Compositions and methods relating to integrin alpha3beta1

Abstract

Compositions and methods comprising antibodies that bind to integrin [alpha] 3 [beta] 1 are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 420,964, filed on October 31, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. Background Art

[0003] Integrin α3β1 is a key integrin found on the surface of cells including podocytes (cells that enclose glomerular capillaries within Bowman's capsule of the kidney). Integrin α3β1 is essential for podocytes to attach to the outside of blood vessels to form healthy glomeruli in the kidney. Antibodies that are allosteric agonists of integrin α3β1 can enhance integrin-dependent ligand binding and cell adhesion. Summary of the Invention

[0004] In one aspect, the disclosure features an isolated antibody that binds to integrin α3β1 or a portion thereof, the isolated antibody comprising:

[0005] (1) a heavy chain complementarity determining region 1 (CDR H1) comprising the sequence of X1X2SGX3TFX4X5YX6X7X8 (SEQ ID NO: 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;

[0006] (2) CDR H2 comprising a sequence having up to two amino acid substitutions relative to the sequence of GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), or the sequence of WISAX1NGNX2N (SEQ ID NO: 39), wherein X1 is Y or N; and X2 is T or S;

[0007] (3) CDR H3 comprising a sequence having up to two amino acid substitutions relative 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 relative to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), or ARGVPSGSGYYLGLDY (SEQ ID NO: 15);

[0008] (4) a light chain complementary determining region 1 (CDR L1) comprising the sequence of X1ASQX2ISX3YLN (SEQ ID NO: 40), or a sequence having up to three amino acid substitutions relative to the sequence of QGDSLRSYYAS (SEQ ID NO: 23) or SGSSSNIGSNYVY (SEQ ID NO: 24), wherein X1 is Q or A; X2 is D or Y; and X3 is N or S;

[0009] (5) CDR L2 comprising a sequence having at most one amino acid substitution relative 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

[0010] (6) CDR L3 comprising the sequence of X1QX2YX3X4PX5T (SEQ ID NO:42), or a sequence having up to two amino acid substitutions relative to the sequence of NSRDSSGNHWV (SEQ ID NO:31) or AAWDDSLSGPV (SEQ ID NO:32), wherein 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.

[0011] In some embodiments of this aspect, (1) CDR H1 comprises the sequence of any one of 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 the sequence of any one of 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 the sequence of any one of VRDDIQLRD (SEQ ID NO: 11), ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREFPGWYFDY (SEQ ID NO: 13), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14). NO: 14) and ARGVPSGSGYYLGLDY (SEQ ID NO: 15); (4) CDR L1 comprises the sequence of any one of QASQDISNYLN (SEQ ID NO: 21), RASQYISSYLN (SEQ ID NO: 22), QGDSLRSYYAS (SEQ ID NO: 23) and SGSSSNIGSNYVY (SEQ ID NO: 24); (5) CDRL2 comprises the sequence of any one of YDASNLET (SEQ ID NO: 25), YGKNNRPS (SEQ ID NO: 26) and YRNNQRPS (SEQ ID NO: 27); and (6) CDR L3 comprises the sequence of any one of 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).

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of any one of SEQ ID NOs: 16-20. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of any one of SEQ ID NOs: 33-37.

[0023] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 1, a HCDR2 having the sequence of SEQ ID NO: 6, a HCDR3 having the sequence of SEQ ID NO: 11, a LCDR1 having the sequence of SEQ ID NO: 21, a LCDR2 having the sequence of SEQ ID NO: 25, and a LCDR3 having the sequence of SEQ ID NO: 28. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 33.

[0024] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 2, a HCDR2 having the sequence of SEQ ID NO: 7, a HCDR3 having the sequence of SEQ ID NO: 12, a LCDR1 having the sequence of SEQ ID NO: 22, a LCDR2 having the sequence of SEQ ID NO: 25, and a LCDR3 having the sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 34.

[0025] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 3, a HCDR2 having the sequence of SEQ ID NO: 8, a HCDR3 having the sequence of SEQ ID NO: 13, a LCDR1 having the sequence of SEQ ID NO: 21, a LCDR2 having the sequence of SEQ ID NO: 25, and a LCDR3 having the sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 35.

[0026] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 4, a HCDR2 having the sequence of SEQ ID NO: 9, a HCDR3 having the sequence of SEQ ID NO: 14, a LCDR1 having the sequence of SEQ ID NO: 23, a LCDR2 having the sequence of SEQ ID NO: 26, and a LCDR3 having the sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 36.

[0027] In some embodiments, the antibody comprises a HCDR1 having the sequence of SEQ ID NO: 5, a HCDR2 having the sequence of SEQ ID NO: 10, a HCDR3 having the sequence of SEQ ID NO: 15, a LCDR1 having the sequence of SEQ ID NO: 24, a LCDR2 having the sequence of SEQ ID NO: 27, and a LCDR3 having the sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises a light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 37.

[0028] In some embodiments, the antibody comprises an Fc polypeptide that is at least 90% identical to the sequence of SEQ ID NO:43.

[0029] In some embodiments of the antibodies described herein, the antibody binds to a cell that expresses integrin α3β1 or a portion thereof. In certain embodiments, the cell is a podocyte, a T cell, a cancer cell, or a neutrophil.

[0030] 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 specific embodiments, the antibody binds to a 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 conformation of α3. In some embodiments, the antibody binds to and stabilizes α3 in a specific conformation.

[0031] 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.

[0032] In another aspect, the present disclosure also provides an isolated nucleic acid encoding the isolated antibody described herein.

[0033] In another aspect, the present disclosure provides an expression vector comprising a nucleic acid encoding the isolated antibody described herein.

[0034] In another aspect, the present disclosure provides an isolated host cell comprising the above-mentioned vector.

[0035] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody described herein and a pharmaceutically acceptable carrier.

[0036] In another aspect, the present disclosure provides a method for treating a disease or condition associated with podocyte loss in a subject in need thereof, the method comprising administering to the subject an isolated antibody described herein. In some embodiments, the disease or condition is kidney disease, autoimmune disease, cancer, or inflammation. In some embodiments, the disease or condition is a transplant procedure.

[0037] In some embodiments of this method, the kidney disease is a glomerular disease, such as nephritic disease, nephrotic disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

[0038] In another aspect, the disclosure features a method for identifying an antibody that binds to integrin α3β1 or a portion thereof, the method comprising:

[0039] 1) removing antibodies that bind to the β1 chain of integrin α3β1 in the presence or absence of ligand-mimicking peptides and / or antibodies;

[0040] 2) selecting an antibody that binds to integrin α3β1 from the remaining antibodies in step 1) in the presence or absence of a β1 agonist antibody;

[0041] 3) counterselecting antibodies that bind to integrin α3β1 against immobilized β1 agonist antibodies or ligand mimetic peptides alone; and

[0042] 4) Repeating the above steps 1), 2) and 3) to enrich antibodies that are allosteric agonists of integrin α3 in the presence of integrin α3β1 expressed on the cell surface.

[0043] In some embodiments of this method, the ligand mimetic peptide is LXY2. In some embodiments of this method, steps 1) and / or 3) are performed using a β1-containing integrin dimer other than α3β1 (e.g., 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.

[0044] In some embodiments of the method, steps 2) and / or 3) are performed using human K562 cells that overexpress α3β1. In some embodiments, steps 1) and / or 2) and / or 3) are performed in the presence of an agent that blocks the ligand binding site or domain of the integrin (e.g., an antibody or ligand).

[0045] In some embodiments, integrin α3β1 is stabilized in a specific conformation by pre-complexing with an activator or inhibitor (such as activating antibodies 9EG7 or TS2 / 16). In some other embodiments, integrin α3β1 is stabilized in a specific conformation by pre-complexing with an agent that selectively binds to the β chain of the integrin dimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figures 1A-1D Characterization of integrin agonist antibody binding by direct integrin ELISA. Plates were coated with bovine serum albumin (BSA), recombinant human integrin α3β1 ECD, recombinant human integrin α4β1 ECD, or recombinant mouse integrin α3β1 ECD and incubated with human anti-α3 Ab or isotype (n = 8 for each coating 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 Ab HRP conjugate and development with a fluorescent substrate, followed by reading the mean fluorescence intensity on a microplate reader.

[0048] Figure 2 Epitope mapping of integrin agonist antibodies by direct integrin ELISA. Recombinant integrin α3β1 domains or bovine serum albumin (BSA) were coated on plates and incubated with human anti-α3 Ab (red) or isotype (blue) (n = 4). Binding of Ab74 A101 was detected by incubation with anti-hIgG1 Ab HRP conjugate, development with a fluorescent substrate, and reading the mean fluorescence intensity on a microplate reader.

[0049] Figures 3A-3DFigure 3: Increased ligand binding of cells expressing mouse integrin α3β1 in the presence of agonist antibodies. K562 cells expressing α3β1 were incubated with the α3β1 ligand mimetic LXY2-biotin conjugate and either an integrin agonist antibody or an isotype control. Cells were then stained with a streptavidin-fluorophore conjugate and measured in a flow cytometer. (A) Ab74 A100, (B) Ab74 A101, (C) Ab74 A102, and (D) Ab74 A104 demonstrated increased LXY2 binding compared to the isotype control alone.

[0050] Figures 4A-4E Figure 3: Reduction of cell migration in the presence of integrin agonist antibodies as determined by wound healing assay. SK-OV-3 cells expressing α3β1 were plated onto ligand-coated wells and allowed to attach for 16 hours at 37°C. (A) Prior to addition of treatment, a scratch injury was created on the cell layer using a sterile pipette tip. (B) Isotype antibody control and (C) blocking anti-α3 antibodies did not reduce cell migration, allowing cells to close the wound. (D) Control anti-β1 agonist antibodies and (E) anti-α3 agonist antibody Ab74 A101 reduced wound closure after 16 hours.

