhancing, activating, or increasing the target's response to the binding of its native ligand.

[0055] As used herein, “chimeric antibody” means (a) an immunoglobulin molecule in which the constant region or part thereof is modified, replaced, or exchanged so as to be bound to the constant region of a different or modified class, effector function and / or species, or to a completely different molecule that confers new characteristics to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) an immunoglobulin molecule in which the variable region or part thereof is modified, replaced, or exchanged by a variable region or part thereof having a different or modified antigen specificity, or by a corresponding sequence derived from another species or another antibody class or subclass.

[0056] As used herein, “humanized antibody” refers to an immunoglobulin molecule in which a donor antibody-derived CDR is grafted onto a human framework sequence. The humanized antibody may also contain donor-derived residues in the framework sequence. The humanized antibody may also contain at least a portion of the human immunoglobulin constant region. The humanized antibody may also contain residues not found in the recipient antibody or in the transferred CDR or framework sequence. Humanization can be carried out using methods known in the art (e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Patent No. 4,816,567), for example, "superhumanization" antibodies (Tan et al., J. Immunol. 169:1119, 2002) and "resurfacing" (e.g., Staelens et al., Mol. Immunol. 43:1243, 2006; and Roguska et al., Proc. Natl. Acad. Sci This includes techniques such as those described in USA 91:969, 1994.

[0057] The term "recombinant," when used, for example, in relation to cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of a different nucleic acid or protein or by modification of a native nucleic acid or protein, or that the cells are derived from such modified cells. For example, recombinant cells express genes not found in the native (non-recombinant) form of cells, or express native genes that are expressed in an unusual manner, underexpressed, or not expressed at all.

[0058] The terms “antigen,” “immunogen,” “antibody target,” “target analyte,” and similar terms are used herein to refer to molecules, compounds, or complexes that are recognized by an antibody, i.e., that can be specifically bound by an antibody. These terms may refer to any molecule that can be specifically recognized by an antibody, such as polypeptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides). Those skilled in the art will understand that these terms do not indicate that the molecule is immunogenic in any context, but merely that it can be targeted by an antibody.

[0059] Antibodies bind to "epitopes" on antigens. An epitope is a localized site on an antigen that is recognized and bound by an antibody. An epitope may contain several amino acids or a portion of several amino acids, e.g., five or six or more, e.g., twenty or more amino acids, or a portion of those amino acids. In some cases, epitopes may contain non-protein components, e.g., derived from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Therefore, for example, if the target is a protein, the epitope may consist of consecutive amino acids or amino acids derived from different parts of the protein that are adjacent due to protein folding (e.g., a discontinuous epitope). The same applies to other types of target molecules that form a three-dimensional structure. Typically, an epitope contains at least three, more commonly, at least five or eight to ten amino acids in its unique spatial structure. Methods for determining the spatial structure of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. For example, see Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0060] The terms "specific to," "specifically binds," and similar terms refer to a molecule (e.g., an antibody or antibody fragment) binding to a target with at least twice the affinity of a non-target compound, for example, at least four times, five times, six times, seven times, eight times, nine times, ten times, twenty times, twenty-five times, fifty times, or 100 times higher affinity. For example, an antibody that specifically binds to a target typically binds to the target with at least twice the affinity of a non-target. Specificity can be measured using standard methods, such as solid-phase ELISA immunoassays (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity).

[0061] The term "binding" in relation to antibody targets (e.g., antigens, analytes, immune complexes) typically indicates that an antibody binds to the majority of antibody targets within a pure population (assuming appropriate molar ratios). For example, an antibody that binds to a given antibody target typically binds to at least two-thirds of the antibody targets in solution (e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). Those skilled in the art will recognize that some variation will occur depending on the method and / or threshold used to measure binding.

[0062] A "control" sample or value refers to a sample that serves as a reference for comparison with the test sample, usually a known reference. For example, the test sample is obtained under test conditions, such as in the presence of the test compound, and can be compared with a sample from known conditions, such as in the absence of the test compound (negative control) or in the presence of a known compound (positive control). A control may also represent a mean or range collected from multiple tests or results. A person skilled in the art will recognize that controls can be designed to evaluate any number of parameters. For example, controls may be designed to compare therapeutic effects based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of efficacy and / or side effects). Controls may be designed for in vitro applications. A person skilled in the art will understand which controls are useful in a given situation and will be able to analyze data based on comparison with control values. Controls are also useful in determining the significance of data. For example, if the value of a given parameter varies significantly in the control, the variation in the test sample is not considered significant.

[0063] In the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “identity” percentage means that two or more sequences or subsequences are identical or have the same amino acid residues or nucleotides at a specific percentage when measured using the BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (i.e., when compared and aligned to maximize the match across the comparison window or designated region, the identity across a specific region is about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater) (see, for example, the NCBI website ncbi.nlm.nih.gov / BLAST / ). Such sequences are said to be “substantially identical.” Preferred algorithms may take gaps, etc., as described below. Preferably, the identity exists over a region of at least about 25 amino acids or nucleotides, or more preferably, over a region of 50 to 100 or more amino acids or nucleotides.

[0064] For sequence comparison, typically one sequence acts as a reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Preferably, default program parameters are used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the reference sequence based on the program parameters.

[0065] When used herein, "comparison window" includes a reference to one segment of consecutive position numbers selected from the group consisting of about 20 to 600, typically about 50 to 200, and more typically about 100 to 150, where a given sequence can be compared to a reference sequence with the same number of consecutive positions after the sequence and the reference sequence have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.

[0066] Suitable algorithms for determining sequence identity percentages and sequence similarity percentages are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used to determine the sequence identity percentages of nucleic acids and proteins in this disclosure, using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match when aligned with a word of the same length in the database sequence or satisfy a positive threshold score T. T is referred to as the neighbor word score threshold (Altschul et al., above). These initial neighbor word hits serve as seeds to initiate the search so that longer HSPs containing them can be found. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using the parameter M (reward score for matching residue pairs; always > 0) and the parameter N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops if: the cumulative alignment score has fallen by X from its maximum achieved value; the cumulative score has become 0 or less due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence has been reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The BLASTN program (for nucleotide sequences) uses the following default settings: word length (W) 11, expected value (E) 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program uses the following default settings: word length 3, expected value (E) 10, BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignment (B) 50, expected value (E) 10, M=5, N=-4, and comparison of both strands.

[0067] The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides, polymers thereof in single-stranded or double-stranded forms, and their complements. The term encompasses synthetic nucleic acids, naturally occurring nucleic acids, and non-naturally occurring nucleic acids, nucleic acids having similar binding properties to a reference nucleic acid, and nucleic acids metabolized in a manner similar to a reference nucleotide, including known nucleotide analogs or modified skeletal residues or linkages. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0068] Unless otherwise specified, a particular nucleic acid sequence implicitly includes, in addition to the explicitly indicated sequence, its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0069] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms encompass amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers.

[0070] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0071] Amino acids may be referred herein by either their widely known three-letter code or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their widely accepted one-letter code.

[0072] III. Antibodies that bind to integrin α3β1 or a portion thereof This specification provides for antibodies (including antibody fragments) that specifically bind to integrin α3β1 or a portion thereof (e.g., a sequence within the thigh-genu region of integrin α3β1). Integrin α3β1 is an integrin heterodimer of an α3 moiety and a β1 moiety. Integrin α3β1 is highly expressed on the surface of renal podocytes and is essential for podocytes to attach to the outside of blood vessels in order to form healthy glomeruli in the kidney. This integrin also promotes T cells (Park et al., Integrin α3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation, Science Immunology, Vol 8(88), 2023), cancer cells (Ke et al., Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer, Cancer Sci., 111(10), p3478, 2020), neutrophils (Lerman et al., Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrin α3β1-dependent, Blood, 2014 Dec 4;124(24):3515-23), and keratinocytes (Has et al, Integrin α3 mutations with kidney, lung, and skin disease, N Engl J Med). It is also expressed on other cells, such as 366:1508-1514, 2012), and may affect the function of cells expressing this integrin. The antibodies described herein act as allosteric agonist antibodies against integrin α3β1, enhancing integrin-dependent ligand binding and cell adhesion, thereby preventing podocyte loss in the urine and protecting against loss of renal function.This antibody can also alleviate autoimmune diseases by suppressing T cell migration and infiltration, reduce tumor growth and metastasis by inhibiting cancer cell migration, and suppress the activation and tissue recruitment of pro-inflammatory neutrophils.

[0073] In some embodiments, the anti-α3β1 antibody is isolated (e.g., separated from its natural environment (e.g., animals, biological specimens)). In some embodiments, the anti-α3β1 antibody is a humanized antibody or its antigen-binding fragment. In some embodiments, the anti-α3β1 antibody is a derivative of a humanized antibody that binds to α3β1 or a portion thereof. In some embodiments, the anti-α3β1 antibody binds to α3β1 under laboratory conditions (e.g., binds to α3β1 in vitro, binds to α3β1 in a flow cytometry assay, binds to α3β1 in an ELISA). In some embodiments, the anti-α3β1 antibody binds to α3β1 under physiological conditions (e.g., binds to α3β1 in cells (e.g., podocytes) in a subject).

[0074] In some embodiments, the α3 portion of heterodimer integrin α3β1 has the following sequence: [Table 1] It has a sequence that has at least 90% identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the given sequence. The thigh-genu region is shown in bold at sequence number 45.

[0075] In some embodiments, the β1 portion of heterodimer integrin α3β1 has the following sequence: The sequence has at least 90% identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to (sequence number 46).

[0076] In certain embodiments, the antibody binds to the α3 portion of integrin α3β1 (e.g., SEQ ID NO: 45). In some embodiments, the antibody binds to a sequence within the thigh-genu region of the α3 portion. In certain embodiments, the antibody binds to the sequence of SEQ ID NO: 44 or a portion within the sequence of SEQ ID NO: 44. VINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQKEC (Sequence ID 44).

[0077] Generally, the anti-α3β1 antibodies provided herein include at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region. In some embodiments, the anti-α3β1 antibodies described herein include two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions. Typically, each immunoglobulin heavy chain variable region of the anti-α3β1 antibody includes first, second, and third heavy chain complementarity determining regions (CDRs; HCDR1, HCDR2, and HCDR3), and each immunoglobulin light chain variable region of the anti-α3β1 antibody includes first, second, and third light chain CDRs (LCDR1, LCDR2, and LCDR3).

[0078] In some embodiments, the antibody is an antibody fragment such as Fab, F(ab')2, Fv, or scFv. The antibody fragment can be produced by any means known in the art, including chemical digestion (e.g., papain or pepsin) and recombination. Methods for isolating and preparing recombinant nucleic acids are known to those skilled in the art (see Sambrook et al., Molecular Cloning. A Laboratory Manual (2nd ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). The antibody can be expressed in a variety of host cells, including Escherichia coli (E. coli), other bacterial hosts, yeast, and various higher eukaryotic cells such as COS, CHO, and HeLa cell lines and myeloma cell lines.

[0079] In some embodiments, the antibodies of this disclosure include the heavy chain complementarity determination region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determination region 1 (LCDR1), LCDR2, LCDR3, and heavy chain variable region (V) as listed in Table 1. H ) and / or light chain variable region (V L It may contain an array of ). [Table 2]

[0080] Isolated antibodies that specifically bind to integrin α3β1 or a portion thereof (for example, a sequence within the thigh-genu region of integrin α3β1) are (1) A heavy chain complementarity determination region 1 (HCDR1) containing the sequence X1X2SGX3TFX4X5YX6X7X8 (sequence number 38), wherein X1 is A or K, X2 is A or T, X3 is F, G, or F, X4 is S or T, X5 is S or N, X6 is G, S, or A, X7 is M or I, and X8 is H, N, or S; (2) HCDR2 having a sequence that includes up to two amino acid substitutions to the sequence GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), or the sequence WISAX1NGNX2N (SEQ ID NO: 39), where X1 is Y or N and X2 is T or S; (3) HCDR3 comprising a sequence having up to two amino acid substitutions to the sequence of VRDDIQLRD (SEQ ID NO: 11) or AREFPGWYFDY (SEQ ID NO: 13), or a sequence having up to four amino acid substitutions to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), or ARGVPSGSGYYLGLDY (SEQ ID NO: 15); (4) A light chain complementarity determination region 1 (LCDR1) comprising the sequence X1ASQX2ISX3YLN (Sequence ID 40), wherein X1 is Q or A, X2 is D or Y, and X3 is N or S, or a sequence having up to three amino acid substitutions relative to the sequence QGDSLRSYYAS (Sequence ID 23) or SGSSSNIGSNYVY (Sequence ID 24); (5) LCDR2 comprising a sequence having at most one amino acid substitution to the sequence of YDASNLET (SEQ ID NO: 25) or the sequence of YX1X2NX3RPS (SEQ ID NO: 41), wherein X1 is G or R, X2 is K or N, and X3 is N or Q; and (6) LCDR3 comprising the sequence X1QX2YX3X4PX5T (SEQ ID NO: 42), where X1 is L or Q, X2 is D or S, X3 is N, S, or R, X4 is Y or T, and X5 is L or P, or a sequence having up to two amino acid substitutions relative to the sequence NSRDSSGNHWV (SEQ ID NO: 31) or AAWDDSLSGPV (SEQ ID NO: 32), It may include.

[0081] In some embodiments, the antibody of the Disclosure includes HCDR1 having one of the sequences of SEQ ID NOs: 1 to 5, or a variant thereof having a sequence with one amino acid substitution over one of the sequences of SEQ ID NOs: 1 to 5. In some embodiments, the antibody of the Disclosure includes HCDR2 having one of the sequences of SEQ ID NOs: 6 to 10, or a variant thereof having a sequence with one amino acid substitution over one of the sequences of SEQ ID NOs: 6 to 10. In some embodiments, the antibody of the Disclosure includes HCDR3 having one of the sequences of SEQ ID NOs: 11 to 15, or a variant thereof having a sequence with one amino acid substitution over one of the sequences of SEQ ID NOs: 11 to 15.

[0082] In some embodiments, the antibody of the Disclosure includes LCDR1 having any one sequence of SEQ ID NOs. 21-24, or a variant thereof having a sequence with one amino acid substitution over any one sequence of SEQ ID NOs. 21-24. In some embodiments, the antibody of the Disclosure includes LCDR2 having any one sequence of SEQ ID NOs. 25-27, or a variant thereof having a sequence with one substitution over any one sequence of SEQ ID NOs. 25-27. In some embodiments, the antibody of the Disclosure includes LCDR3 having any one sequence of SEQ ID NOs. 28-32, or a variant thereof having a sequence with one amino acid substitution over the sequences of SEQ ID NOs. 28-32.

[0083] HCDR1-3 and V H In some embodiments, the antibodies of the present disclosure may include HCDR1 having the sequence of SEQ ID NO: 1 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 1, HCDR2 having the sequence of SEQ ID NO: 6 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 6, and HCDR3 having the sequence of SEQ ID NO: 11 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 11. In some embodiments, the antibodies of the present disclosure may include HCDR1 having the sequence of SEQ ID NO: 2 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 2, HCDR2 having the sequence of SEQ ID NO: 7 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 7, and HCDR3 having the sequence of SEQ ID NO: 12 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 12. In some embodiments, the antibodies of the present disclosure may include HCDR1 having the sequence of SEQ ID NO: 3 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 3, HCDR2 having the sequence of SEQ ID NO: 8 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 8, and HCDR3 having the sequence of SEQ ID NO: 13 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 13. In some embodiments, the antibodies of the present disclosure may include HCDR1 having the sequence of SEQ ID NO: 4 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 4, HCDR2 having the sequence of SEQ ID NO: 9 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 9, and HCDR3 having the sequence of SEQ ID NO: 14 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 14.In some embodiments, the antibodies of the present disclosure may include HCDR1 having the sequence of SEQ ID NO: 5 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 5, HCDR2 having the sequence of SEQ ID NO: 10 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 10, and HCDR3 having the sequence of SEQ ID NO: 15 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 15.

[0084] The antibodies disclosed herein have heavy chain variable regions (V) having HCDR1, HCDR2, and HCDR3 as described herein. H) may include. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the sequences of SEQ ID NOs. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having HCDR1 of SEQ ID NOs.1, HCDR2 of SEQ ID NOs.6, and HCDR3 of SEQ ID NOs.11, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NOs. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 12, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 17. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 13, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 18. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having HCDR1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 14, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 19. In certain embodiments, the antibody of the Disclosure may include a heavy chain variable region having HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 15, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 20.

[0085] LCDR1~3 and V L In some embodiments, the antibodies of the present disclosure may include LCDR1 having the sequence of SEQ ID NO: 21 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 28 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 28. In some embodiments, the antibodies of the present disclosure may include LCDR1 having the sequence of SEQ ID NO: 22 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 22, LCDR2 having the sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 29 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 29. In some embodiments, the antibodies of the present disclosure may include LCDR1 having the sequence of SEQ ID NO: 21 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO: 30 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 30. In some embodiments, the antibodies of the present disclosure may include LCDR1 having the sequence of SEQ ID NO: 23 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 23, LCDR2 having the sequence of SEQ ID NO: 26 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 26, and LCDR3 having the sequence of SEQ ID NO: 31 or a variant thereof having a sequence with one amino acid substitution to the sequence of SEQ ID NO: 31.In some embodiments, the antibodies of the present disclosure may include LCDR1 having the sequence of SEQ ID NO: 24 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 24, LCDR2 having the sequence of SEQ ID NO: 27 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 27, and LCDR3 having the sequence of SEQ ID NO: 32 or a variant thereof having a sequence having one amino acid substitution to the sequence of SEQ ID NO: 32.

[0086] The antibodies disclosed herein have light chain variable regions (V) having LCDR1, LCDR2, and LCDR3 as described herein. L) may include. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the sequences of SEQ ID NOs.33-37. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having LCDR1 of SEQ ID NOs.21, LCDR2 of SEQ ID NOs.25, and LCDR3 of SEQ ID NOs.28, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NOs. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 29, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of SEQ ID NO: 34. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having LCDR1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 30, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of SEQ ID NO: 35. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having LCDR1 of SEQ ID NO: 23, LCDR2 of SEQ ID NO: 26, and LCDR3 of SEQ ID NO: 31, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of SEQ ID NO: 36. In certain embodiments, the antibody of the Disclosure may include a light chain variable region having LCDR1 of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 27, and LCDR3 of SEQ ID NO: 32, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of SEQ ID NO: 37.

[0087] A100 In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 1 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 1; (2) HCDR2 having the sequence of SEQ ID NO: 6 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 6; (3) HCDR3 having the sequence of SEQ ID NO: 11 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 11; (4) LCDR1 having the sequence of SEQ ID NO: 21 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 21; (5) LCDR2 having the sequence of SEQ ID NO: 25 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 25; and (6) LCDR3 having the sequence of SEQ ID NO: 28 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 28. In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 1, (2) HCDR2 having the sequence of SEQ ID NO: 6, (3) HCDR3 having the sequence of SEQ ID NO: 11, (4) LCDR1 having the sequence of SEQ ID NO: 21, (5) LCDR2 having the sequence of SEQ ID NO: 25, and (6) LCDR3 having the sequence of SEQ ID NO: 28.

[0088] In some embodiments, the antibody may include (1) a heavy chain variable region having HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 6, and HCDR3 of SEQ ID NO: 11, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 16; and (2) a light chain variable region having LCDR1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 28, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 33.

[0089] In a particular embodiment, the antibody is SEQ ID NO: 47: EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWLSGISGSADTTYYADSVKGRFTISRDNSKNTLYLQMTSLRAEDTAVYYCVRDDIQLRDWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS A heavy chain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Sequence ID No. 48: The light chain comprises having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFALTISSLQPEDFATYYCLQDYNYPLTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0090] In a particular embodiment, the antibody comprises (i) a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO: 16 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 47, and (ii) a light chain having a light chain variable region having the sequence of SEQ ID NO: 33 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 48.

[0091] A101 In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 2 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 2, (2) HCDR2 having the sequence of SEQ ID NO: 7 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 7, (3) HCDR3 having the sequence of SEQ ID NO: 12 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 12, (4) LCDR1 having the sequence of SEQ ID NO: 22 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 22, (5) LCDR2 having the sequence of SEQ ID NO: 25 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 25, and (6) LCDR3 having the sequence of SEQ ID NO: 29 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 29. In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 2, (2) HCDR2 having the sequence of SEQ ID NO: 7, (3) HCDR3 having the sequence of SEQ ID NO: 12, (4) LCDR1 having the sequence of SEQ ID NO: 22, (5) LCDR2 having the sequence of SEQ ID NO: 25, and (6) LCDR3 having the sequence of SEQ ID NO: 29.

[0092] In some embodiments, the antibody may include (1) a heavy chain variable region having HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 12, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 17; and (2) a light chain variable region having LCDR1 of SEQ ID NO: 22, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 29, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 34.

[0093] In a particular embodiment, the antibody is SEQ ID NO: 49: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYSGSWYPSNGPALDYWGQGTMVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV A heavy chain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Sequence ID No. 50: The light chain comprises having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of DIQMTQSPSSLSASVGDRVTITCRASQYISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQDYSYPLTFGGGIKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0094] In a particular embodiment, the antibody comprises (i) a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO: 17 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 49, and (ii) a light chain having a light chain variable region having the sequence of SEQ ID NO: 34 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 50.

[0095] A102 In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 3 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 3, (2) HCDR2 having the sequence of SEQ ID NO: 8 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 8, (3) HCDR3 having the sequence of SEQ ID NO: 13 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 13, (4) LCDR1 having the sequence of SEQ ID NO: 21 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 21, (5) LCDR2 having the sequence of SEQ ID NO: 25 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 25, and (6) LCDR3 having the sequence of SEQ ID NO: 30 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 30. In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 3, (2) HCDR2 having the sequence of SEQ ID NO: 8, (3) HCDR3 having the sequence of SEQ ID NO: 13, (4) LCDR1 having the sequence of SEQ ID NO: 21, (5) LCDR2 having the sequence of SEQ ID NO: 25, and (6) LCDR3 having the sequence of SEQ ID NO: 30.

[0096] In some embodiments, the antibody may include (1) a heavy chain variable region having HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 13, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 18; and (2) a light chain variable region having LCDR1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 30, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 35.

[0097] In a particular embodiment, the antibody is SEQ ID NO: 51: EVQLVQSGAEVKKPGASVKVSCKTSGFTFTNYGISWVRQAPGQGLEWMGWISANNGNSNYAQDHQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREFPGWYFDYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV A heavy chain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Sequence ID No. 52: The light chain comprises having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPDDFATYYCQQSYRTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0098] In a particular embodiment, the antibody comprises (i) a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO: 18 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 51, and (ii) a light chain having a light chain variable region having the sequence of SEQ ID NO: 35 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 52.

[0099] A103 In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 4 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 4, (2) HCDR2 having the sequence of SEQ ID NO: 9 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 9, (3) HCDR3 having the sequence of SEQ ID NO: 14 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 14, (4) LCDR1 having the sequence of SEQ ID NO: 23 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 23, (5) LCDR2 having the sequence of SEQ ID NO: 26 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 26, and (6) LCDR3 having the sequence of SEQ ID NO: 31 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 31. In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 4, (2) HCDR2 having the sequence of SEQ ID NO: 9, (3) HCDR3 having the sequence of SEQ ID NO: 14, (4) LCDR1 having the sequence of SEQ ID NO: 23, (5) LCDR2 having the sequence of SEQ ID NO: 26, and (6) LCDR3 having the sequence of SEQ ID NO: 31.

[0100] In some embodiments, the antibody may include (1) a heavy chain variable region having HCDR1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 9, and HCDR3 of SEQ ID NO: 14, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 19; and (2) a light chain variable region having LCDR1 of SEQ ID NO: 23, LCDR2 of SEQ ID NO: 26, and LCDR3 of SEQ ID NO: 31, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 36.

[0101] In a particular embodiment, the antibody is SEQ ID NO: 53: EVQLVESGGGLVQPGGSLRLSCAASGFTFSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAREYYDFWSGYPSGYAFDIWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV A heavy chain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Sequence ID No. 54: The light chain comprises having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of QSALTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0102] In a particular embodiment, the antibody comprises (i) a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO: 19 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 53, and (ii) a light chain having a light chain variable region having the sequence of SEQ ID NO: 36 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 54.