[0051] Figure 5 : Schematic representation of domain-swapped mammalian expression constructs.

[0052] Figures 6A-6C Antibody staining of podocytes shows that the novel anti-integrin α3 antibody stains integrin α3β1 expressed on podocytes. Kidney sections from C57B / L6 wild-type mice were immunofluorescently stained with various antibodies (5 μg / mL) and imaged using confocal microscopy. Representative images of staining with Ab74_A100 (A), 9EG7 (B), or a human anti-mouse IgG1 isotype control antibody (C) are shown. Detailed Description of the Invention

[0054] I. Introduction

[0055] The present inventors have discovered new antibodies that bind to integrin α3β1 (e.g., sequences within the thigh-genu region of integrin α3β1). Such antibodies act as agonists of integrin α3β1 and can enhance integrin-dependent functions such as ligand binding and cell adhesion. In particular, given that integrin α3β1 is a key integrin on the surface of podocytes, the antibodies can be used to treat diseases and / or conditions associated with podocyte loss, for example, kidney diseases such as nephritic disease, renal disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

[0056] The inventors have also discovered that the novel anti-integrin α3 allosteric antibody induces intracellular signaling in the presence of external ligands. For example, when cells expressing integrin α3 are incubated with the novel anti-α3 integrin antibody in the absence of an integrin ligand, the level of phosphorylated focal adhesion kinase (pFAK) remains unchanged. However, co-incubation of cells with the novel antibody and the ligand laminin increases the relative level of pFAK.

[0057] II. Definitions

[0058] As used herein, the term "antibody" includes antibody fragments that retain binding specificity. For example, there are many well-characterized antibody fragments. Thus, for example, pepsin digests the C-terminus of an antibody at the disulfide bond in the hinge region to produce a dimer of F(ab)'2 (Fab, which itself is bound to V by disulfide bonds). H -C H 1 connected light chain). F(ab) '2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, edited by W.E. Paul, Raven Press, NY (1993)). Although various antibody fragments are defined based on the digestion of intact antibodies, those skilled in the art will appreciate that fragments can be synthesized de novo chemically or by utilizing recombinant DNA methods. Therefore, as used herein, the term antibody also includes antibody fragments produced by modifying whole antibodies or antibody fragments synthesized using recombinant DNA methods.

[0059] Antibodies as described herein can be composed of one or more polypeptides that are substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. The immunoglobulin genes confirmed include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, and a large number of immunoglobulin variable region genes. Light chain is classified as kappa or lambda. Heavy chain is classified as gamma, mu, alpha, delta or epsilon, which define 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.

[0060] It is known that the typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (VH ) refer to these light and heavy chains, respectively.

[0061] In an antibody, a substitution variant has at least one amino acid residue removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework changes are also contemplated. Examples of conservative substitutions are described above.

[0062] Substantial modifications to the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the region of the substitution, for example as a beta-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following categories:

[0063] (1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0064] (2) Polarity without charge: Cys, Ser, Thr, Asn, Gln;

[0065] (3) Acidic (negatively charged): Asp, Glu;

[0066] (4) Basic (positively charged): Lys, Arg;

[0067] (5) Residues that affect chain orientation: Gly, Pro; and

[0068] (6) Aromatic: Trp, Tyr, Phe, His.

[0069] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0070] A type of substitution that can be carried out is to change one or more cysteines that may be chemically reactive in the antibody into another residue, such as, but not limited to, alanine or serine. For example, there may be a substitution of non-classical cysteine. The substitution can be carried out in the CDR or framework region of the variable domain or the constant region of the antibody. In some embodiments, cysteine ​​is classical (for example, participating in the formation of disulfide bonds). Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can also be substituted, usually with serine, to improve the oxidative stability of the molecule and to prevent abnormal cross-linking. On the contrary, one or more cysteine ​​bonds can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0071] Antibodies include V H -V LDimers include single-chain antibodies (antibodies that exist as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv), in which the variable heavy chain and variable light chain regions are linked together (directly or through a peptide linker) to form a continuous polypeptide. Single-chain Fv antibodies are covalently linked V H -V L , which may consist of Vs linked directly or via a peptide-encoded linker H - and V L - nucleic acid expression of the coding sequence (eg, Huston et al., Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). H and V L When linked together as a single polypeptide chain, V H and V L The domains are non-covalently associated. Alternatively, the antibody may be another fragment. Other fragments may also be generated, for example, using recombinant technology, as soluble proteins or as fragments obtained from display methods. Antibodies may also include diabodies and minibodies. Antibodies of the present disclosure also include heavy chain dimers, such as antibodies from camels. In some embodiments, the antibodies are dimeric. In other embodiments, the antibodies may be in a monomeric form with active isoforms. In some embodiments, the antibodies are in a multivalent form, for example, a trivalent or tetravalent form.

[0072] As used herein, the terms "variable region" and "variable domain" refer to the portion of the amino acid sequence of the light and heavy chains of an antibody that includes the complementarity determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR3) and framework regions (FRs). The variable regions of the heavy and light chains are usually designated V H and V L The variable region is included in the Fab, F(ab')2, Fv and scFv antibody fragments described herein and is involved in specific antigen recognition.

[0073] As used herein, "complementarity determining regions (CDRs)" refer to the three hypervariable regions in each chain, which are interrupted by four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially starting from the N-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the antibody heavy chain in which it is present, while V L CDR1 is the CDR1 from the variable domain of the antibody light chain in which it is present.

[0074] The sequences of the framework regions of different light chains or heavy chains are relatively conserved within a species.The framework region of an antibody (ie, the combined framework regions of the constituent light and heavy chains) is used to position and arrange the CDRs in three-dimensional space.

[0075] The amino acid sequences of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, the North method (see, e.g., North et al., J Mol Biol. 406(2):228-256, 2011), Chothia, the International ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; 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 Hsegments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). The definition of antigen combining sites is also described in the following literature: 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).

[0076] As used herein, the term "allosteric agonist" refers to a molecule (e.g., an antibody) that binds to its 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 that is not the active site of the target to enhance, activate, or increase the response of the target to binding of its natural ligand.

[0077] As used herein, "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region or a portion thereof is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged with a variable region or a portion thereof having a different or altered antigenic specificity; or is altered, replaced or exchanged with a corresponding sequence from another species or from another antibody class or subclass.

[0078] As used herein, "humanized antibody" refers to an immunoglobulin molecule in which the CDRs of a donor antibody are grafted onto human framework sequences. Humanized antibodies may also contain residues of donor origin in the framework sequences. Humanized antibodies may also contain at least a portion of a human immunoglobulin constant region. Humanized antibodies may also contain residues that are neither present in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed 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. Pat. No. 4,816,567), including techniques such as "superhumanized" 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 USA 91:969, 1994).

[0079] The term "recombinant" when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0080] The terms "antigen," "immunogen," "antibody target," "target analyte," and similar terms are used herein to refer to a molecule, compound, or complex that is recognized by an antibody (i.e., can be specifically bound by an antibody). The terms can refer to any molecule that can be specifically recognized by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or a combination thereof (e.g., a phosphorylated or glycosylated polypeptide, etc.). One skilled in the art will understand that the terms do not indicate that the molecule is immunogenic in every case, but simply indicate that it can be targeted by an antibody.

[0081] Antibodies bind to "epitopes" on antigens. An epitope is a localization site on an antigen that is recognized and bound by an antibody. An epitope can include several amino acids or portions of several amino acids, such as 5 or 6 or more, such as 20 or more amino acids, or portions of those amino acids. In some cases, an epitope includes non-protein components, such as from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, when the target is a protein, the epitope can be composed of continuous amino acids, or composed of amino acids from different parts of the protein that are approached by protein folding (e.g., discontinuous epitopes). This is also true for other types of target molecules that form three-dimensional structures. An epitope typically includes at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0082] The terms "specific for," "specifically binds," and similar terms refer to a molecule (e.g., an antibody or antibody fragment) that binds to a target with an affinity that is at least 2-fold greater than that of a non-target compound, e.g., at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater. For example, an antibody that specifically binds to a target typically binds to the target with an affinity that is at least 2-fold greater than that of a non-target. Specificity can be determined using standard methods, such as solid phase ELISA immunoassays (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0083] The term "binds" with respect to an antibody target (e.g., an antigen, an analyte, an immune complex) typically indicates that the antibody binds to the majority of the antibody target in a pure population (assuming an appropriate molar ratio). For example, an antibody that binds to a given antibody target typically binds to at least 2 / 3 of the antibody target in solution (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). One skilled in the art will recognize that some variability will occur depending on the method and / or threshold for determining binding.

[0084] A "control" sample or value refers to a sample that serves as a reference (usually a known reference) for comparison with a test sample. For example, a test sample can be obtained from a test condition (e.g., in the presence of a test compound) and compared with a sample from a known condition (e.g., in the absence of a test compound (negative control) or in the presence of a known compound (positive control)). A control can also represent an average value or range collected from multiple tests or results. Those skilled in the art will recognize that a control can be designed to evaluate any number of parameters. For example, a control can be designed to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of benefits and / or side effects). A control can be designed for in vitro applications. Those skilled in the art will understand which controls are valuable in a given situation and can analyze data based on comparison with control values. Controls are also valuable for determining the significance of data. For example, if the value of a given parameter varies greatly in a control, the change in the test sample will not be considered significant.

[0085] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" means that two or more sequences or subsequences are identical or have a specified percentage of identical (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity, when compared and aligned for maximum correspondence over a comparison window or specified region) amino acid residues or nucleotides as measured using the BLAST 2.0 sequence comparison algorithm using the default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website ncbi.nlm.nih.gov / BLAST / , etc.). Such sequences are then said to be "substantially identical." Preferred algorithms may take into account gaps, etc., as described below. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 or more amino acids or nucleotides in length.