[0103] A104 In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 5 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 5; (2) HCDR2 having the sequence of SEQ ID NO: 10 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 10; (3) HCDR3 having the sequence of SEQ ID NO: 15 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 15; (4) LCDR1 having the sequence of SEQ ID NO: 24 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 24; (5) LCDR2 having the sequence of SEQ ID NO: 27 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 27; and (6) LCDR3 having the sequence of SEQ ID NO: 32 or a sequence having one amino acid substitution over the sequence of SEQ ID NO: 32. In certain embodiments, the antibodies of the present disclosure may include (1) HCDR1 having the sequence of SEQ ID NO: 5, (2) HCDR2 having the sequence of SEQ ID NO: 10, (3) HCDR3 having the sequence of SEQ ID NO: 15, (4) LCDR1 having the sequence of SEQ ID NO: 24, (5) LCDR2 having the sequence of SEQ ID NO: 27, and (6) LCDR3 having the sequence of SEQ ID NO: 32.

[0104] In some embodiments, the antibody may include (1) a heavy chain variable region having HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 15, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 20; and (2) a light chain variable region having LCDR1 of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 27, and LCDR3 of SEQ ID NO: 32, and having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with respect to the sequence of SEQ ID NO: 37.

[0105] In a particular embodiment, the antibody is SEQ ID NO: 55: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGVPSGSGYYLGLDYWGQGTMVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV A heavy chain having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Sequence ID No. 56: The light chain comprises having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of QSELTQPPSASGAPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPVFSGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0106] In a particular embodiment, the antibody comprises (i) a heavy chain having a heavy chain variable region having the sequence of SEQ ID NO: 20 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 55, and (ii) a light chain having a light chain variable region having the sequence of SEQ ID NO: 37 and a sequence having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with the sequence of SEQ ID NO: 56.

[0107] IV. Fc polypeptide The anti-α3β1 antibodies provided herein may contain a fragment crystallizable region (Fc region), also referred to herein as an Fc polypeptide. The Fc polypeptide is part of each of the two heavy chains in the antibody and can interact with specific cell surface receptors and specific components of the complement system. Typically, the Fc polypeptide comprises CH2 and CH3 domains, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in the antibodies described herein may be wild-type Fc polypeptides, such as human IgG1 Fc polypeptide. In certain embodiments, the antibodies described herein may be SEQ ID NO: 43: It may contain wild-type Fc polypeptides having the sequence APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. In other embodiments, the antibodies described herein may include variants of the wild-type Fc polypeptide having at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) to the sequence of the wild-type Fc polypeptide (e.g., SEQ ID NO: 43) and having at least one amino acid substitution relative to the sequence of the wild-type Fc polypeptide (e.g., SEQ ID NO: 43).

[0108] In some embodiments, the Fc polypeptide comprises one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions compared to an equivalent wild-type Fc region). Antibodies containing modified Fc polypeptides typically have altered phenotypes compared to antibodies containing wild-type Fc polypeptides. For example, antibodies containing modified Fc polypeptides may have altered serum half-life, altered stability, altered sensitivity to cellular enzymes, and / or altered effector function (e.g., assayed in NK-dependent or macrophage-dependent assays).

[0109] In some embodiments, the Fc polypeptides in the antibodies described herein may include amino acid substitutions that modulate effector function. In certain embodiments, the Fc polypeptides in the antibodies described herein may include amino acid substitutions that reduce or eliminate effector function. Examples of Fc polypeptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in the CH2 domain, e.g., substitutions at positions 4 and 5 (position numbers relative to the sequence of SEQ ID NO: 43) (see, for example, Lund et al., J Immunol. 147(8):2657-62, 1991). For example, in some embodiments, one or both of the Fc polypeptides in the antibodies described herein may include L4A substitutions and L5A substitutions.

[0110] Further Fc polypeptide amino acid substitutions that modulate effector function include, for example, substitutions at position 99 (position number relative to the sequence of SEQ ID NO: 43). For example, in some embodiments, one or both Fc polypeptides in the antibodies described herein may include a P99G substitution. In certain embodiments, one or both Fc polypeptides in the antibodies described herein may have an L4A substitution, an L5A substitution, and a P99G substitution.

[0111] In some embodiments, the Fc polypeptide includes one or more modifications that alter the ratio of the affinity of the modified Fc polypeptide to an active FcγR (e.g., FcγRIIA or FcγRIIIA) to the affinity of the modified Fc polypeptide to an inhibitory FcγR (e.g., FcγRIIB) (relative to the wild-type Fc polypeptide).

number

[0112] When the affinity ratio of the modified Fc polypeptide is greater than 1, the anti-α3β1 antibody described herein may be particularly useful in providing therapeutic or prophylactic treatment for diseases, disorders, or infections, such as cancer or infections, where enhancement of the efficacy of FcγR-mediated effector cell function (e.g., ADCC) is desired, or in improving the symptoms thereof. When the affinity ratio of the modified Fc region is less than 1, the anti-α3β1 antibody described herein may be particularly useful in providing therapeutic or prophylactic treatment for diseases or disorders, such as autoimmune or inflammatory disorders, where a decrease in the efficacy of FcγR-mediated effector cell function is desired, or in improving the symptoms thereof. Table 2 lists examples of single, double, triple, quadruple, and quintuple amino acid substitutions in Fc polypeptides with affinity ratios greater than or less than 1 (see, for example, International Publications 04 / 063351; 06 / 088494; 07 / 024249; 06 / 113665; 07 / 021841; 07 / 106707; and 2008 / 140603). Amino acid positions are numbered according to the EU numbering scheme. [Table 3]

[0113] V. Antibodies that competitively bind to anti-α3β1 antibodies Anti-α3β1 antibodies (e.g., competing antibodies) that competitively bind to or can competitively bind to one or more anti-α3β1 antibodies described herein are also provided herein. In certain cases, an antibody (i.e., a competing antibody) may be considered to compete for binding to α3β1 if the competitor binds to the same approximate α3β1 region as the anti-α3β1 antibodies described herein. In certain cases, an antibody (i.e., a competing antibody) may be considered to compete for binding to α3β1 if the competitor binds to the exact same α3β1 region as the anti-α3β1 antibodies described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conceptual epitope)). In certain cases, an antibody (i.e., a competing antibody) may be considered to be able to compete for binding to α3β1 if the competitor binds to the same approximate α3β1 region as the anti-α3β1 antibodies described herein under preferred assay conditions (i.e., a sequence within the thigh-genu region of integrin α3β1). In certain cases, an antibody (i.e., a competitor) may be considered capable of competing for binding to α3β1 if the competitor binds to the exact same α3β1 region (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (structural epitope)) as the anti-α3β1 antibody described herein under preferred assay conditions.

[0114] In certain cases, an antibody (i.e., a competing antibody) may be considered to compete for binding to α3β1 if the competitor blocks the binding of one or more anti-α3β1 antibodies described herein to α3β1, for example, under preferred assay conditions. Whether a competitor blocks the binding of one or more anti-α3β1 antibodies described herein to α3β1 can be determined using a preferred competition assay or blocking assay (e.g., the blocking assay described herein). A competing antibody may block the binding of one or more anti-α3β1 antibodies described herein to α3β1 by 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) in a competitive or blocking assay. Conversely, one or more anti-α3β1 antibodies described herein may block the binding of a competing antibody to α3β1 by approximately 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%) in a competitive or blocking assay.

[0115] In certain cases, an antibody (i.e., a competing antibody) may be considered to compete for binding to α3β1 if the competitor binds to α3β1 with an affinity similar to that of one or more anti-α3β1 antibodies described herein, for example, under preferred assay conditions. In some embodiments, an antibody (i.e., a competing antibody) may be considered to compete for binding to α3β1 if the competitor binds to α3β1 with an affinity that is at least about 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the affinity of one or more anti-α3β1 antibodies described herein.

[0116] Also provided herein are anti-α3β1 antibodies that bind to, or can bind to, the same epitopes as one or more anti-α3β1 antibodies described herein. In particular, anti-α3β1 antibodies that compete with one or more anti-α3β1 antibodies described herein for binding to the same epitope on α3β1 (e.g., the same peptide (linear epitope) or the same surface amino acid (conceptual epitope)) are provided herein. Such antibodies that bind to the same epitope may be referred to as epitope competitors.

[0117] VI. Polyclonal antibodies and monoclonal antibodies Polyclonal antibodies can be produced in animals (vertebrates or invertebrates (including mammals), birds and fish (including cartilaginous fish)) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen with a protein or other carrier that is immunogenic in the immunized species, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or a soy trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide (conjugation via a cysteine ​​residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR (where R and R1 are different alkyl groups). Non-protein carriers (e.g., gold colloid) may also be used for antibody production.

[0118] For example, animals can be immunized to an antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with three times the volume of Freund's complete adjuvant and intradermally injecting this solution at multiple sites. After one month, the animals are boosted by subcutaneously injecting 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant at multiple sites. After 7 to 14 days, blood is collected from the animals and the antibody titer of the serum is assayed. Booster immunization is performed on the animals until the titer reaches a plateau. In many cases, animals are boosted with a conjugate of the same antigen, but the conjugate may be conjugated to a different protein and / or conjugated via a different crosslinking reagent. Conjugates can also be prepared in recombinant cell cultures as protein fusions. Agglutinants such as alum are also suitably used to enhance the immune response.

[0119] Monoclonal antibodies may be produced using hybridomas, for example, the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by other methods such as recombinant DNA (see, for example, U.S. Patent No. 4,816,567). In the hybridoma method, immunization of mice or other suitable host animals (e.g., hamsters or macaques) induces lymphocytes that produce or are capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0120] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that may contain one or more substances that inhibit the proliferation or survival of unfused parental myeloma cells. For example, if parental myeloma cells lack the hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT) enzyme, the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the proliferation of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium. Of these, preferred myeloma cell lines are mouse myeloma lines such as SP-2 or X63-Ag8-653 cells, available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma cell lines and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0121] The culture medium in which hybridoma cells are proliferating is assayed for the production of monoclonal antibodies against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation, in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or by flow cytometry analysis of cells expressing membrane antigens. The binding affinity of monoclonal antibodies can be determined, for example, by scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0122] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution and proliferated by standard methods (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640 medium. Furthermore, hybridoma cells can be proliferated in vivo as ascites tumors in animals. Monoclonal antibodies can be suitably separated from culture media, ascites, or serum by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0123] The DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Alternatively, cDNA may be prepared from mRNA and then subjected to DNA sequencing. Hybridoma cells serve as a preferred source of such genomic DNA or RNA for cDNA preparation. Once the DNA is isolated, it can be placed in an expression vector well known in the art and then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would normally produce immunoglobulin proteins but do not, to obtain the synthesis of monoclonal antibodies in recombinant host cells.

[0124] VII. Humanization and Amino Acid Variants Common methods for humanizing antibodies include, for example, U.S. Patent Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, and 5714350. This is described in Nos. 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671, 6329511, 5225539, 6548640, and 5624821. In certain embodiments, it may be desirable to create amino acid sequence variants of these humanized antibodies, particularly to improve the binding affinity or other biological properties (e.g., half-life) of the antibody.

[0125] In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody but has low immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart. See, for example, Morrison et al., PNAS USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994). Techniques for humanizing antibodies are well known in the art and are described, for example, in U.S. Patents Nos. 4,816,567, 5,530,101, 5,859,205, 5,585,089, 5,693,761, 5,693,762, 5,777,085, 6,180,370, 6,210,671, and 6,329,511, International Publication No. 87 / 02671, European Patent No. 0173494, Jones et al. (1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534. Humanized antibodies are further described, for example, in Winter and Milstein (1991) Nature 349:293. For example, a polynucleotide comprising a first sequence encoding a humanized immunoglobulin framework region and a second sequence set encoding a desired immunoglobulin complementarity-determining region can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated from various human cells according to well-known procedures. CDRs for producing the immunoglobulins of this disclosure can similarly be derived from monoclonal antibodies capable of specifically binding to α3β1.

[0126] Amino acid sequence variants of anti-α3β1 antibodies can be prepared by introducing appropriate nucleotide changes into anti-α3β1 antibody DNA or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the anti-α3β1 antibodies of the examples herein. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, as long as the final construct has the desired properties. Amino acid changes can also modify the post-translational processes of humanized or variant anti-α3β1 antibodies, such as by changing the number or location of glycosylation sites.

[0127] One method for identifying specific residues or regions of an anti-α3β1 antibody that are favorable sites for mutagenesis is called "alanine scanning mutagenesis," as described, for example, by Cunningham and Wells, Science, 244:1081-1085 (1989). Hereinafter, target residues or groups of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with neutral or charged amino acids (most preferably Ala or poly-Ala) to affect the interaction between the amino acid and the α3β1 antigen (e.g., sequences within the thigh-genu region of integrin α3β1). These amino acid positions, functionally sensitive to substitution, are then modified by introducing further or other variants at or in place of the substitution site. Thus, while the sites for introducing amino acid sequence diversity are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed on the target codon or region, and the expressed anti-α3β1 antibody variant is screened for desired activity. Amino acid insertions include amino and / or carboxyl terminal fusions across polypeptides ranging in length from one residue to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies fused to an N-terminal methionyl residue or an epitope tag. Other insertion variants include fusions of enzymes or polypeptides that increase the serum half-life of the antibody to the N or C-terminus of the antibody.

[0128] Another type of variant is amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The most important sites for substitution mutagenesis include the hypervariable region, but FR modifications are also attempted. Conservative substitutions are preferred, but more substantial changes may be introduced, and the product may be screened. Examples of substitutions are listed below. Ala(A): Val; Leu; Ile; Val Arg(R):Lys;Gln;Asn;Lys Asn(N):Gln;His;Asp,Lys;Gln;Arg Asp(D):Glu;Asn Cys(C):Ser;Ala Gln(Q):Asn;Glu Glu(E):Asp;Gln Gly(G):Ala His(H):Asn;Gln;Lys;Arg Ile(I):Leu;Val;Met;Ala;Leu;Phe;Norleucine Leu(L): norleucine; Ile; Val; Ile; Met; Ala; Phe Lys(K):Arg;Gln;Asn Met(M):Leu;Phe;Ile Phe(F):Leu;Val;Ile;Ala;Tyr Pro(P):Ala Ser(S):Thr Thr(T):Ser Trp(W):Tyr;Phe Tyr(Y):Trp;Phe;Thr;Ser Val(V):Ile;Leu;Met;Phe;Ala;Norleucine

[0129] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the polypeptide backbone in the substitution region, such as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain. Naturally occurring residues are grouped based on the following common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basicity: Asn, Gln, His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0130] Non-conservative substitution involves swapping one member of one of the above classes with one of another.

[0131] Any cysteine ​​residues that do not contribute to maintaining the proper three-dimensional structure of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0132] One type of substitution variant involves substituting one or more hypervariable region residues of the parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties compared to the parent antibody from which they were generated. A convenient means of generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated can be displayed in a monovalent manner from filamentous phage particles as fusions with the gene III product of M13, packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants may be screened as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0133] Another type of amino acid variant in an antibody alters the antibody's original glycosylation pattern. Alteration means the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites that were not present in the antibody. Antibody glycosylation is typically either N-linked and / or O-linked. N-linked glycosylation refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are the most common recognition sequences for the enzymatic binding of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The addition of a glycosylation site to an antibody can be achieved by modifying the amino acid sequence to include one or more of the tripeptide sequences described above (in the case of an N-linked glycosylation site). This modification can also be achieved by adding or substituting one or more serine or threonine residues into the original antibody sequence (in the case of an O-linked glycosylation site).

[0134] VIII. Other Modifications Other modifications of anti-α3β1 antibodies are intended. For example, the techniques herein also relate to immunoconjugates comprising anti-α3β1 antibodies described herein that are conjugated to toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), radioisotopes (e.g., radioconjugates), or cytotoxic agents such as cytotoxic drugs. Such conjugates may also be referred to as “antibody-drug conjugates” or “ADCs”. Conjugates can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0135] The anti-α3β1 antibodies disclosed herein (e.g., anti-α3β1 antibodies) can be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation times are disclosed in U.S. Patent 5,013,556. For example, liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). By extruding the liposomes through a filter of a specified pore size, liposomes with a desired diameter can be obtained. The Fab' fragment of the antibody provided herein can be conjugated into liposomes via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem. 257:286-288 (1982). Another active ingredient may be optionally contained within the liposome.

[0136] Enzymes or other polypeptides can be covalently bound to anti-α3β1 antibodies by techniques well known in the art (e.g., the use of the heterobifunctional crosslinking reagents described above). In some embodiments, a fusion protein comprising at least the antigen-binding region of the antibody provided herein, linked to at least a functionally active portion of the enzyme, can be constructed using recombinant DNA techniques well known in the art (see, for example, Neuberger et al., Nature 312:604-608 (1984)).

[0137] In certain embodiments, for example, it may be desirable to use antibody fragments rather than intact antibodies to enhance penetration into target tissues and cells. In such cases, it may be desirable to modify the antibody fragment to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor-binding epitope into the antibody fragment (for example, by mutation in an appropriate region of the antibody fragment, or by incorporating the epitope into a peptide tag and then fusing it to any end or middle of the antibody fragment, for example, by DNA or peptide synthesis; see, for example, International Publication No. 96 / 32478, published on October 17, 1996).

[0138] In some embodiments, modifications such as PEGylation or incorporation of long-chain polyethylene glycol polymer (PEG) can be optionally introduced into the antibody (e.g., within the polypeptide chain, or at either the N-terminus or C-terminus) to extend the in vivo half-life, for example. The introduction of PEG or long-chain polymers of PEG increases the effective molecular weight of the polypeptide, preventing, for example, rapid filtration into urine. In some embodiments, lysine residues in the sequence are conjugated to PEG directly or via a linker. Such linkers may be, for example, Glu residues or acyl residues containing thiol functional groups for linking to a suitably modified PEG chain. Another method for introducing a PEG chain is to first introduce a Cys residue to the C-terminus or to a solvent-exposed residue (e.g., substitution of an Arg or Lys residue). This Cys residue is then site-specifically bound to the PEG chain (e.g., containing a maleimide functional group). Methods for incorporating PEG or long-chain polymers of PEG are known in the art (for example, described in Veronese, FM, et al., Drug Disc. Today 10:1451-8 (2005); Greenwald, RB, et al., Adv. Drug Deliv. Rev. 55:217-50 (2003); Roberts, MJ, et al., Adv. Drug Deliv. Rev., 54:459-76 (2002)) (these contents are incorporated herein by reference).

[0139] Covalent modification of anti-α3β1 antibodies is also included within the scope of this technology. For example, the modification may be carried out by chemical synthesis or by enzymatic or chemical cleavage of the anti-α3β1 antibody. Other types of covalent modification of antibodies are introduced intramolecularly by reacting a target amino acid residue of the antibody with an organic derivatizing agent that can react with a selected side chain or N-terminal or C-terminal residue. Examples of covalent modification of polypeptides are described in U.S. Patent No. 5,534,615, which is specifically incorporated herein by reference. Preferred types of covalent modification of antibodies include linking the antibody to one of various non-proteinoid polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene) in the manner described in, for example, U.S. Patents No. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0140] IX. Nucleic acids, vectors, host cells, and recombination methods This disclosure also provides isolated nucleic acids encoding anti-α3β1 antibodies, vectors and host cells containing said nucleic acids, and recombinant techniques for producing said antibodies. The nucleic acids herein may each comprise one or more subsequences called polynucleotides.

[0141] Nucleic acids (e.g., isolated nucleic acids) comprising a nucleotide sequence encoding an anti-α3β1 antibody or a fragment thereof are provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin heavy chain variable domain of the anti-α3β1 antibody provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin light chain variable domain of the anti-α3β1 antibody provided herein. In some embodiments, the nucleic acid encodes both the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the anti-α3β1 antibody provided herein. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 37.

[0142] For the recombinant production of anti-α3β1 antibodies, the nucleic acid encoding the anti-α3β1 antibody can be isolated, inserted into a replicable vector, and then further cloned (DNA amplification) or expressed. In certain cases, anti-α3β1 antibodies can be produced by homologous recombination. The DNA encoding the anti-α3β1 antibody can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences and origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0143] Suitable host cells for cloning or expressing DNA in the vectors described herein may be prokaryotes, yeasts, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens. This includes marcescans and Shigella, as well as bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas such as Pseudomonas aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) may also be suitable. These examples are illustrative and not limiting.

[0144] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for cloning or expressing vectors encoding anti-α3β1 antibodies. Saccharomyces cerevisiae or common baker's yeast are among the most commonly used host microorganisms for lower eukaryotes. Many other genera, species, and strains, such as fission yeast (Schizosaccharomyces pombe); Kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), and Kluyveromyces drosophilarum (ATCC 16,500). 36,906), Kluiveromyces thermotolerans and Kluiveromyces marxianus; Yarrowia (European Patent No. 402,226); Pichia pastoris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces osidentalis Schwanniomyces such as occidentalis; as well as filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger are commonly available and useful herein.

[0145] Host cells suitable for the expression of anti-α3β1 antibodies may also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm), have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are generally available and such viruses can be used as the viruses of this specification according to the technique of the present invention, particularly for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0146] Suitable host cells for the expression of anti-α3β1 antibodies may include vertebrate cells (e.g., mammalian cells). Vertebrate cells can be grown in culture (tissue culture). Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocytes (Hep G2, HB 8065); Mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and Human hepatocarcinoma cell line (HepG2).

[0147] Host cells may be transformed with the expression vectors or cloning vectors described above for antibody production and cultured in conventional nutrient media appropriately modified for promoter induction, transformant selection, or amplification of genes encoding desired sequences. The host cells used to produce the antibodies provided herein may be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, see Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in al., Analyst Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, International Publication Nos. 90 / 03430, 87 / 00195, or U.S. Reissue Patent No. 30,985 may be used as culture media for host cells. Any of these media may, as needed, contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., The culture may be supplemented with sodium chloride, calcium, magnesium, and phosphates, buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used in the host cells selected for expression and will be obvious to those skilled in the art.

[0148] When recombinant techniques are used, antibodies can be produced intracellularly, in the perimembranous space, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the perimembranous space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant of such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit protein degradation, and antibiotics may be included to prevent the growth of accidental contaminants.

[0149] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, but other matrices are also available. Mechanically stable matrices such as control-pore glass or poly(styrenedivinyl)benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody being recovered.

[0150] After any optional pre-purification steps, the mixture containing the antibody of interest and impurities may be subjected to low-pH hydrophobic interaction chromatography, which may be carried out at a low salt concentration (e.g., about 0–0.25 M salt) using an elution buffer with a pH of about 2.5–4.5, for example.

[0151] X. Pharmaceutical preparations, administration, and routes of administration This disclosure provides anti-α3β1 antibodies and related compositions, which may be useful, for example, for the elimination of α3β1-expressing pathogens from the body, and for example, for the identification and quantification of α3β1-expressing pathogens in biological samples.

[0152] Anti-α3β1 antibodies can be formulated into pharmaceutical compositions useful for a variety of purposes, including the treatment of diseases or disorders. A pharmaceutical composition containing one or more anti-α3β1 antibodies can be administered to a patient in need using a pharmaceutical device, and according to one embodiment of this technology, a kit containing such a device is provided. Such devices and kits may be designed for the routine administration (including self-administration) of the pharmaceutical compositions herein.

[0153] Antibody therapeutic formulations can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing a drug or antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin Proteins such as methyl, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0154] The formulations described herein may contain two or more active compounds as necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0155] Formulations for in vivo administration are generally sterile. This can be achieved, for example, by filtering through a sterile filtration membrane.

[0156] Sustained-release preparations may be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing a drug / antibody, these matrices in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow for molecular release for more than 100 days, while certain hydrogels release proteins for shorter periods. If encapsulated drugs / antibodies remain in the body for extended periods, exposure to moisture at 37°C can result in denaturation or aggregation, leading to loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular disulfide bond formation via thio-disulfide exchange, stabilization can be achieved through modification of sulfhydryl residues, freeze-drying from acidic solutions, control of water content, use of appropriate additives, and development of specific polymer matrix compositions.

[0157] For therapeutic use, the anti-α3β1 antibodies provided herein are administered to mammals, such as humans, in pharmaceutically acceptable dosage forms, including, for example, those administered intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-sacral, intrathecal, oral, topical, or inhalation routes. For the prevention or treatment of disease, the appropriate dosage of the drug or antibody depends on the type of disease being treated as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is preferably administered to the patient as a single dose or over a series of treatments.

[0158] Depending on the type and severity of the disease, an antibody dose of approximately 1 μg / kg to 50 mg / kg (e.g., 0.1 to 20 mg / kg) may be the initial candidate dose for administration to a patient, whether by single or multiple separate doses or by continuous infusion. A typical daily or weekly dose may range from approximately 1 μg / kg to 20 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or more, the treatment is repeated, depending on the patient's condition, until the desired suppression of disease symptoms occurs. However, other drug regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays, including, for example, radiographic imaging. Detection methods using antibodies to determine α3β1 levels in body fluids or tissues may be used to optimize patient exposure to the therapeutic antibody.