[0086] For sequence comparison, typically a sequence serves as a reference sequence, and a test sequence is compared thereto. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0087] As used herein, a "comparison window" includes a segment of any number of contiguous positions selected from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art.

[0088] Suitable algorithms for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are 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 together with the parameters described herein to determine percentages of sequence identity for nucleic acids and proteins disclosed herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for starting searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction stops when the following situation occurs: the cumulative alignment score decreases by an amount X from its maximum achieved value; due to the accumulation of one or more negative-scoring residue alignments, the cumulative score becomes zero or lower; or reaches the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The BLASTN program (for nucleotide sequences) defaults to using a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of two chains. For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains.

[0089] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, and their complements, in either single-strand or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0090] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (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)).

[0091] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms encompass amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0092] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the 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-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

[0093] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0094] III. Antibodies that bind to integrin α3β1 or a portion thereof

[0095] Provided herein are 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 α3 and β1 portions. Integrin α3β1 is highly expressed on the surface of renal podocytes and is essential for podocytes to attach to the outside of blood vessels to form healthy glomeruli in the kidney. This integrin is also expressed on other cells, such as T cells (Park et al., Integrinα3 promotes TH17 cell polarization and extravasation during autoimmune neuroinflammation, Science Immunology, Vol. 8(88), 2023), cancer cells (Ke et al., Novel monoclonal antibody against integrinα3 shows therapeutic potential for ovarian cancer, Cancer Sci., 111(10), p. 3478, 2020) and neutrophils (Lerman et al., Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrinα3β1-dependent, Blood, Dec 4, 2014; 124(24): 3515-23) as well as keratinocytes (Has et al., Integrin a3 mutations with kidney, lung, and skin disease. N Engl J Med 366:1508-1514, 2012), and can affect the function of cells expressing this integrin. The antibodies described herein act as allosteric agonist antibodies to integrin α3β1 and can enhance integrin-dependent ligand binding and cell adhesion, thereby preventing podocyte loss in urine and preventing renal function loss. These antibodies can also reduce T cell migration and infiltration to reduce autoimmune diseases; reduce cancer cell migration to reduce tumor growth and metastasis; and reduce pro-inflammatory neutrophil activation and tissue recruitment.

[0096] In some embodiments, the anti-α3β1 antibody is isolated (e.g., separated from a component of its natural environment (e.g., an animal, a biological sample)). In some embodiments, the anti-α3β1 antibody is a humanized antibody, or an antigen-binding fragment thereof. 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 a subject's cells (e.g., podocytes)).

[0097] In some embodiments, the α3 portion of the heterodimeric integrin α3β1 has a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to:

[0098]

[0099] The thigh-genu region is in bold in SEQ ID NO:45.

[0100] In some embodiments, the β1 portion of the heterodimeric integrin α3β1 has a sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: (SEQ ID NO: 46).

[0101] 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 specific embodiments, the antibody binds to the sequence of SEQ ID NO: 44 or a portion within the sequence of SEQ ID NO: 44.

[0102] VINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFA

[0103] GSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQKEC (SEQ ID NO: 44).

[0104] Generally speaking, the anti-α3β1 antibodies provided herein comprise 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 comprise 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 comprises the 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 comprises the first, second, and third light chain CDRs (LCDR1, LCDR2, and LCDR3).

[0105] In some embodiments, the antibody is an antibody fragment, such as Fab, F(ab')2, Fv or scFv. Antibody fragments can be generated using any means known in the art, including chemical digestion (e.g., papain or pepsin) and recombinant methods. Methods for separating and preparing recombinant nucleic acids are known to those skilled in the art (see Sambrook et al., Molecular Cloning.A Laboratory Manual (2nd Edition, 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). Antibodies 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).

[0106] In some embodiments, the antibodies of the present disclosure may comprise a heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementary determining region 1 (LCDR1), LCDR2, LCDR3, a heavy chain variable region (V H ), and / or light chain variable region (V L ) sequence.

[0107]

[0108] An isolated antibody that specifically binds to integrin α3β1 or a portion thereof (e.g., a sequence within the thigh-genu region of integrin α3β1) can comprise:

[0109] (1) a heavy chain complementarity determining region 1 (HCDR1) comprising the sequence of X1X2SGX3TFX4X5YX6X7X8 (SEQ ID NO: 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;

[0110] (2) HCDR2 comprising a sequence having up to two amino acid substitutions relative to the sequence of GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), or the sequence of WISAX1NGNX2N (SEQ ID NO: 39), wherein X1 is Y or N; and X2 is T or S;

[0111] (3) HCDR3 comprising a sequence having up to two amino acid substitutions relative 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 relative to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO: 12), AREYYDFWSGYPSGYAFDI (SEQ ID NO: 14), or ARGVPSGSGYYLGLDY (SEQ ID NO: 15);

[0112] (4) a light chain complementary determining region 1 (LCDR1) comprising the sequence of X1ASQX2ISX3YLN (SEQ ID NO: 40), or a sequence having up to three amino acid substitutions relative to the sequence of QGDSLRSYYAS (SEQ ID NO: 23) or SGSSSNIGSNYVY (SEQ ID NO: 24), wherein X1 is Q or A; X2 is D or Y; and X3 is N or S;

[0113] (5) LCDR2 comprising a sequence having at most one amino acid substitution relative 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

[0114] (6) LCDR3 comprising the sequence of X1QX2YX3X4PX5T (SEQ ID NO:42), or a sequence having up to two amino acid substitutions relative to the sequence of NSRDSSGNHWV (SEQ ID NO:31) or AAWDDSLSGPV (SEQ ID NO:32), wherein 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.

[0115] In some embodiments, the antibodies of the present disclosure comprise a HCDR1 having a sequence of any one of SEQ ID NOs: 1-5, or a variant thereof having a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOs: 1-5. In some embodiments, the antibodies of the present disclosure comprise a HCDR2 having a sequence of any one of SEQ ID NOs: 6-10, or a variant thereof having a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOs: 6-10. In some embodiments, the antibodies of the present disclosure comprise a HCDR3 having a sequence of any one of SEQ ID NOs: 11-15, or a variant thereof having a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOs: 11-15.

[0116] In some embodiments, the antibodies of the present disclosure comprise a LCDR1 having a sequence of any one of SEQ ID NOs: 21-24, or a variant thereof having a sequence having one amino acid substitution relative to a sequence of any one of SEQ ID NOs: 21-24. In some embodiments, the antibodies of the present disclosure comprise a LCDR2 having a sequence of any one of SEQ ID NOs: 25-27, or a variant thereof having a sequence having one substitution relative to a sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the antibodies of the present disclosure comprise a LCDR3 having a sequence of any one of SEQ ID NOs: 28-32, or a variant thereof having a sequence having one amino acid substitution relative to a sequence of SEQ ID NOs: 28-32.

[0117] HCDR1-3 and V H

[0118] In some embodiments, an antibody of the present disclosure may comprise a HCDR1 having a sequence of SEQ ID NO: 1 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 1, a HCDR2 having a sequence of SEQ ID NO: 6 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 6, and a HCDR3 having a sequence of SEQ ID NO: 11 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 11. In some embodiments, an antibody of the present disclosure may comprise a HCDR1 having a sequence of SEQ ID NO: 2 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 2, a HCDR2 having a sequence of SEQ ID NO: 7 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 7, and a HCDR3 having a sequence of SEQ ID NO: 12 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 12. In some embodiments, an antibody of the present disclosure may comprise a HCDR1 having a sequence of SEQ ID NO: 3 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 3, a HCDR2 having a sequence of SEQ ID NO: 8 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 8, and a HCDR3 having a sequence of SEQ ID NO: 13 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 13. In some embodiments, an antibody of the present disclosure may comprise a HCDR1 having a sequence of SEQ ID NO: 4 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 4, a HCDR2 having a sequence of SEQ ID NO: 9 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 9, and a HCDR3 having a sequence of SEQ ID NO: 14 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 14. In some embodiments, the antibodies of the present disclosure may comprise a HCDR1 having the sequence of SEQ ID NO: 5 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 5, a HCDR2 having the sequence of SEQ ID NO: 10 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 10, and a HCDR3 having the sequence of SEQ ID NO: 15 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 15.

[0119] The antibodies of the present disclosure may comprise a heavy chain variable region (V HV ) having HCDR1, HCDR2, and HCDR3 as described herein. H In certain embodiments, the antibodies of the present disclosure may comprise a heavy chain variable region that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of any one of SEQ ID NOs: 16-20. In certain embodiments, the antibodies of the present disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 6, and 11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 16. In certain embodiments, an antibody of the present disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 17. In certain embodiments, an antibody of the present disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 3, 8, and 13, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 18. In certain embodiments, an antibody of the present disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4, 9, and 14, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 19. In certain embodiments, an antibody of the present disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 5, 10, and 15, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 20.

[0120] LCDR1-3 and V L

[0121] In some embodiments, an antibody of the present disclosure may comprise a LCDR1 having a sequence of SEQ ID NO: 21 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 21, a LCDR2 having a sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 25, and a LCDR3 having a sequence of SEQ ID NO: 28 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 28. In some embodiments, an antibody of the present disclosure may comprise a LCDR1 having a sequence of SEQ ID NO: 22 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 22, a LCDR2 having a sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 25, and a LCDR3 having a sequence of SEQ ID NO: 29 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 29. In some embodiments, an antibody of the present disclosure may comprise a LCDR1 having a sequence of SEQ ID NO: 21 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 21, a LCDR2 having a sequence of SEQ ID NO: 25 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 25, and a LCDR3 having a sequence of SEQ ID NO: 30 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 30. In some embodiments, an antibody of the present disclosure may comprise a LCDR1 having a sequence of SEQ ID NO: 23 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 23, a LCDR2 having a sequence of SEQ ID NO: 26 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 26, and a LCDR3 having a sequence of SEQ ID NO: 31 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID NO: 31.In some embodiments, an antibody of the present disclosure may comprise a LCDR1 having the sequence of SEQ ID NO: 24 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 24, a LCDR2 having the sequence of SEQ ID NO: 27 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 27, and a LCDR3 having the sequence of SEQ ID NO: 32 or a variant thereof having a sequence having one amino acid substitution relative to the sequence of SEQ ID NO: 32.

[0122] The antibodies of the present disclosure may comprise a light chain variable region (V LCR) having LCDR1, LCDR2, and LCDR3 as described herein. LIn certain embodiments, the antibodies of the present disclosure may comprise a light chain variable region that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of any one of SEQ ID NOs: 33-37. In certain embodiments, the antibodies of the present disclosure may comprise a light chain variable region that has LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 21, 25, and 28, respectively, and is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 33. In certain embodiments, an antibody of the present disclosure may comprise a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 22, 25, and 29, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 34. In certain embodiments, an antibody of the present disclosure may comprise a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 21, 25, and 30, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 35. In certain embodiments, an antibody of the present disclosure may comprise a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 23, 26, and 31, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 36. In certain embodiments, an antibody of the present disclosure may comprise a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 24, 27, and 32, respectively, and having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 37.

[0123] A100

[0124] In certain embodiments, the antibodies of the present disclosure may comprise: (1) HCDR1 having a sequence of SEQ ID NO: 1 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 1; (2) HCDR2 having a sequence of SEQ ID NO: 6 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 6; (3) HCDR3 having a sequence of SEQ ID NO: 11 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 11; (4) LCDR1 having a sequence of SEQ ID NO: 21 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 21; (5) LCDR2 having a sequence of SEQ ID NO: 25 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 25; and (6) LCDR3 having a sequence of SEQ ID NO: 28 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 28. In certain embodiments, the antibodies of the present disclosure may comprise: (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.

[0125] In some embodiments, the antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1, 6, and 11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 16, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 21, 25, and 28, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 33.

[0126] In certain embodiments, the antibody comprises a heavy chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:47:

[0127] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWLSGISGSADTTYYADSVK

[0128] GRFTISRDNSKNTLYLQMTSLRAEDTAVYYCVRDDIQLRDWGQGTLVTVSSASTKGPSVFPLAPSS

[0129] KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

[0130] CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

[0131] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

[0132] PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following SEQ ID NO:48 sequence:

[0133] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

[0134] GTDFALTISSLQPEDFATYYCLQDYNYPLTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCL

[0135] LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0136] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 16 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 47; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO: 33 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID No: 48.

[0137] A101

[0138] In certain embodiments, the antibodies of the present disclosure may comprise: (1) HCDR1 having a sequence of SEQ ID NO: 2 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 2; (2) HCDR2 having a sequence of SEQ ID NO: 7 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 7; (3) HCDR3 having a sequence of SEQ ID NO: 12 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 12; (4) LCDR1 having a sequence of SEQ ID NO: 22 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 22; (5) LCDR2 having a sequence of SEQ ID NO: 25 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 25; and (6) LCDR3 having a sequence of SEQ ID NO: 29 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 29. In certain embodiments, the antibodies of the present disclosure may comprise: (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.

[0139] In some embodiments, the antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 7, and 12, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 17, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 22, 25, and 29, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 34.

[0140] In certain embodiments, the antibody comprises a heavy chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:49:

[0141] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQK

[0142] LQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDYSGSWYPSNGPALDYWGQGTMVTVSSAS

[0143] TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

[0144] TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

[0145] RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

[0146] EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:50:

[0147] DIQMTQSPSSSLSASVGDRVTITCRASQYISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

[0148] GTDFTFTISSLQPEDIATYYCLQDYSYPLTFGGGIKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

[0149] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0150] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 17 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 49; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO: 34 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 50.

[0151] A102

[0152] In certain embodiments, the antibodies of the present disclosure may comprise: (1) HCDR1 having a sequence of SEQ ID NO: 3 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 3; (2) HCDR2 having a sequence of SEQ ID NO: 8 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 8; (3) HCDR3 having a sequence of SEQ ID NO: 13 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 13; (4) LCDR1 having a sequence of SEQ ID NO: 21 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 21; (5) LCDR2 having a sequence of SEQ ID NO: 25 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 25; and (6) LCDR3 having a sequence of SEQ ID NO: 30 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO: 30. In certain embodiments, the antibodies of the present disclosure may comprise: (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.

[0153] In some embodiments, the antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 3, 8, and 13, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 18, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 21, 25, and 30, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 35.

[0154] In certain embodiments, the antibody comprises a heavy chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:51:

[0155] EVQLVQSGAEVKKPGASVKVSCKTSGFTFTNYGISWVRQAPGQGLEWMGWISANNGNSNYAQD

[0156] HQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREFPGWYFDYWGQGTLVTVSSASTKGPSVFP

[0157] LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

[0158] TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

[0159] VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0160] NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following SEQ ID NO:52 sequence:

[0161] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

[0162] GTDFTFTISSLQPDDFATYYCQQSYRTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

[0163] NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0164] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 18 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 51; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO: 35 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 52.

[0165] A103

[0166] In certain embodiments, the antibodies of the present disclosure may comprise: (1) HCDR1 having a sequence of SEQ ID NO:4 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:4; (2) HCDR2 having a sequence of SEQ ID NO:9 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:9; (3) HCDR3 having a sequence of SEQ ID NO:14 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:14; (4) LCDR1 having a sequence of SEQ ID NO:23 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:23; (5) LCDR2 having a sequence of SEQ ID NO:26 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:26; and (6) LCDR3 having a sequence of SEQ ID NO:31 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:31. In certain embodiments, the antibodies of the present disclosure may comprise: (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.

[0167] In some embodiments, the antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 4, 9, and 14, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 19, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 23, 26, and 31, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 36.

[0168] In certain embodiments, the antibody comprises a heavy chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:53:

[0169] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKG

[0170] RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAREYYDFWSGYPSGYAFDIWGQGTLVTVSSASTKG

[0171] PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

[0172] SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

[0173] VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

[0174] KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:54:

[0175] QSALTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSSG

[0176] NTASLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASV

[0177] VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0178] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 53; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO: 36 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO: 54.

[0179] A104

[0180] In certain embodiments, the antibodies of the present disclosure may comprise: (1) HCDR1 having a sequence of SEQ ID NO:5 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:5; (2) HCDR2 having a sequence of SEQ ID NO:10 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:10; (3) HCDR3 having a sequence of SEQ ID NO:15 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:15; (4) LCDR1 having a sequence of SEQ ID NO:24 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:24; (5) LCDR2 having a sequence of SEQ ID NO:27 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:27; and (6) LCDR3 having a sequence of SEQ ID NO:32 or a sequence having an amino acid substitution relative to the sequence of SEQ ID NO:32. In certain embodiments, the antibodies of the present disclosure may comprise: (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.

[0181] In some embodiments, the antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 5, 10, and 15, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 20, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 24, 27, and 32, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO: 37.

[0182] In certain embodiments, the antibody comprises a heavy chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:55:

[0183] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGTANYAQKFQ

[0184] GRVTITADKSTSTAYMELSSLRSEDTAVYYCARGVPSGSGYYLGLDYWGQGTMVTVSSASTKGPS

[0185] VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

[0186] LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

[0187] TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

[0188] VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and a light chain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the following SEQ ID NO:56 sequence:

[0189] QSELTQPPSASGAPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSK

[0190] SGTSASLAISGLRSEDEADYYCAAWDDSLSGPVFSGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTAS

[0191] VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0192] In certain embodiments, the antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO:20 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:55; and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO:37 and a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of SEQ ID NO:56.

[0193] IV. Fc polypeptides

[0194] The anti-α3β1 antibodies provided herein may comprise a crystallizable fragment region (Fc region), also referred to herein as an Fc polypeptide. The Fc polypeptide is a portion of each of the two heavy chains in an antibody and can interact with certain cell surface receptors and certain components of the complement system. The Fc polypeptide typically includes a CH2 domain and a CH3 domain, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in the antibodies described herein may be a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide. In certain embodiments, the antibodies described herein may comprise a wild-type Fc polypeptide having the sequence of the following SEQ ID NO: 43:

[0195] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0196] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN

[0197] QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0198] In other embodiments, the antibodies described herein may comprise a variant of a wild-type Fc polypeptide having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) identity 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).

[0199] In some embodiments, the Fc polypeptide comprises one or more modifications (e.g., one or more amino acid substitutions, insertions, or deletions relative to a comparable wild-type Fc region). Antibodies comprising modified Fc polypeptides typically have an altered phenotype relative to an antibody comprising a wild-type Fc polypeptide. For example, antibodies comprising modified Fc polypeptides may have altered serum half-life, altered stability, altered sensitivity to cellular enzymes, and / or altered effector function (e.g., as determined in NK-dependent or macrophage-dependent assays).

[0200] 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. Exemplary Fc polypeptide amino acid substitutions that reduce effector function include, but are not limited to, substitutions in the CH2 domain, for example, at positions 4 and 5 (relative to the position numbering of 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 two Fc polypeptides in the antibodies described herein may include L4A and L5A substitutions.

[0201] Additional Fc polypeptide amino acid substitutions that modulate effector function include, for example, substitutions at position 99 (numbered relative to the position of SEQ ID NO: 43). For example, in some embodiments, one or both Fc polypeptides in the antibodies described herein may comprise a P99G substitution. In certain embodiments, one or both Fc polypeptides in the antibodies described herein may have L4A, L5A, and P99G substitutions.