[0159] In some embodiments, the composition comprising the anti-α3β1 antibody described herein can be administered as monotherapy, and in some embodiments, the composition comprising the anti-α3β1 antibody can be administered as part of a combination therapy. In some cases, the effectiveness of the antibody in the prevention or treatment of a disease can be improved by administering the antibody sequentially or in combination with another drug effective for those purposes (e.g., a chemotherapeutic agent for the treatment of cancer or microbial infections). In other cases, the anti-α3β1 antibody may act to enhance cells or increase their sensitivity to chemotherapeutic treatment, thus enabling effectiveness at lower doses and with lower toxicity. Certain combination therapies involve administering a composition comprising an antibody that reduces the number of α3β1-expressing cells, in addition to performing a second treatment regimen selected from the group consisting of chemotherapeutic agents, radiotherapy, surgery, and any combination of the foregoing. Such other agents may be present in the administered composition or administered separately. The anti-α3β1 antibody can also be suitably administered sequentially or in combination with other drugs or modalities, such as chemotherapeutic agents or radiotherapy for the treatment of cancer, infections, etc., or immunosuppressants.

[0160] XI. Method As described herein, integrin α3β1 is a crucial integrin on the surface of podocytes, which are cells in Bowman's capsule of the kidney that surround the glomerular capillaries. Integrin α3β1 is essential for podocytes to adhere to the outside of blood vessels in order to form healthy glomeruli in the kidney. The antibodies described herein act as allosteric agonist antibodies against integrin α3β1 and can enhance integrin-dependent ligand binding and cell adhesion, thereby preventing cell loss in the urine and protecting against loss of renal function. Furthermore, methods are provided herein for treating diseases and / or conditions associated with podocyte loss in subjects requiring treatment by administering the anti-α3β1 antibodies described herein that bind to integrin α3β1 or a portion thereof (e.g., sequences within the thigh-genu region of integrin α3β1) to the subject. In some embodiments, the diseases and / or conditions associated with podocyte loss may be diseases and / or conditions caused by podocyte loss (i.e., loss of cell number and / or loss of cellular function). In some embodiments, diseases and / or conditions associated with podocyte loss may be diseases and / or conditions affecting the kidneys, and therefore, podocyte loss (i.e., loss of cell number and / or loss of cellular function) may be a result or manifestation of kidney disease and / or condition.

[0161] In some embodiments of this method, the subjects have a kidney disease associated with podocyte loss (i.e., loss of cell number and / or loss of cellular function). The kidney disease may be a glomerular disease, such as nephritis, nephrotic disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

[0162] In some embodiments, the subjects are those who have undergone, have undergone, or are planning to undergo a transplant. In some embodiments, the transplant is a kidney transplant. In certain embodiments, the antibody is administered after the transplant. In certain embodiments, the antibody is administered after a kidney transplant to protect, maintain, and / or improve kidney function and health.

[0163] In some embodiments, the disease or condition associated with the loss of podocytes in the subject is an autoimmune disease. In some embodiments, the autoimmune disease affects renal function and / or kidney health. In some embodiments, the autoimmune disease is lupus nephritis. In some embodiments, the autoimmune disease is Goodpasture syndrome. In some embodiments, the autoimmune disease is anti-glomerular basement membrane disease (anti-GBM). In some embodiments, the autoimmune diseases are ANCA-associated vasculitis and glomerulonephritis.

[0164] In other embodiments, the disease or condition associated with podocyte loss is cancer, particularly cancer affecting kidney function and / or health. In certain embodiments, the cancer is renal cancer. In certain embodiments, the cancer is renal cell carcinoma, urothelial carcinoma, nephrosarcoma, Wilms' tumor, or lymphoma.

[0165] In some embodiments, the disease or condition associated with podocyte loss is inflammation, particularly inflammation affecting kidney function and / or health. In particular, the inflammation is glomerulonephritis. In some embodiments, the inflammation is membranoproliferative glomerulonephritis (MPGN), interstitial nephritis, IgA nephropathy (Berger's disease), pyelonephritis, lupus nephritis, or Wegener's granulomatosis.

[0166] This disclosure also relates to a method for identifying antibodies that bind to integrin α3β1 or a portion thereof, 1) Removing an antibody that binds to the β1 chain of integrin α3β1 in the presence or absence of a ligand-mimicking peptide and / or the antibody; 2) Select an antibody from the remaining antibodies in step 1) that binds to integrin α3β1 in the presence or absence of the β1 agonist antibody; 3) Counter-selection of an antibody that binds to integrin α3β1 against an immobilized β1 agonist antibody or ligand-mimicking peptide alone; and 4) Repeat steps 1), 2), and 3) above in the presence of cell surface-expressed integrin α3β1 to enrich the antibody which is an integrin α3 allosteric agonist. The method is characterized by including the following:

[0167] In some embodiments of this method, the ligand-mimicking peptide is LXY2. In some embodiments of this method, steps 1) and / or 3) are performed using human K562 cells that primarily express human α5β1 integrin and do not overexpress α3β1.

[0168] In some embodiments of this method, steps 2) and / or 3) are carried out using human K562 cells overexpressing α3β1. In some embodiments, steps 1) and / or 2) and / or 3) are carried out in the presence of an agent that blocks the ligand-binding site or domain of the integrin, such as an antibody and a ligand.

[0169] In some embodiments, integrin α3β1 is stabilized to a specific conformation by pre-complexing with an activator or inhibitor, such as the activating antibody 9EG7 or TS2 / 16. In some other embodiments, integrin α3β1 is stabilized to a specific conformation by pre-complexing with an active agent that selectively binds to the β-chain of the integrin dimer.

[0170] The inventors clear the β1 binder by clearing "other" β1 integrins, either as a recombinant protein or using cell lines such as K562 that express α5β1, etc.

[0171] The inventors prepare an α3β1 complex by pre-conjugating α3β1 (recombinant or expressed on a cell line) with a β1-activating antibody, thereby creating an α3β1 complex that exhibits a more "open" or "active" conformation. This allows for easier identification of antibodies that bind to or activate active α3β1. The inventors further increase the probability of identifying the activating antibody by adding a ligand or ligand mimetic to this complex. This prepared pre-complex is used for the selection of the activating antibody.

[0172] The inventors further advance the identification of novel antibodies against allosteric sites by blocking the ligand-binding site of the integrin complex using ligand mimetic or blocking antibodies. [Examples]

[0173] Example 1 - Identification of antibody fragments using phage display To identify variable short-chain fragments (scFv), a phage display library of naive human scFv was subjected to a novel selection strategy to identify allosteric agonists. This strategy helps identify allosteric agonist binders, binders that are more specific to one integrin chain than the other, and binders that increase ligand binding. Furthermore, this strategy relies on structurally stabilized integrins (e.g., integrin α3β1, stabilized in an "active" conformation by complexing with a β1-activating antibody) to help identify structure-sensitive binders. In addition, this strategy uses ligand blockers (e.g., integrin α3β1 complexed with ligand-mimicking peptide LXY2 or ligand laminin, with or without a β1-activating antibody, to block binders into the ligand-binding pocket) to intentionally exclude binders from the ligand-binding pocket. In this specification, the inventors used two selection methods, referred to as Selection 1 and Selection 2, to identify binders. Selection 1 utilized recombinant integrins, while Selection 2 utilized cell surface-expressed integrins. For Selection 1, the screening round used three steps: pre-depletion, selection, and counter-selection. The pre-depletion step was used to remove binders that bind to the β1 chain of the α3β1 dimer by removing phage binders from the screening pool using negative selection against immobilized recombinant human integrin α4β1. Furthermore, some steps included a commercially available β1 agonist antibody (TS2 / 16) and a ligand-mimicking peptide (LXY2) along with immobilized integrin α4β1 to further remove any phage binders against these agents. Unbound phages were used for positive selection in the subsequent selection step. In this process, phages were incubated with immobilized recombinant human or mouse integrin α3β1 in the presence or absence of a β1 agonist antibody (antibody clone TS2 / 16 for human α3β1, and antibody clone 9EG7 for mouse α3β1) and ligand-mimicking peptide LXY2. Unbound phages were removed and discarded.The bound phages were eluted and used in the final step. In this step, both the β1 agonist antibody and the binder for LXY2 were removed using counter-selection against the immobilized β1 agonist antibody and LXY2 alone. All unbound phages were considered enriched as anti-integrin α3 allosteric binders. Furthermore, the target phage clone can be further enriched by repeating this process multiple times.

[0174] Next, the enriched phage pool from Selection 1 was amplified by choice, and then proceeded to Selection 2, enriching anti-integrin α3 allosteric agonists that bind to cell surface-expressed integrins, similar to the method described above. Herein, the screening rounds consist of: 1) a pre-depletion step against K562 cells (primarily expressing human α5β1 integrin and not overexpressing α3β1) in the presence or absence of a β1 agonist antibody and ligand-mimicking peptide LXY2; 2) positive selection against human K562 cells expressing integrin α3β1 in the presence or absence of the β1 agonist antibody TS2 / 16, and counter-selection against immobilized β1 agonist antibody and LXY2 alone. Cell line generation is described in the Methods.

[0175] The concentrated phage pool from Option 2 was further amplified by choice and plated using standard methods. 184 individual clones were identified and isolated for the clone amplification step. Each clone was purified to obtain a periplasmic extract (PE) solution containing the soluble parent clone scFv. These extracts were tested directly against human α3β1, mouse α3β1, and human α4β1, respectively, in or without LXY2, by integrin ELISA (as described in the method). The extracts were then tested against human α3-expressing K562 and α3-non-expressing K562 by flow cytometry.

[0176] After characterization of PE, the DNA of the variable domain of each scFv was sequenced. The CDR sequence was assigned to each of the 184 clones, then aligned and clustered to eliminate duplicates. The 184 clones isolated from selection yielded 25 unique sets of scFv CDR sequences. The sequencing data was combined with assay data from PE ELISA and FACS to enable selection of top hits.

[0177] Example 2 - Production of full-length IgG antibodies Using datasets generated from sequencing and characterization assays, the top five sequences were selected for reformatting. The heavy chain variable region DNA sequence of each scFv was added to full-length human heavy chain constant IgG1 DNA (IGHC1 gene transcript) by gene synthesis and cloning. The light chain human kappa variable region and lambda variable region DNA sequences of each scFv were added to full-length human light chain constant IGKC1 and IGLC1 DNA, respectively, while retaining their heavy / light chain pairs at the scFv stage. The heavy chain and light chain DNA constructs were cloned into separate cloning vectors and then transferred to mammalian expression vectors.

[0178] Each of the five paired constructs was transfected and expressed in 10 mL of mammalian cells, and antibodies were then isolated using protein A purification. Antibody samples were subjected to reducing and non-reducing SDS-PAGE and SEC-HPLC for quality control. Antibody samples were found at the expected molecular weight on the SDS-PAGE gel, and SEC-HPLC peaks indicated that the samples were reasonably pure.

[0179] Example 3 - Verification of antibody binding using ELISA Five full-length IgG antibodies were named Ab74 A100-A104, or simply Ab74. First, the binding of the five Ab74 antibodies to ECD was characterized by direct integrin ELISA. Briefly, BSA, recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD were coated onto plates overnight and then incubated with each of the five Ab74 antibodies individually or with an isotype human IgG1 antibody-negative control or a commercially available anti-human α3 antibody-positive control. Binding detection was performed by incubation with an anti-human IgG1 antibody horseradish peroxidase (HRP) conjugate, followed by treatment with a fluorescent substrate to develop the reaction product, and then reading the average fluorescence intensity using a plate reader.

[0180] ELISA results showed that the five Ab74 antibodies preferentially bound to human α3β1 ECD over all other antigens coated on the plate (Figures 1A-1D). Isotype-negative and anti-α3-positive controls yielded the expected negative and positive results, confirming low assay background and a positive signal against human α3, respectively. Two of the five Ab74 antibodies showed low binding to mouse α3, while all five Ab74 antibodies showed only background signals against coated BSA and human α4β1 ECD. ELISA confirmed that Ab74 binds to human α3 ECD but not to human β1 ECD.

[0181] To further improve the binding site, individual human α3 domains were recombinantly expressed and purified in mammalian cells. Ab74 was tested against BSA, soluble human α3 Thigh-Genu, human α3 Calf1-Calf2, or human α3β1 ECD by direct integrin ELISA. The data suggest that Ab74 A101 binds to the Thigh-Genu region in the sequence of the expressed construct (Figure 2).