[0202] In some embodiments, the Fc polypeptide comprises one or more modifications that alter (relative to a wild-type Fc polypeptide) the ratio of the affinity of the modified Fc polypeptide for an activating FcγR (e.g., FcγRIIA or FcγRIIIA) relative to an inhibitory FcγR (e.g., FcγRIIB):

[0203]

[0204] Where the modified Fc polypeptide has an affinity ratio greater than 1, the anti-α3β1 antibodies herein may be particularly useful for providing therapeutic or prophylactic treatment of a disease, disorder, or infection, or amelioration of a symptom of a disease, disorder, or infection, where enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcγRs is desired, such as cancer or infectious diseases. Where the modified Fc region has an affinity ratio less than 1, the anti-α3β1 antibodies herein may be particularly useful for providing therapeutic or prophylactic treatment of a disease or disorder, or amelioration of a symptom of a disease or disorder, where reduced efficacy of effector cell function mediated by FcγRs is desired, such as autoimmune or inflammatory disorders. Table 2 lists examples of single, double, triple, quadruple, and five amino acid substitutions in Fc polypeptides that provide affinity ratios greater than 1 or less than 1 (see, e.g., PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603). Amino acid positions are numbered according to the EU numbering scheme.

[0205] Table 2

[0206]

[0207]

[0208] V. Antibodies that Compete for Binding with Anti-α3β1 Antibodies

[0209] Also provided herein are anti-α3β1 antibodies (e.g., competing antibodies) that competitively bind to, or are capable of competitively binding to, one or more anti-α3β1 antibodies described herein. In some cases, when a competitor binds to the same general region of α3β1 as an anti-α3β1 antibody described herein, the antibody (i.e., competing antibody) can be considered to competitively bind to α3β1. In some cases, when a competitor binds to the exact same region of α3β1 as an anti-α3β1 antibody described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acids (conformational epitope)), the antibody (i.e., competing antibody) can be considered to competitively bind to α3β1. In some cases, under appropriate assay conditions, when a competitor binds to the same general region of α3β1 as an anti-α3β1 antibody described herein (i.e., a sequence within the thigh-genu of integrin α3β1), the antibody (i.e., competing antibody) can be considered to competitively bind to α3β1. In certain cases, an antibody (i.e., competitor) can be considered capable of competitive binding to α3β1 when the competitor binds to the exact same region of α3β1 (e.g., the exact same peptide (linear epitope) or the exact same surface amino acids (conformational epitope)) as an anti-α3β1 antibody described herein under appropriate assay conditions.

[0210] In certain cases, for example, under appropriate assay conditions, an antibody (i.e., a competing antibody) can be considered to compete for binding to α3β1 when the competitor blocks the binding of one or more anti-α3β1 antibodies described herein to α3β1. Whether a competitor blocks the binding of one or more anti-α3β1 antibodies described herein to α3β1 can be determined using an appropriate competition assay or blocking assay (e.g., a blocking assay as described herein). In a competition or blocking assay, a competing antibody can 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%). Conversely, in a competition or blocking assay, one or more anti-α3β1 antibodies described herein can block the binding of a competing antibody to α3β1 by about 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%).

[0211] In certain cases, for example, under appropriate assay conditions, an antibody (i.e., a competing antibody) can be considered to competitively bind to α3β1 when the competitor binds to α3β1 with a similar affinity as one or more anti-α3β1 antibodies described herein. In some embodiments, an antibody (i.e., a competing antibody) is considered to competitively bind to α3β1 when 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.

[0212] Also provided herein are anti-α3β1 antibodies that bind to the same epitope as one or more anti-α3β1 antibodies described herein or are capable of binding to the same epitope as one or more anti-α3β1 antibodies described herein. In particular, provided herein are 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 acids (conformational epitope)). Such antibodies that bind to the same epitope can be referred to as epitope competitors.

[0213] VI. Polyclonal and Monoclonal Antibodies

[0214] 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 an adjuvant. It may be useful to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional agent or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR (wherein R and R1 are different alkyl groups). Non-protein carriers (such as colloidal gold) can also be used for antibody production.

[0215] Animals can be immunized against antigens, immunogenic conjugates, or derivatives by, for example, mixing 100 μg or 5 μg (for rabbits or mice, respectively) of the protein or conjugate with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animal is bled and the antibody titer of the serum is determined. The booster immunization of the animal is continued until the titer is stable. Typically, animals are boosted with conjugates of the same antigen but conjugated to different proteins and / or by different cross-linking reagents. Conjugates can also be prepared as protein fusions in recombinant cell culture. Moreover, coagulants (such as alum) are suitable for enhancing the immune response.

[0216] Monoclonal antibodies can be made using hybridomas (e.g., the hybridoma method first described by Kohler et al., Nature, 256:495 (1975)), or can be made by other methods such as recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). In the hybridoma method, mice or other suitable host animals (such as hamsters or macaques) are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0217] The hybridoma cell so made is seeded in a suitable culture medium and grown, and this culture medium can contain one or more substances that suppress the growth or survival of non-fusion parental myeloma cells. For example, if the parental myeloma cell lacks hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), then the hybridoma culture medium typically includes the substances (HAT culture medium) that these hinder HGPRT defective cell growth of hypoxanthine, aminopterin and thymidine. Preferred myeloma cells are that fusion efficiency is high, support selected antibody production cell to stably and highly produce antibody and to culture medium such as those cells that HAT culture medium is sensitive. Wherein, preferred myeloma cell line is mouse myeloma cell line, such as SP-2 or X63-Ag8-653 cell (available from American Type Culture Collection (American Type Culture Collection), Rockville, Md.USA). Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0218] The culture medium of the growing hybridoma cells is assayed for the production of monoclonal antibodies against the antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation, in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), or flow cytometric analysis of cells expressing membrane antigens. For example, the binding affinity of the monoclonal antibodies can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0219] After hybridoma cells have been identified as producing antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). For this purpose, suitable culture media include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0220] Using conventional procedures (for example, by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of the monoclonal antibodies), the DNA encoding the monoclonal antibodies can be easily isolated and sequenced. Alternatively, cDNA can be prepared from mRNA, and then DNA sequencing is performed on the cDNA. Hybridoma cells serve as the preferred source of genomic DNA or RNA for such preparation of cDNA. Once separated, DNA can be placed in an expression vector well known in the art, which is then transfected into a host cell (such as Escherichia coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or hybridoma cells) that does not originally produce immunoglobulin, thereby obtaining the synthesized monoclonal antibody in the recombinant host cell.

[0221] VII. Humanization and Amino Acid Variants

[0222] General methods for antibody humanization are described, for example, in U.S. Patent Nos. 5,861,155, 6,479,284, 6,407,213, 6,639,055, 6,500,931, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, 5,714,350, 6,350,861, 5,777,085, 5,834,597, 5,882,644, 5,932,448, 6,013,256, 6,129,914, 6,210,671, 6,329,511, 5,225,539, 6,548,640, and 5,624,821. In certain embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly where these improve the binding affinity or other biological properties (e.g., half-life) of the antibody.

[0223] In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts. See, e.g., 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. Patent 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; WO 87 / 02671; EP Patent Application 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 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 a variety of human cells according to well-known procedures. The CDRs used to generate the immunoglobulins of the present disclosure can similarly be derived from monoclonal antibodies that specifically bind to α3β1.

[0224] 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 within the amino acid sequence of the anti-α3β1 antibodies of the examples herein. As long as the final construct has the desired characteristics, any combination of deletions, insertions, and substitutions can be performed to obtain the final construct. Amino acid changes can also alter the post-translational processing of humanized or variant anti-α3β1 antibodies, such as changing the number or position of glycosylation sites.

[0225] One method for identifying specific residues or regions in anti-α3β1 antibodies that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described, for example, by Cunningham and Wells, Science, 244: 1081-1085 (1989). Here, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably Ala or poly-Ala) to affect the interaction of the amino acids with the α3β1 antigen (e.g., sequences within the thigh-genu region of integrin α3β1). Amino acid positions that exhibit functional sensitivity to the substitutions are then refined by introducing further or additional variants at or against the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed anti-α3β1 antibody variants are screened for the desired activity. Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertion variants include enzymes or polypeptides that increase the serum half-life of the antibody fused to the N-terminus or C-terminus of the antibody.

[0226] Another type of variant is an 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 interesting sites for substitution mutagenesis include the hypervariable regions, but FR changes are also contemplated. Conservative substitutions are preferred, but more substantial changes can be introduced and the products can be screened. Examples of substitutions are listed below:

[0227] Ala(A): Val; Leu; Ile; Val

[0228] Arg(R): Lys; Gln; Asn; Lys

[0229] Asn(N): Gln; His; Asp, Lys; Gln; Arg

[0230] Asp(D):Glu;Asn

[0231] Cys(C):Ser;Ala

[0232] Gln(Q):Asn;Glu

[0233] Glu(E):Asp;Gln

[0234] Gly(G):Ala

[0235] His(H): Asn; Gln; Lys; Arg

[0236] Ile(I): Leu; Val; Met; Ala; Leu; Phe; norleucine

[0237] Leu(L): norleucine; Ile; Val; Ile; Met; Ala; Phe

[0238] Lys(K):Arg;Gln;Asn

[0239] Met(M):Leu;Phe;Ile

[0240] Phe(F): Leu; Val; Ile; Ala; Tyr

[0241] Pro(P):Ala

[0242] Ser(S):Thr

[0243] Thr(T):Ser

[0244] Trp(W):Tyr;Phe

[0245] Tyr(Y): Trp; Phe; Thr; Ser

[0246] Val(V): Ile; Leu; Met; Phe; Ala; Norleucine

[0247] Substantial modifications to the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of ​​the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following categories:

[0248] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0249] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0250] (3) Acidic: Asp, Glu;

[0251] (4) Basic: Asn, Gln, His, Lys, Arg;

[0252] (5) Residues that affect chain orientation: Gly, Pro; and

[0253] (6) Aromatic: Trp, Tyr, Phe

[0254] Non-conservative substitutions entail exchanging a member of one of the above classes for another class.