[0182] Example 4 - Verification of antibody binding using a flow cytometry-based assay To verify antibody binding on cells, human mouse integrin α3 or mouse integrin α3-expressing K562 cells were generated as described in the method.

[0183] K562 cells overexpressing either human mouse integrin α3 or mouse integrin α3 were treated, and after both treatments and secondary antibody staining with anti-human IgG1 antibody conjugated with a fluorophore, all full-length human IgG antibodies were detected by flow cytometry. The results in Table 3 show high detection of Ab74 in both human integrin α3-expressing K562 cells and mouse integrin α3-expressing K562 cells compared to negative control isotype antibodies. [Table 4]

[0184] Example 5 - Increased ligand binding by human integrin α3β1-expressing cells in the presence of an agonist antibody. To investigate ligand-binding agonization, human α3-expressing K562 cells were treated with anti-α3 Ab74, a negative control isotype antibody, or a positive control commercially available β1 agonist antibody TS2 / 16 in the presence of the biotinylated ligand-mimicking peptide LXY2 in low-affinity Ca2+ / Mg2+ buffer. LXY2 binding was detected by treating cells with streptavidin fluorophore conjugate and reading the cells using a flow cytometer. Negative and positive controls accurately showed low to non-binding and high ligand binding, respectively, in the low-affinity buffer. The results in Table 4 show that Ab74 increases LXY2 binding in the low-affinity buffer compared to the isotype antibody. [Table 5]

[0185] Example 6 - Increased ligand binding by mouse integrin α3β1-expressing cells in the presence of an agonist antibody. Cross-reactivity with mouse α3 was characterized by repeated ligand experiments against mouse α3-expressing K562. Briefly, cells were treated with anti-α3 Ab74 or a negative control isotype antibody or a positive control commercially available β1 agonist antibody 9EG7 in the presence of the biotinylated ligand-mimicking peptide LXY2 in low-affinity Ca2+ / Mg2+ buffer. LXY2 binding was detected by treating the cells with streptavidin fluorophore conjugate and reading the cells with a flow cytometer. Negative and positive controls accurately showed low to non-binding and high ligand binding, respectively, in the low-affinity buffer. The results in Figures 3A to 3D show that Ab74 also increases LXY2 binding in the low-affinity buffer.

[0186] Example 7 - Reduction of cell migration in the presence of an integrin agonist antibody. The adherent human ovarian cancer cell line SK-OV-3 expresses high levels of integrin α3β1, which mediates ligand binding to laminin-511. Tissue culture-treated 96-well plates were coated with the integrin α3 ligand laminin-511 and incubated overnight. The following day, SK-OV-3 cells in serum-free medium were plated into the wells to allow adhesion. After 16 hours, scratching was performed using a sterile plastic P200 pipette tip, and anti-α3 Ab74, a negative control isotype antibody, or a positive control β1 agonist antibody was added in complete medium. Changing from serum-free medium to a medium containing fetal bovine serum (FBS) promotes cell migration, thereby facilitating cell motility and wound closure through migration. After 48 hours, the medium was removed, cells were fixed with 4% paraformaldehyde, and then stained with 0.2% crystal violet. The results in Figures 4A to 4E show inhibition of wound closure in wells treated with all Ab74 antibodies or positive control anti-β1 agonist antibodies compared to isotype-treated negative controls or blocking anti-α3 antibodies that resulted in wound closure.

[0187] Example 8: Integrin agonist antibody targeting the -thigh-genu domain To further improve the antibody binding epitope, several α3 integrin domains were individually "exchanged" with their homologous counterparts in a similar protein, human integrin α7 (described in the methods), which also contains thigh-genu, calf 1, and calf 2 domains.

[0188] For this purpose, integrin DNA constructs were created in which the Thigh-genu, Calf 1, and Calf 2 domains on integrin α7 replaced equivalent domains on integrin α3 in mammalian expression plasmids. These three constructs were individually transfected into HEK-293 cells using lipofectamine and grown under culture conditions for 48 hours. These cells were then treated with Ab74 or a negative control isotype antibody or a positive control commercially available α3 antibody P1B5, stained with fluorophore conjugate anti-human IgG1, and detected by flow cytometry.

[0189] The results in Table 5 show that Ab74 detection was observed in cells transfected with either full-length integrin α3 DNA, a construct containing integrin α3 with calf-2 of integrin α7, or a construct containing integrin α3 with calf-1 of integrin α7. However, little to no binding occurred in integrin α3 constructs replaced with the thigh-genu domain of integrin α7, suggesting that the five Ab74 antibodies recognize the epitope in the thigh-genu region of α3.

[0190] As shown in Table 5, for each integrin domain, DNA constructs were created in which each domain was swapped with its homologous integrin α7 counterpart. These constructs had ITGA7 Thigh-genu, Calf 1, and Calf 2 individually inserted into α3, replacing the wild-type sequence. These DNA constructs were then cloned into mammalian expression vectors, individually transfected into mammalian cells for 48 hours, and then incubated with activated human anti-α3 antibody. Antibody binding was detected by staining the cells with an anti-hIgG1 antibody APC conjugate and reading the results using a flow cytometer. [Table 6]

[0191] Example 9 - Method Cell culture and transient protein expression in the HEK293 cell line HEK293 cells were cultured in serum-free CD medium (Sino Biological catalog number SMM 293-TI) until the optimal cell density was reached. The expression vector was added to the cells in the presence of TF1 transfection reagent, and serum-free feeder solution (Sino Biological catalog number M293-SUPI-100) was added to the culture on days 1, 3, and 5 of transfection. Cells were harvested on day 7 of culture and proceeded to protein purification.

[0192] Protein purification from HEK293 cells The cells were removed by centrifugation, and the culture supernatant was collected for protein purification.

[0193] Affinity purification: The column was equilibrated with loading buffer, and the culture supernatant was loaded onto the column. The column was re-equilibrium, and the target protein was eluted by a buffer gradient containing imidazole (Ni affinity) or glycine and NaCl (protein A affinity or FLAG affinity). The protein was subjected to further buffer exchange to remove excess imidazole or other salts. The protein solution was concentrated, and the protein concentration and purity were assayed by the corresponding method. The protein concentration was measured by UV, and its purity was assayed by SDS-PAGE and Western blotting.

[0194] Cloning in expression vectors Restriction site 1 - Kozak sequence - signal peptide - target protein - stop codon - restriction site 2.

[0195] Signal peptide N-terminus-MGWSCIILFLVATATGVHS-(SEQ ID NO: 57)

[0196] Protein tags and characteristics Some constructs have an N-terminal FLAG, a C-terminal 6XHis, three Gly4Ser linkers, and 3C protease cleavage sites.

[0197] N-terminal FLAG tag MDYKDDDDK (sequence number 58)

[0198] C-terminal 6X His tag HHHHHH (sequence number 59)

[0199] (Gly4Ser)3 Linker GGGGSGGGGSGGGGS (Sequence No. 60)

[0200] Prescission protease (3C) LEVLFQGP (SEQ ID NO: 61) cleavage site (LEVLFQ / GP (SEQ ID NO: 61) (" / " indicates the cleavage site))

[0201] Domain-swapped mammalian expression constructs To test the antibody for domain specificity, individual domains derived from ITGA3 were replaced in the DNA sequence with homologous domains derived from ITGA7 (Figure 5). These were then inserted into a CMV-driven mammalian expression vector (pCMV6-Neo) and transient transfection was performed.

[0202] Sequence ID 62: Full-length human ITGA3 swapped with ITGA7 Thigh-Genu: *

[0203] Sequence ID 63: Full-length human ITGA3 swapped in ITGA7 Calf 1: *

[0204] Sequence ID 64: Full-length human ITGA3 in which Calf 2 is replaced by ITGA7 Calf 2: *

[0205] Cell line generation K562 cells from ATCC were transfected with a linear expression plasmid containing human integrin α3 by electroporation and maintained for 2 weeks under G418 selectivity at 0.5 mg / mL. Cells were enriched for high integrin expression by fluorescence-activated cell sorting (FACS) according to a staining protocol for anti-integrin α3 antibody staining using the commercially available antibody P1B5 (Millipore Sigma, MA Waltham, MA, USA).

[0206] K562 mouse α3β1 and K562 cynomolgus monkey α3β1:ATCC cells were transfected by electroporation with linear expression plasmids containing either a C-terminal FLAG tag, mouse or cynomolgus monkey integrin α3, and maintained for 2 weeks under 0.8 mg / mL puromycin selectivity. Cells were enriched for high integrin expression by fluorescence-activated cell sorting (FACS) according to a staining protocol for anti-FLAG antibody (Sino Biological, China).

[0207] Cell adhesion assay, fluorescent reporter Target integrin-expressing K562 cells expressing human integrin α3β1 were washed with TBS, and 50,000 cells / well were placed in ligand-coated wells of a highly bounded, transparent 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) with a total of 90 μL of assay buffer (indicated as HEPES-CaMg, Ca 2+ and Mg 2+Cells were transferred in 20 mM HEPES / 2 mg / mL glucose / 140 mM NaCl (containing 1 mM of each antibody). The plates were incubated at 37°C for 30 minutes in the presence of the antibody. To induce detachment of non-adherent cells, the plates were gently inverted and maintained at room temperature for 45 minutes. The plates were returned to an upright position, and the wells were rapidly aspirated using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). Adherent cells were quantified using CyQuantNF (Invitrogen, Waltham, MA, USA). For min-max normalization, a negative control assay buffer (HEPES buffer containing 10 mM EDTA, labeled HEPES-EDTA) and a positive control assay buffer (1 mM Mn, labeled TBS-Mn) were used. 2+ and 200uM Ca 2+ The plate contained TBS (which included [unclear]).

[0208] Cell adhesion assay, automated imaging Target integrin-expressing K562 cells expressing human integrin α3β1 were washed with TBS, and 50,000 cells / well were placed in ligand-coated wells of a highly bounded, transparent 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) with a total of 90 μL of assay buffer (indicated as HEPES-CaMg, Ca 2+ and Mg 2+Cells were transferred in 20 mM HEPES / 2 mg / mL glucose / 140 mM NaCl (containing 1 mM of each antibody). The plates were incubated at 37°C for 30 minutes in the presence of antibodies. To induce detachment of non-adherent cells, the plates were gently inverted and maintained at room temperature for 45 minutes. The plates were returned to an upright position, and the wells were fixed at room temperature for 10 minutes with a paraformaldehyde stock solution to a final concentration of 2% while inverted. The plates were returned to an upright position and rapidly aspirated using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). Adherent cells were quantified using DAPI and an automated imaging system with a nuclear segmentation algorithm. In min-max normalization, negative control assay buffer (TBS containing 10 mM EDTA, labeled TBS-EDTA) and positive control assay buffer (TBS containing 1 mM Mn2+ and 200 μM Ca2+, labeled TBS-Mn) were included in the plates.