[0255] Any cysteine ​​residue that is not involved in maintaining the correct conformation of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0256] A type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody. Generally speaking, one or more variants selected for further development will have improved biological properties relative to the parent antibody that generates them. A convenient way to generate such substitution variants is affinity maturation using phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants generated in this way are displayed on filamentous phage particles as a fusion with the gene III product of the M13 packaged within each particle in a monovalent manner. Then, as disclosed herein, phage-displayed variants are screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that make a significant contribution to antigen binding. Alternatively or in addition, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. According to the technology described herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0257] Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. "Altering" means deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is typically N-linked and / or O-linked. N-linked refers to the attachment of the 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 except proline) are the most common recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide forms a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, i.e., N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Adding glycosylation sites to an antibody can be achieved by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0258] VIII. Other Modifications

[0259] Other modifications of anti-α3β1 antibodies are contemplated. For example, the technology herein also relates to immunoconjugates comprising an anti-α3β1 antibody described herein conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof) or a radioactive isotope (e.g., a radioconjugate) or a cytotoxic drug. Such conjugates are sometimes referred to as "antibody-drug conjugates" or "ADCs." Conjugates can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazido compounds (such as bis-(p-azidobenzoyl)hexanediamine), diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and diactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0260] The anti-α3β1 antibodies disclosed herein (e.g., anti-α3β1 antibodies) can be formulated as immunoliposomes. Antibodies containing liposomes are prepared by methods known in the art, such as Epstein et al., Proc.Natl.Acad.Sci.USA 82:3688 (1985); Hwang et al., Proc.Natl.Acad.Sci.USA 77:4030 (1980); and described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556. For example, liposomes can be generated by reverse phase evaporation of a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of limited pore size to produce liposomes with a desired diameter. Fab' fragments of the antibodies provided herein can be conjugated to liposomes via a disulfide interchange reaction as described in Martin et al., J. Biol. Chem. 257:286-288 (1982). Optionally, the liposomes contain another active ingredient.

[0261] Enzymes or other polypeptides can be covalently bound to anti-α3β1 antibodies using techniques well known in the art, such as using heterobifunctional cross-linkers as discussed above. In some embodiments, recombinant DNA techniques well known in the art can be used to construct fusion proteins comprising at least the antigen-binding region of an antibody provided herein linked to at least one functionally active portion of an enzyme (see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

[0262] In certain embodiments, it may be desirable to use antibody fragments rather than intact antibodies to increase penetration of, for example, target tissues and cells. In this case, it may be desirable to modify the antibody fragment in order to increase its serum half-life. This can be achieved, for example, by introducing a salvage receptor binding epitope into the antibody fragment (e.g., by mutating appropriate regions in the antibody fragment, or by introducing the epitope into a peptide tag, which is then fused to both ends or the middle of the antibody fragment by, for example, DNA or peptide synthesis; see, for example, WO 96 / 32478, published October 17, 1996).

[0263] In some embodiments, modification can be optionally introduced into the antibody (for example, within the polypeptide chain or at either the N-terminus or the C-terminus) to, for example, extend half-life in vivo, such as pegylation or incorporation of long-chain polyethylene glycol polymers (PEG). The introduction of PEG or PEG long-chain polymers increases the effective molecular weight of the polypeptide, for example, to prevent rapid filtration into urine. In some embodiments, the lysine residues in the sequence are conjugated to PEG directly or through a linker. Such a linker can be, for example, a Glu residue or an acyl residue containing a thiol functional group for connection to a suitably modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the C-terminus or a residue exposed to a solvent (such as a substitute for an Arg or Lys residue). The Cys residue is then site-specifically connected to a PEG chain containing, for example, a maleimide functional group. Methods for incorporating PEG or long-chain polymers of PEG are known in the art (e.g., as 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)), the contents of which are incorporated herein by reference.

[0264] Covalent modification of anti-α3β1 antibodies is also included within the scope of this technology. For example, modification can be performed by chemical synthesis or by enzymatic or chemical cleavage of anti-α3β1 antibodies. Other types of covalent modifications of antibodies are introduced into the molecule by reacting the targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues. Examples of covalent modifications of polypeptides are described in U.S. Patent No. 5,534,615, which is specifically incorporated herein by reference. A preferred type of covalent modification of an antibody comprises linking the antibody to one of a variety of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene) in a manner such as described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. IX. Nucleic Acids, Vectors, Host Cells and Recombinant Methods

[0265] The present disclosure also provides isolated nucleic acids encoding anti-α3β1 antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies. A nucleic acid herein may include one or more subsequences, each of which is referred to as a polynucleotide.

[0266] Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising a nucleotide sequence encoding an anti-α3β1 antibody, or a fragment thereof. 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 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 the amino acid sequence of any one of SEQ ID NOs: 1-37.

[0267] In order to recombinantly produce anti-α3β1 antibodies, nucleic acids encoding anti-α3β1 antibodies can be isolated and inserted into replication vectors for further cloning (amplification of DNA) or expression. In some cases, anti-α3β1 antibodies can be produced by homologous recombination. 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), DNA encoding anti-α3β1 antibodies can be easily isolated and sequenced. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, and an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0268] Suitable host cells for cloning or expressing DNA in vectors herein can be prokaryotes, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include true bacteria, such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae such as Escheriachia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), and Enterobacter. typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., Pseudomonas aeruginosa), and Streptomyces. A 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.

[0269] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for anti-α3β1 antibody encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used lower eukaryotic host microorganism. Numerous other genera, species or strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, for example, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wicheramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, for example, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0270] Suitable host cells for expressing anti-α3β1 antibodies can also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (cattail), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silk moth). A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used herein, in accordance with the present technology, as viruses, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0271] Suitable host cells for expressing anti-α3β1 antibodies may also include vertebrate cells (eg, mammalian cells). Vertebrate cells can be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subcloned 293 cells grown 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

[0272] Host cell can be transformed with above-mentioned expression or cloning vector that are used to produce antibody, and cultivate in conventional nutrient medium, this substratum is suitably improved with inducible promoter, selects transformant or the gene of amplification coding desired sequence.The host cell that is used to produce the antibody that this paper provides can be cultivated in multiple substratum.Commercially available substratum such as HamF10 (Sigma), minimum essential medium (MEM, Sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle's medium ((DMEM), Sigma) are applicable to cultivating host cell. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 can be used as culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™), trace elements (defined as inorganic compounds generally present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art.Culture conditions such as temperature, pH, and the like are those previously used to select the host cell for expression, and will be apparent to the skilled artisan.

[0273] When using recombinant technology, antibodies can be produced in the cell, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced in the cell, then as the first step, granular debris (host cells or cleavage fragments) is removed by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) have described the program for separating the antibodies secreted into the periplasmic space of E. coli. In short, the cell paste is melted 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. In the case where the antibody is secreted into the culture medium, usually, the supernatant derived from such an expression system is concentrated with a commercially available protein concentration filter (for example, Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to stop the growth of foreign contaminants.

[0274] The antibody composition obtained by the cell can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type 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 can be recommended for all mouse isotypes and human γ 3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is often agarose, but other matrices are also available. Compared with what can be achieved with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times. In the case of antibodies containing a CH3 domain, Bakerbond ABX.TM. resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques such as ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica gel column chromatography, heparin SEPHAROSE can be used depending on the antibody to be recovered. TM Chromatography on ELISA, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also useful.

[0275] Following any one or more preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between, for example, about 2.5-4.5, and can be performed at low salt concentrations (e.g., about 0-0.25 M salt).

[0276] X. Pharmaceutical Formulations, Dosage, and Administration Routes

[0277] The present disclosure provides anti-α3β1 antibodies and related compositions that can be used, for example, to eliminate α3β1-expressing pathogens from the body, and for example, to identify and quantify the amount of α3β1-expressing pathogens in a biological sample.

[0278] Anti-α3β1 antibodies can be formulated into pharmaceutical compositions that can be used for a variety of purposes, including treating diseases or conditions. Pharmaceutical compositions containing one or more anti-α3β1 antibodies can be administered to patients in need thereof using a pharmaceutical device, and according to one embodiment of the technology, a kit comprising such a device is provided. Such devices and kits can be designed for routine administration of the pharmaceutical compositions herein, including self-administration.

[0279] The antibody therapeutic formulation can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing the reagent or antibody having the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer for storage (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, 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 dextrins; 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®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0280] The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0281] Preparations for in vivo administration are generally sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

[0282] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent / antibody in the form of shaped objects, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron, and the like. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable sustained release of molecules for over 100 days, certain hydrogels release proteins over shorter time periods. When encapsulated agents / antibodies remain in the body for extended periods, they may denature or aggregate due to exposure to moisture at 37°C, leading to loss of bioactivity and possible changes in immunogenicity. Depending on the mechanism involved, rational stabilization strategies can be designed. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiol-disulfide interchange, stabilization can be achieved by modifying thiol residues, lyophilizing from acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0283] For therapeutic applications, the anti-α3β1 antibodies provided herein are administered to mammals, such as humans, in pharmaceutically acceptable dosage forms (such as those discussed above, including those that can be administered to humans intravenously as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes). For the prevention or treatment of disease, the appropriate dose of the agent or antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient once or over a series of treatments.

[0284] Depending on the type and severity of the disease, whether for example by one or more separate administrations or by continuous infusion, an antibody of about 1 μg / kg to about 50 mg / kg (e.g., 0.1-20 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily or weekly dose may range from about 1 μg / kg to about 20 mg / kg or more, depending on the factors mentioned above. For repeated administration for several days or longer (depending on the condition), treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of such therapy is easily monitored by conventional techniques and assays (including, for example, radiographic imaging). Detection methods utilizing antibodies to determine α3β1 levels in body fluids or tissues can be used to optimize patient exposure to therapeutic antibodies.

[0285] In some embodiments, the composition comprising the anti-α3β1 antibody herein can be administered as a 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 preventing or treating the disease can be improved by continuously administering the antibody or administering the antibody in combination with another drug that is effective for those purposes (such as a chemotherapy drug for treating cancer or microbial infection). In other cases, the anti-α3β1 antibody can be used to enhance the effect of chemotherapy on cells or to sensitize cells to chemotherapy treatment, thereby allowing efficacy to be produced at lower doses and lower toxicity. In addition to administering a composition comprising an antibody that reduces the number of cells expressing α3β1, certain combination therapies also include delivering a second treatment regimen selected from the following: chemotherapeutic agents, radiotherapy, surgery, and any combination of the foregoing. Such other agents may be present in the composition administered, or may be administered separately. In addition, the anti-α3β1 antibody may be appropriately administered continuously or in combination with other agents or forms (e.g., chemotherapy drugs or radiation for treating cancer, infection, etc., or immunosuppressive drugs).

[0286] XI. Methods

[0287] As described herein, integrin α3β1 is a key integrin on the surface of podocytes (cells that wrap glomerular capillaries in the Bowman's capsule of the kidney). Integrin α3β1 is crucial for podocytes to attach to the outside of blood vessels to form healthy glomeruli in the kidney. The antibodies described herein can serve as allosteric agonist antibodies for integrin α3β1, and can enhance integrin-dependent ligand binding and cell adhesion, thereby preventing cell loss in urine and preventing renal function loss. Also provided herein is a method for treating diseases and / or conditions associated with podocyte loss in subjects in need thereof by administering to the subject anti-α3β1 antibodies described herein that are combined with integrin α3β1 or a portion thereof (e.g., a sequence within the thigh-genu region of integrin α3β1). In some embodiments, the disease and / or condition associated with podocyte loss can be a disease and / or condition caused by podocyte loss (i.e., cell number loss and / or cell function loss). In some embodiments, the disease and / or condition associated with podocyte loss can be a disease and / or condition affecting the kidney, and thus podocyte loss (i.e., loss of cell number and / or loss of cell function) can be a result or manifestation of a kidney disease and / or condition.

[0288] In some embodiments of the method, the subject has a kidney disease associated with podocyte loss (i.e., loss of cell number and / or loss of cell function). The kidney disease can be a glomerular disease, such as a nephritic disease, renal disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

[0289] In some embodiments, the subject is currently undergoing, has undergone, or is about to undergo a transplant. In some embodiments, the transplant is a kidney transplant. In certain embodiments, the antibody is administered after the transplant. In specific embodiments, the antibody is administered after a kidney transplant to protect, maintain, and / or improve kidney function and health.

[0290] In some embodiments, the disease or condition associated with podocyte loss in a subject is an autoimmune disease. In some embodiments, the autoimmune disease affects kidney 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 (anti-GBM) disease. In some embodiments, the autoimmune disease is ANCA-associated vasculitis and glomerulonephritis.

[0291] In other embodiments, the disease or condition associated with podocyte loss is cancer, particularly a cancer that affects kidney function and / or health. In certain embodiments, the cancer is kidney cancer. In certain embodiments, the cancer is renal cell carcinoma, urothelial carcinoma, renal sarcoma, Wilms tumor, or lymphoma.

[0292] In some embodiments, the disease or condition associated with podocyte loss is inflammation, particularly inflammation that affects 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.

[0293] The disclosure also features a method for identifying an antibody that binds to integrin α3β1 or a portion thereof, the method comprising:

[0294] 1) removing antibodies that bind to the β1 chain of integrin α3β1 in the presence or absence of ligand-mimicking peptides and / or antibodies;

[0295] 2) selecting an antibody that binds to integrin α3β1 from the remaining antibodies in step 1) in the presence or absence of a β1 agonist antibody;

[0296] 3) counterselecting antibodies that bind to integrin α3β1 against immobilized β1 agonist antibodies or ligand mimetic peptides alone; and

[0297] 4) Repeating the above steps 1), 2) and 3) to enrich antibodies that are allosteric agonists of integrin α3 in the presence of integrin α3β1 expressed on the cell surface.

[0298] In some embodiments of the method, the ligand mimetic peptide is LXY2. In some embodiments of the method, steps 1) and / or 3) are performed using human K562 cells that primarily express human α5β1 integrin and do not overexpress α3β1.

[0299] In some embodiments of the method, steps 2) and / or 3) are performed using human K562 cells that overexpress α3β1. In some embodiments, steps 1) and / or 2) and / or 3) are performed in the presence of an agent that blocks the ligand binding site or domain of the integrin (e.g., an antibody or ligand).

[0300] In some embodiments, integrin α3β1 is stabilized in a specific conformation by pre-complexing with an activator or inhibitor (such as activating antibodies 9EG7 or TS2 / 16). In some other embodiments, integrin α3β1 is stabilized in a specific conformation by pre-complexing with an agent that selectively binds to the β chain of the integrin dimer.

[0301] We deplete β1 binders by targeting “other” β1 integrins (either as recombinant proteins or using cell lines such as K562 expressing α5β1).

[0302] We prepare α3β1 complexes by pre-complexing α3β1 (recombinant or expressed in a cell line) with a β1-activating antibody, placing α3β1 in a more "open" or "active" conformation. This allows for easier identification of antibodies that bind to active or activated α3β1. We further increase the chances of identifying activating antibodies by adding a ligand or ligand mimetic to this complex. This pre-complex is used to select for activating antibodies.

[0303] We further facilitated the identification of new antibodies targeting allosteric sites by blocking the ligand-binding face of the integrin complex using ligand mimetics or blocking antibodies. Example

[0304] Example 1 - Identification of Antibody Fragments Using Phage Display

[0305] To identify short-chain variable fragments (scFvs), a phage-displayed library of naive human scFvs was run through a novel selection strategy to identify allosteric agonists. This strategy facilitates the identification of allosteric agonist binders, binders specific for one integrin chain relative to the other, and binders that enhance ligand binding. Furthermore, the strategy relies on conformationally stabilized integrins (such as integrin α3β1 complexed with a β1-activating antibody to stabilize the integrin in an "active" conformation) to facilitate the identification of conformationally sensitive binders. Furthermore, the strategy uses ligand blockers (such as integrin α3β1 complexed with the ligand-mimicking peptide LXY2 or the ligand laminin to block the highly antigenic ligand-binding pocket and the MIDAS site, in the presence or absence of a β1-activating antibody) to intentionally exclude binders targeting the ligand-binding pocket. Here, we used two selection approaches (designated Selection 1 and Selection 2) to identify binders. Selection 1 utilizes recombinant integrins, while Selection 2 utilizes cell-surface-expressed integrins. For selection 1, a round of screening used three steps: pre-depletion, selection, and counter-selection. The pre-depletion step was used to remove binders to the β1 chain of the α3β1 dimer by using negative selection, during which phage binders to immobilized recombinant human integrin α4β1 were selected from the screening pool. In addition, in some steps, a commercial β1 agonist antibody (TS2 / 16) and a ligand-mimicking peptide (LXY2) were included with the immobilized integrin α4β1 to further remove any phage binders of these agents. Non-binding phage were used in the next step, the selection step for positive selection, in which phage were incubated with immobilized recombinant human or mouse integrin α3β1 in the presence or absence of β1 agonist antibodies (antibody clone TS2 / 16 for human α3β1 and antibody clone 9EG7 for mouse α3β1) and the ligand-mimicking peptide LXY2. Non-binding phage were removed and discarded. Bound phage are eluted and used in the final step, where counterselection against immobilized β1 agonist antibody and LXY2 alone is used to remove any binders to the β1 agonist antibody and LXY2. All non-binding phage are considered enriched for anti-integrin α3 allosteric binders. Furthermore, this process can be repeated for multiple rounds to further enrich for phage clones of interest.

[0306] Next, the enriched phage library from selection 1 is optionally amplified and then brought into selection 2 to enrich for anti-integrin α3 allosteric agonists that bind to integrins expressed on the cell surface (similar to the above method). Here, one round of screening consists of: 1) a pre-depletion step against K562 cells (which primarily express human α5β1 integrin and do not overexpress α3β1) in the presence or absence of β1 agonist antibodies and ligand mimetic peptide LXY2; 2) positive selection against human K562 cells expressing integrin α3β1 in the presence or absence of β1 agonist antibodies TS2 / 16, and counter-selection against immobilized β1 agonist antibodies and LXY2 alone. Cell line generation is described in this method.

[0307] The enriched phage pool from selection 2 was optionally further amplified and plated using standard methods. 184 individual clones were identified and selected for clonal expansion steps. Each clone was purified to produce a periplasmic extract (PE) solution containing soluble parental clone scFv. These extracts were tested in the presence or absence of LXY2 via direct integrin ELISA for each of human α3β1, mouse α3β1, and human α4β1 (as described in the methods). The extracts were then tested for K562 expressing human α3 and K562 not expressing α3 by flow cytometry.

[0308] After PE characterization, the DNA of the variable domain of each scFv was sequenced. The CDR sequence was assigned to each of the 184 clones, and then compared and clustered to eliminate duplication. The 184 clones separated from the selection produced 25 groups of unique scFv CDR sequences. The sequencing data was combined with the assay data from PE ELISA and FACS to select the best hit.