[0209] Direct integrin ELISA A highly bound black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated overnight at 4°C with 30 μL of TBS containing 3 μg / mL recombinant integrin. The plate was shaken to remove the liquid, and 90 μL of TBS containing 5% bovine serum albumin (w / v), 0.05% Triton X-100 (v / v), and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA) was added and blocked by incubation for 1 hour. After incubation, the plate was washed three times with 100 μL of TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL of assay buffer (indicated as TBS-T, containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68) was added to each well. 1 μL of the test antibody stock solution was added to each corresponding well, the plate was centrifuged at 1000 g for 1 minute, and incubated at room temperature for 1 hour. The plate was washed three times with 100 μL of TBS using an automated plate washer. 30 μL of staining buffer (TBS-T containing 1:2000 dilution of anti-IgG HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated for 30 minutes. The plate was washed three times with 100 μL using an automated plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added and incubated at room temperature for 30 minutes. The plate was analyzed at 563 / 587 nm using a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0210] Integrin sandwich ELISA A highly bound black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated overnight at 4°C with 30 μL of TBS containing 4 μg / mL of anti-integrin antibody. Assay buffer (indicated as TBS-T, containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA)) was prepared before the following steps. The plate was shaken to remove the liquid, and 90 μL of TBS-T containing 5% bovine serum albumin (w / v) (Sigma-Aldrich, St. Louis, MO, USA) was added and blocked by incubation for 1 hour. After incubation, the plate was washed three times with 100 μL of TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL of assay buffer (TBS-T containing 4 μg / mL tagged recombinant integrin) was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 1 hour. The plate was washed three times with 100 μL of TBS using an automated plate washer. 30 μL of staining buffer (TBS-T containing 1:2000 dilution of anti-tagged antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. The plate was washed three times with 100 μL using an automated plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and allowed to develop color at room temperature for 30 minutes. The plates were analyzed at 563 / 587 nm using a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0211] Recombinant integrin function assay (SoLISA), integrin detection A highly bound black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated overnight at 4°C with 30 μL of TBS containing 8 μg / mL of ligand. Assay buffer (indicated as TBS-T, containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA)) was prepared before the following steps. The plate was shaken to remove the liquid, and 90 μL of TBS-T containing 5% bovine serum albumin (w / v) (Sigma-Aldrich, St. Louis, MO, USA) was added and blocked by incubation for 1 hour. After incubation, the plate was washed three times with 100 μL of TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL of assay buffer (containing 4 μg / mL tagged recombinant integrin and 1 mM Ca 2+ / 1mM Mg 2+ , 1mM Mn 2+ / 200μM Ca 2+TBS-T containing one of the following: or 10 mM EDTA, was added to each well. 1 μL of an appropriate concentration of agonist antibody (or isotype control) stock solution was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 3 hours. The plate was washed three times with 100 μL of TBS using an automated plate washer. 30 μL of staining buffer (TBS-T containing a 1:2000 dilution of anti-tag antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. The plate was washed three times with 100 μL using an automated plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and allowed to develop at room temperature for 30 minutes. The plates were analyzed at 563 / 587 nm using a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0212] Recombinant integrin function assay (SoLISA), ligand detection A highly bound black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated overnight at 4°C with 30 μL of TBS containing 4 μg / mL of anti-tag antibody. Assay buffer (indicated as TBS-T, containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA)) was prepared before the following steps. The plate was shaken to remove the liquid, and 90 μL of TBS-T containing 5% bovine serum albumin (w / v) was added and blocked by incubation for 1 hour. After incubation, the plate was washed three times with 100 μL of TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL of capture buffer (TBS-T containing 4 μg / mL of tagged recombinant integrin) was added to each well. 30 μL of assay buffer (containing 8 μg / mL of tagged recombinant ligand and 1 mM Ca) was added. 2+ / 1mM Mg 2+ , 1mM Mn 2+ / 200μM Ca 2+TBS-T containing either 10 mM EDTA or 10 mM EDTA was added to each well. 1 μL of an appropriate concentration of agonist antibody (or isotype control) stock solution was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 3 hours. The plate was washed three times with 100 μL of TBS using an automated plate washer. 30 μL of staining buffer (TBS-T containing 1:2000 dilution of anti-ligand antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. The plate was washed three times with 100 μL using an automated plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and allowed to develop at room temperature for 30 minutes. The plates were analyzed at 563 / 587 nm using a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0213] Recombinant integrin function assay (SoLISA), antibody detection A highly bound black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated overnight at 4°C with 30 μL of TBS containing 8 μg / mL of ligand. Assay buffer (indicated as TBS-T, containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA)) was prepared before the following steps. The plate was shaken to remove the liquid, and 90 μL of TBS-T containing 5% bovine serum albumin (w / v) (Sigma-Aldrich, St. Louis, MO, USA) was added and then blocked by incubation for 1 hour. After incubation, the plate was washed three times with 100 μL of TBS using an automated plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL of assay buffer (containing 4 μg / mL tagged recombinant integrin and 1 mM Ca 2+ / 1mM Mg 2+ 1mM Mn 2+ / 200μM Ca 2+TBS-T containing either 10 mM EDTA or 10 mM EDTA was added to each well. 1 μL of an appropriate concentration of agonist antibody (or isotype control) stock solution was added to each well. The plate was centrifuged at 1000 g for 1 minute and incubated at room temperature for 3 hours. The plate was washed three times with 100 μL of TBS using an automated plate washer. 30 μL of staining buffer (TBS-T containing 1:2000 dilution of anti-IgG antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. The plate was washed three times with 100 μL using an automated plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and allowed to develop at room temperature for 30 minutes. The plates were analyzed at 563 / 587 nm using a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0214] Flow cytometry assay for soluble ligand (laminin 511) binding The day before the assay, integrin-expressing K562 cells were counted in antibiotic-containing medium, washed with 10 mL of PBS, centrifuged, and resuspended in complete medium without the positive-selective antibiotic. These were cultured overnight at 37°C and 5% CO2. Laminin-511 E8 fragment Fc (Acro Biosystems, Newark, DE, USA) was conjugated with anti-human IgG Alexa Fluor 647 conjugate (Jackson Immuno Research Labs, West Grove, PA, USA) at a molar ratio of 1:1.5 (5 μg of laminin-511 E8 Fc per test group), and incubated in the dark at room temperature for 30 minutes. The cells were centrifuged and resuspended at 10 million cells / mL in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, Hampton, NH, USA). Human Fc block was added to the cells (BD Biosciences, Franklin Lakes, NJ, USA) to a final concentration of 25 μg / mL, and incubated on ice for 15 minutes. After incubation, the concentration was diluted to 1 million cells / mL by adding FACS buffer. The cells were transferred to a V-bottom 96-well plate [catalog number 290-8116-01V], and then 1 mM Ca was added. 2+ / 1mM Mg 2+ or 1mM Mn 2+ / 200μM Ca 2+40 μL of assay buffer containing one of either 1 mM Ca 2+ / 1 mM Mg 2+ or 1 mM Mn 2+ / 200 μM Ca 2+ or 10 mM EDTA was added to each well, followed by the addition of agonist antibody or isotype and incubation for 5 minutes at room temperature. Next, 5 μL of laminin 511 E8 Fc / Ab-AF647 solution was added to each well and incubated for 25 minutes at room temperature. The plate was washed with 200 μL of assay buffer. To detect the binding of the agonist antibody, the cells were resuspended in 100 μL of detection buffer (assay buffer containing 2.5 - 5.0 μg / mL anti-IgG (BD Pharmigen)) and incubated for 30 minutes at room temperature in the dark. The plate was washed with 200 μL of assay buffer, and the pellet was resuspended in 100 μL of freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [Catalog number AA47377-9M] and incubated for 10 minutes on ice. Finally, the cells were resuspended in PBS and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA).

[0215] Flow Cytometry-based Soluble Ligand (Ligand Mimetic) Binding Assay One day prior to the assay, integrin-expressing K562 cells in antibiotic-containing medium were counted, washed with 10 mL of PBS, centrifuged, and resuspended in complete medium without the positive selection antibiotic. These were cultured overnight at 37 °C and 5% CO2. The cells were centrifuged and resuspended at 10 million cells / mL in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, Hampton, NH, USA). Human Fc block was added to the cells (BD Biosciences, Franklin Lakes, NJ, USA) to a final concentration of 25 μg / mL and incubated for 15 minutes on ice. After incubation, FACS buffer was added to dilute the concentration to 1 million cells / mL. After transferring the cells to a V-bottom 96-well plate [Catalog number 290-8116-01V], 1 mM Ca 2+ / 1 mM Mg 2+ or 1 mM Mn 2+ / 200 μM Ca 2+Alternatively, 40 μL of assay buffer containing one of the 10 mM EDTAs was added to each well, followed by the addition of the agonist antibody or isotype, and incubated at room temperature for 5 minutes. Next, 5 μL of 10X biotinylated ligand mimetic was added to each well and incubated at room temperature for 25 minutes. The plate was washed with 200 μL of assay buffer. To detect agonist antibody binding, the cells were resuspended in 100 μL of detection buffer (assay buffer containing 2.5–5.0 ug / mL of anti-IgG (BD Pharmigen)). The biotinylated ligand mimetic was detected by fluorescently tagged streptavidin and incubated in the dark at room temperature for 30 minutes. The plate was washed with 200 μL of assay buffer, and the pellet was resuspended in 100 μL of freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [catalog no. AA47377-9M] and incubated on ice for 10 minutes. Finally, the cells were resuspended in PBS and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA).

[0216] Wound healing / scratch assay The day before, a tissue culture-treated flat 96-well plate was coated with 100 μL of 2.0 μg / mL laminin-511 (iMatrix) prepared in 1×PBS under sterile conditions and left overnight at 4°C. The following day, the coating solution was aspirated and the plate was blocked at room temperature for 1 hour with 100 μL of sterile 2% FBS. To detach and seed cells for the scratch assay, the SKOV3 cell layer was treated with 0.25% trypsin-EDTA and seeded at a rate of 30,000 cells per well in 100 μL of warm serum-free medium. The plate was centrifuged at 500 g for 5 minutes to settle the cells and incubated overnight in a cell incubator. The following day, vertical wounds / scratches were created in the center of each well using a sterile p200 tip. Agonist or isotype antibody treatment was prepared in warm complete medium, and 100 μL of the treatment was added to each well. Finally, Ca 2+ Mg 2+ or Mn 2+It was added to a final concentration of 0.5 mM in 10 μL per well. The wound healing process was observed for wound closure at the 24-hour time point when treatment with only complete medium had almost closed the wound. The medium was aspirated, the cell layer was washed with 200 μL of PBS, and fixed with 4% PFA at 4 °C for 10 minutes. 200 μL of 0.5% crystal violet was added to each well and stained at room temperature for 30 minutes.

[0217] Transient transfection of 293HEK with integrin α subunit chimeric DNA constructs 293HEK cells were plated in 6-well plates at 500,000 cells / well. On the day of transfection, the complete medium was aspirated, the cell layer was washed with 2 mL of PBS, and then 800 μL of Opti-MEM was gently added to the cells. A transfection agent was prepared using 2.5 μg of DNA and 3 μL of Lipofectamine 2000 in 250 μL of Opti-MEM and then incubated at room temperature for 5 minutes. The solution was dropped into the wells, and after incubation overnight, it was replaced with complete medium. The cells were analyzed for Ab74 binding by flow cytometry and detected with an anti-human IgG1 antibody conjugated to a fluorophore.

[0218] Additional sequences Human recombinant integrin α3β1: ITGA3 sp|P26006|33-99; Protein sequence: 1034 amino acids ITGB1 sp|P05556|21-728; Protein sequence: 781 amino acids (Sequence number 66).

[0219] Human recombinant integrin α3β1 domain Calf1-Calf2: ESNLQMRAAFVSEQQQQKLSRLQYSRDVRKLLLSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSVRPPGACQANETIFCELGNPFKRNQRMELLIAFEVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLLVDYTLQTSLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLEWPYEVSNGKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGPPPVTLAAAKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENHHHHHH(Sequence ID 67).

[0220] Human recombinant integrin α3β1 domain Thigh: MGWSCIILFLVATATGVHSMDYKDDDDKGGGGSGGGGSGGGGSLEVLFQGPLRARPVINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQLEVLFQGPGGGGSGGGGSGGGGSHHHHHH(Sequence ID 68).

[0221] Mouse recombinant integrin α3β1: Mouse α3 ECD sequence; protein sequence: 1036 amino acids Mouse β1 ECD sequence; protein sequence: 781 amino acids (Sequence ID 70).

[0222] Mouse recombinant integrin α3β1 domain Calf1-Calf2: MGWSCIILFLVATATGVHSDSNLQMRAAFLSEQLQPLSRLQYSRDTKKLFLSINVTNSPSSQRAGEDAHEALLTLEVPSALLLSSVRPSGTCQANNETILCELGNPFKRNQRMELLIAFEVIGVTLHTRDLPVLLQLSTSSHQDNLQPVLLTLQVDYTLQASLSLMNHRLQSFFGGTVMGEAAMKTAEDVGSPLKYEFQVSPVGDGLAALGTLVLGLEWPYEVTNGKWLLYPTEITIHSNGSWPCQPSGNLVNPLNLTLSDPGVTPLSPQRRRRQLDPGGDQSSPPVTLAAAKKAKSETVLTCSNGRARCVWLECPLPDTSNITNVTVKARVWNSTFIEDYKDFDRVRVDGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGENHHHHHH(Sequence ID 71).

[0223] The embodiments described above are provided to illustrate the present disclosure, but are not intended to limit its scope. Other variations of the present disclosure will be readily apparent to those skilled in the art and are included in the appended claims. All publications, databases, internet sources, patents, patent applications, and accession numbers referenced herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An isolated antibody that binds to integrin α3β1 or a portion thereof, (1) X １ X ２ SGX ３ TFX ４ X ５ YX ６ X ７ X ８ The heavy chain complementarity-determining region 1 (CDR H1) containing the sequence of (Sequence No. 38), wherein X １ is A or K, and X ２ is A or T, and X ３ is F, G, or F, and X ４ is S or T, and X ５ is S or N, and X ６ is G, S, or A, and X ７ is M or I, and X ８ is H, N, or S, CDR H1; (2) CDR H2 containing the sequence GISGSADTTY (SEQ ID NO: 6), SISSSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), or WISAX １ NGNX ２ The sequence contains up to two amino acid substitutions relative to the sequence of N (SEQ ID NO: 39), and X １ If Y or N, X ２ CDR H2 is T or S; (3) CDR H3 containing a sequence having up to two amino acid substitutions to the sequence of VRDDIQLRD (SEQ ID NO: 11) or AREFPGWYFDY (SEQ ID NO: 13), or containing a sequence having up to four amino acid substitutions to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), or ARGVPPSGSGYYYLGLDY (SEQ ID NO: 15); (4) Light chain complementarity determination region 1 (CDR L1), X １ ASQX ２ ISX ３ Includes the sequence YLN (sequence number 40), X １ is Q or A, and X ２ is D or Y, and X ３ CDR L1 includes a sequence in which is N or S, or has up to three amino acid substitutions in the sequence QGDSLRSYYAS (SEQ ID NO: 23) or SGSSSNIGSNYVY (SEQ ID NO: 24); (5) CDR L2, which has the sequence YDASNLET (sequence number 25) or YX １ X ２ NX ３ The sequence includes a sequence having at most one amino acid substitution relative to the sequence of RPS (SEQ ID NO: 41), X １ is G or R, X ２ is K or N, X ３ CDR L2 is N or Q; and (6) CDR L3, X １ QX ２ YX ３ X ４ PX ５ Includes the sequence T (sequence number 42), X １ is L or Q, and X ２ is D or S, X ３ is N, S, or R, and X ４ is Y or T, X ５ CDR L3, which includes a sequence in which is L or P, or a sequence having up to two amino acid substitutions to the sequence of NSRDSSGNHWV (SEQ ID NO: 31) or AAWDDSLSGPV (SEQ ID NO: 32), Isolated antibodies containing [the specified substance].

2. An isolated antibody according to claim 1, (1) The CDR H1 includes one of the sequences AASGFTFSSYGMH (SEQ ID NO: 1), KASGYTFTSYGIS (SEQ ID NO: 2), KTSGFTFFTNYGIS (SEQ ID NO: 3), AASGFTFSSSYSMN (SEQ ID NO: 4), and KASGGTFSSYAIN (SEQ ID NO: 5), (2) The CDR H2 contains one of the sequences GISGSADTTY (SEQ ID NO: 6), WISAYNGNTN (SEQ ID NO: 7), WISANNGNSN (SEQ ID NO: 8), SISSSSSSYIY (SEQ ID NO: 9), and GIIPIFGTAN (SEQ ID NO: 10), (3) The CDR H3 contains one of the sequences VRDDIQLRD (SEQ ID NO: 11), ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREFPGWYFDY (SEQ ID NO: 13), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), and ARGVPGSGSGYYYLGLDY (SEQ ID NO: 15), (4) The CDR L1 includes one of the sequences QASQDISNYLN (SEQ ID NO: 21), RASQYISSYLN (SEQ ID NO: 22), QGDSLRSYYAS (SEQ ID NO: 23), and SGSSSNIGSNYVY (SEQ ID NO: 24), (5) The CDR L2 contains one of the sequences YDASNLET (SEQ ID NO: 25), YGKNNNRPS (SEQ ID NO: 26), and YRNNQRPS (SEQ ID NO: 27), (6) The CDR L3 includes one of the sequences LQDYNYPLT (SEQ ID NO: 28), LQDYSYPLT (SEQ ID NO: 29), QQSYRTPPT (SEQ ID NO: 30), NSRDSSGNHWV (SEQ ID NO: 31), and AAWDDSLSGPV (SEQ ID NO: 32). The isolated antibody according to claim 1.

3. The isolated antibody according to claim 1 or 2, wherein CDR H1 contains the sequence of SEQ ID NO: 1, CDR H2 contains the sequence of SEQ ID NO: 6, and CDR H3 contains the sequence of SEQ ID NO:

11.

4. The isolated antibody according to claim 1 or 2, wherein CDR H1 contains the sequence of SEQ ID NO: 2, CDR H2 contains the sequence of SEQ ID NO: 7, and CDR H3 contains the sequence of SEQ ID NO:

12.

5. The isolated antibody according to claim 1 or 2, wherein CDR H1 contains the sequence of SEQ ID NO: 3, CDR H2 contains the sequence of SEQ ID NO: 8, and CDR H3 contains the sequence of SEQ ID NO:

13.

6. The isolated antibody according to claim 1 or 2, wherein CDR H1 contains the sequence of SEQ ID NO: 4, CDR H2 contains the sequence of SEQ ID NO: 9, and CDR H3 contains the sequence of SEQ ID NO:

14.

7. The isolated antibody according to claim 1 or 2, wherein CDR H1 contains the sequence of SEQ ID NO: 5, CDR H2 contains the sequence of SEQ ID NO: 10, and CDR H3 contains the sequence of SEQ ID NO:

15.

8. The isolated antibody according to any one of claims 1 to 7, wherein CDR L1 contains the sequence of SEQ ID NO: 21, CDR L2 contains the sequence of SEQ ID NO: 25, and CDR L3 contains the sequence of SEQ ID NO:

28.

9. The isolated antibody according to any one of claims 1 to 7, wherein CDR L1 contains the sequence of SEQ ID NO: 22, CDR L2 contains the sequence of SEQ ID NO: 25, and CDR L3 contains the sequence of SEQ ID NO:

29.

10. The isolated antibody according to any one of claims 1 to 7, wherein CDR L1 contains the sequence of SEQ ID NO: 21, CDR L2 contains the sequence of SEQ ID NO: 25, and CDR L3 contains the sequence of SEQ ID NO:

30.

11. The isolated antibody according to any one of claims 1 to 7, wherein CDR L1 contains the sequence of SEQ ID NO: 23, CDR L2 contains the sequence of SEQ ID NO: 26, and CDR L3 contains the sequence of SEQ ID NO:

31.

12. The isolated antibody according to any one of claims 1 to 7, wherein CDR L1 contains the sequence of SEQ ID NO: 24, CDR L2 contains the sequence of SEQ ID NO: 27, and CDR L3 contains the sequence of SEQ ID NO:

32.

13. An isolated antibody according to any one of claims 1 to 12, comprising a heavy chain variable region having at least 90% identity with any one of sequence numbers 16 to 20.

14. An isolated antibody according to any one of claims 1 to 13, comprising a light chain variable region having at least 90% identity with any one sequence of sequence numbers 33 to 37.

15. An isolated antibody according to any one of claims 1 to 14, comprising HCDR1 having the sequence of SEQ ID NO: 1, HCDR2 having the sequence of SEQ ID NO: 6, HCDR3 having the sequence of SEQ ID NO: 11, LCDR1 having the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO:

28.

16. The isolated antibody according to claim 15, comprising a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO:

16.

17. An isolated antibody according to claim 15 or 16, comprising a light chain variable region having at least 90% identity with the sequence of SEQ ID NO:

33.

18. An isolated antibody according to any one of claims 1 to 14, comprising HCDR1 having the sequence of SEQ ID NO: 2, HCDR2 having the sequence of SEQ ID NO: 7, HCDR3 having the sequence of SEQ ID NO: 12, LCDR1 having the sequence of SEQ ID NO: 22, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO:

29.

19. The isolated antibody according to claim 18, comprising a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO:

17.

20. An isolated antibody according to claim 18 or 19, comprising a light chain variable region having at least 90% identity with the sequence of SEQ ID NO:

34.

21. An isolated antibody according to any one of claims 1 to 14, comprising HCDR1 having the sequence of SEQ ID NO: 3, HCDR2 having the sequence of SEQ ID NO: 8, HCDR3 having the sequence of SEQ ID NO: 13, LCDR1 having the sequence of SEQ ID NO: 21, LCDR2 having the sequence of SEQ ID NO: 25, and LCDR3 having the sequence of SEQ ID NO:

30.

22. The isolated antibody according to claim 21, comprising a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO:

18.

23. An isolated antibody according to claim 21 or 22, comprising a light chain variable region having at least 90% identity with the sequence of SEQ ID NO:

35.

24. An isolated antibody according to any one of claims 1 to 14, comprising HCDR1 having the sequence of SEQ ID NO: 4, HCDR2 having the sequence of SEQ ID NO: 9, HCDR3 having the sequence of SEQ ID NO: 14, LCDR1 having the sequence of SEQ ID NO: 23, LCDR2 having the sequence of SEQ ID NO: 26, and LCDR3 having the sequence of SEQ ID NO:

31.

25. The isolated antibody according to claim 24, comprising a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO:

19.

26. An isolated antibody according to claim 24 or 25, comprising a light chain variable region having at least 90% identity with the sequence of SEQ ID NO:

36.

27. An isolated antibody according to any one of claims 1 to 14, comprising HCDR1 having the sequence of SEQ ID NO: 5, HCDR2 having the sequence of SEQ ID NO: 10, HCDR3 having the sequence of SEQ ID NO: 15, LCDR1 having the sequence of SEQ ID NO: 24, LCDR2 having the sequence of SEQ ID NO: 27, and LCDR3 having the sequence of SEQ ID NO:

32.

28. The isolated antibody according to claim 27, comprising a heavy chain variable region having at least 90% identity with the sequence of SEQ ID NO:

20.

29. An isolated antibody according to claim 27 or 28, comprising a light chain variable region having at least 90% identity with the sequence of SEQ ID NO:

37.

30. An isolated antibody according to any one of claims 1 to 29, comprising an Fc polypeptide having at least 90% identity with the sequence of SEQ ID NO:

43.

31. An isolated antibody according to any one of claims 1 to 30, which binds to cells expressing integrin α3β1 or a part thereof.

32. The isolated antibody according to claim 31, wherein the cells are podocytes or neutrophils.

33. An isolated antibody according to any one of claims 1 to 30, which binds to the α3 portion of the integrin α3β1.

34. The isolated antibody according to claim 33, which binds to a sequence within the high-genu region of the α3 portion.

35. An isolated antibody according to any one of claims 1 to 34, which binds to the sequence of SEQ ID NO: 44 or a sequence within the sequence of SEQ ID NO:

44.

36. The isolated antibody according to any one of claims 1 to 35, wherein the antibody is a monoclonal antibody.

37. The isolated antibody according to any one of claims 1 to 36, wherein the antibody is a humanized antibody.

38. The isolated antibody according to any one of claims 1 to 37, wherein the antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, or a single-stranded Fv(scFv) antibody.

39. The isolated antibody according to any one of claims 1 to 38, wherein the antibody is a bispecific antibody.

40. An isolated nucleic acid encoding an isolated antibody according to any one of claims 1 to 38.

41. An expression vector comprising the nucleic acid described in claim 40.

42. An isolated host cell containing the vector according to claim 41.

43. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

44. A method for treating a disease or condition associated with podocyte loss in a subject requiring treatment, comprising administering to the subject an isolated antibody according to any one of claims 1 to 38.

45. The method according to claim 44, wherein the disease or condition is a kidney disease, an autoimmune disease, cancer, or inflammation.

46. The method according to claim 44, wherein the disease or condition is a transplant surgery.

47. The method according to claim 44 or 45, wherein the kidney disease is a glomerular disease.

48. The method according to claim 47, wherein the glomerular disease is nephrotic disease, nephrotic disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

49. A method for identifying an antibody that binds to integrin α3β1 or a part thereof, 1) Removing the antibody that binds to the β1 chain of integrin α3β1 in the presence or absence of the ligand-mimicking peptide and / or the antibody; 2) Select from the remaining antibodies in step 1) an antibody that binds to integrin α3β1 in the presence or absence of the β1 agonist antibody; 3) Counter-selecting an antibody that binds to integrin α3β1 against an immobilized β1 agonist antibody or ligand-mimicking peptide alone; and 4) Repeat steps 1), 2), and 3) above in the presence of cell surface-expressed integrin α3β1 to enrich the antibody which is an integrin α3 allosteric agonist. Methods that include...

50. The method according to claim 49, wherein the ligand-mimetic peptide is LXY2.

51. The method according to claim 49 or 50, wherein step 1) and / or 2) are performed using human K562 cells that primarily express human α5β1 integrin and do not overexpress α3β1.

Images