[0309] Example 2 - Generation of full-length IgG antibodies

[0310] Using the dataset generated by sequencing and characterization, the top five sequences were selected for remodeling. The heavy chain variable region DNA sequences of each scFv were attached to the full-length human heavy chain constant IgG1 DNA (IGHC1 gene transcript) via gene synthesis and cloning. The light chain human kappa and lambda variable region DNA sequences of each scFv were attached to the full-length human light chain constant IGKC1 and IGLC1 DNA, respectively, thereby preserving their heavy chain / light chain pairing at the scFv level. The heavy and light chain DNA constructs were cloned into separate cloning vectors and then shuttled into a mammalian expression vector.

[0311] Each of the five paired constructs was transfected and expressed in 10 mL of mammalian cells, and the antibodies were then isolated using protein A purification. Antibody samples were run on reducing and non-reducing SDS-PAGE and SEC-HPLC for quality control. Antibody samples of the expected molecular weight were found on the SDS-PAGE gel, and the SEC-HPLC peak confirmed that the samples were quite pure.

[0312] Example 3 - Verification of Antibody Binding Using ELISA

[0313] The five full-length IgG antibodies are referred to as Ab74 A100 to A104, or simply Ab74. First, the binding of the five Ab74 to the 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 on plates overnight and then incubated with each of the five Ab74s or with an isotype human IgG1 antibody negative control or a commercial anti-human α3 antibody positive control. Binding was detected by incubation with an anti-human IgG1 antibody horseradish peroxidase (HRP) conjugate, followed by treatment with a fluorescent substrate, developing the reaction, and reading the mean fluorescence intensity using a microplate reader.

[0314] ELISA results showed that the five Ab74 preferentially bound to human α3β1 ECD rather than all other antigens coated on the plate ( Figures 1A-1D Isotype negative controls and anti-α3 positive controls gave the expected negative and positive results, respectively, confirming the low background and positive signal for human α3 of the assay. Two of the five Ab74 antibodies demonstrated low binding to mouse α3, while all five Ab74 antibodies showed only background signal for coated BSA and human α4β1 ECD, confirming by ELISA that Ab74 binds to the human α3 ECD but not the human β1 ECD.

[0315] To further refine 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 showed that Ab74 A101 bound to the Thigh-Genu region ( Figure 2 ).

[0316] Example 4 - Validation of Antibody Binding Using Flow Cytometry-Based Assays

[0317] To validate antibody binding on cells, K562 cells expressing human or mouse integrin α3 were generated as described in the methods.

[0318] After treatment of K562 cells overexpressing human or mouse integrin α3, all full-length human IgG antibodies were detected by flow cytometry after treatment with a fluorophore-conjugated anti-human IgG1 antibody and secondary antibody staining. The results in Table 3 show that Ab74 had higher detection in both human and mouse integrin α3 expressing K562 cells when compared to a negative control isotype antibody.

[0319] Table 3: Characterization of integrin agonist antibodies. The Ab74 clone binds to both human and mouse integrin α3β1 (as measured by ELISA and flow cytometry). α3β1 domain localization was performed by combining ELISA and flow cytometry using recombinant protein domains and domain-swapped integrin-expressing cell lines, respectively.

[0320]

[0321] Example 5 - Increased ligand binding by cells expressing human integrin α3β1 in the presence of agonist antibodies

[0322] To explore ligand binding agonism, K562 cells expressing human α3 were treated with anti-α3 Ab74, a negative control isotype antibody, or a positive control commercial β1 agonist antibody, TS2 / 16, in the presence of the biotinylated ligand-mimetic peptide LXY2 in a low-affinity Ca2+ / Mg2+ buffer. LXY2 binding was detected by treating the cells with a streptavidin-fluorophore conjugate and reading them on a flow cytometer. Negative and positive controls correctly demonstrated low to no and high ligand binding, respectively, in the low-affinity buffer. The results in Table 4 show that Ab74 increased LXY2 binding in the low-affinity buffer compared to the isotype antibody.

[0323] Table 4: Increased ligand binding of cells expressing human integrin α3β1 in the presence of agonist antibodies. K562 cells expressing α3β1 were incubated with the α3β1 ligand mimetic LXY2-biotin conjugate and integrin agonist antibodies or isotype controls. Cells were then stained with a streptavidin-fluorophore conjugate and measured by flow cytometry.

[0324]

[0325]

[0326] Example 6 - Increased ligand binding by cells expressing mouse integrin α3β1 in the presence of agonist antibodies

[0327] Cross-reactivity with mouse α3 was characterized by repeating the ligand assay on K562 cells expressing mouse α3. Briefly, cells were treated with anti-α3 Ab74, a negative control isotype antibody, or a positive control commercial β1 agonist antibody, 9EG7, in the presence of the biotinylated ligand-mimetic peptide LXY2 in low-affinity Ca2+ / Mg2+ buffer. LXY2 binding was detected by treating the cells with a streptavidin-fluorophore conjugate and reading them on a flow cytometer. Negative and positive controls correctly demonstrated low to no and high ligand binding, respectively, in low-affinity buffer. Figures 3A-3D The results in also showed that Ab74 increased LXY2 binding in low affinity buffer.

[0328] Example 7 - Reduction of cell migration in the presence of integrin agonist antibodies

[0329] The adherent human ovarian cancer cell line SK-OV-3 expresses integrin α3β1 at high levels and mediates the binding of ligands to laminin-511. Tissue culture treated 96-well plates were coated with the integrin α3 ligand laminin-511 and incubated overnight. The next day, the wells were plated with SK-OV-3 cells in serum-free medium and the cells were allowed to adhere. After 16 hours, scratches (injuries) were made with sterile plastic P200 pipette tips before adding anti-α3Ab74 or negative control isotype antibodies or positive control β1 agonist antibodies to complete medium. Changing from serum-free medium to medium containing fetal bovine serum (FBS) promoted cell migration and therefore promoted wound closure via cell movement and migration. After 48 hours, the medium was removed and the cells were fixed with 4% paraformaldehyde and then stained with 0.2% crystal violet. Figures 4A-4E The results in demonstrate that wound closure was inhibited in wells treated with all Ab74 antibodies or a positive control anti-β1 agonist antibody compared to an isotype treated negative control or blocking anti-α3 which produced wound closure.

[0330] Example 8 - Integrin agonist antibodies targeting the thigh-genu domain

[0331] To further refine the binding epitope of the antibody, several α3 integrin domains were individually “swapped” with their cognate counterparts in the analogous protein, human integrin α7 (which also contains Thigh-Genu, Calf 1, and Calf 2 domains) (as described in the Methods).

[0332] To this end, integrin DNA constructs were created in which the Thigh-genu, Calf 1, and Calf 2 domains of integrin α7 replaced the equivalent domains of integrin α3 within a mammalian expression plasmid. Using Lipofectamine, these three constructs were individually transfected into HEK-293 cells and allowed to expand in culture for 48 hours. These cells were then treated with Ab74, a negative control isotype antibody, or a positive control commercial α3 antibody, P1B5, before staining with fluorophore-conjugated anti-human IgG1 for detection in a flow cytometer.

[0333] The results in Table 5 show that Ab74 was detected in cells transfected with full-length integrin α3 DNA, as well as constructs of integrin α3 with calf-2 containing integrin α7 and constructs of integrin α3 with calf-1 containing integrin α7, whereas little to no binding occurred in integrin α3 constructs in which the Thigh-genu domain of integrin α7 was replaced, indicating that the five Ab74 antibodies recognize epitopes in the Thigh-Genu region of α3.

[0334] For the results in Table 5, for each integrin domain, a DNA construct was created in which the corresponding domain was replaced with its cognate integrin α7 counterpart. These were ITGA7 Thigh-genu, Calf 1, and Calf 2, each inserted into α3 in place of the wild-type sequence. These DNA constructs were then cloned into mammalian expression vectors and individually transfected into mammalian cells within 48 hours, followed by incubation with an activated human anti-α3 Ab. Antibody binding was detected by staining the cells with an anti-hIgG1 antibody APC conjugate and reading on a flow cytometer.

[0335] Table 5: Epitope domain mapping of integrin agonist antibodies by flow cytometry.

[0336]

[0337]

[0338] Example 9 - Method

[0339] Cell culture and transient protein expression in HEK293 cell line

[0340] HEK293 cells were cultured in serum-free CD medium (Sino Biological catalog number SMM 293-TI) until they reached optimal cell density. 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 after transfection. Cells were harvested on day 7 of culture and protein purification was performed.

[0341] Protein purification from HEK293 cells

[0342] Cells were removed by centrifugation and the culture supernatant was collected for protein purification.

[0343] Affinity purification: The column is equilibrated with loading buffer and the culture supernatant is loaded onto the column. The column is re-equilibrated and the target protein is eluted using a gradient of buffer containing imidazole (Ni-affinity) or glycine and NaCl (Protein A affinity or FLAG affinity). The protein is further buffer exchanged to remove excess imidazole or other salts. The protein solution is concentrated and the protein concentration and purity are determined by appropriate methods. Protein concentration is determined by UV, while its purity is determined by SDS-PAGE and Western blotting.

[0344] Cloning in expression vectors

[0345] Restriction site 1-Kozak sequence-signal peptide-target protein-stop codon-restriction site 2.

[0346] signal peptide

[0347] N-terminal-MGWSCIILFLVATATGVHS-(SEQ ID NO:57)

[0348] Protein tags and characteristics

[0349] Some constructs had an N-terminal FLAG, a C-terminal 6XHis and three Gly4Ser linkers and a 3C protease cleavage site.

[0350] N-terminal FLAG tag MDYKDDDDK (SEQ ID NO: 58)

[0351] C-terminal 6X His tag HHHHHH (SEQ ID NO: 59)

[0352] (Gly4Ser) 3 linkers GGGGSGGGGSGGGGS (SEQ ID NO: 60)

[0353] PreScission protease (3C) LEVLFQGP (SEQ ID NO: 61) cleavage site (LEVLFQ / GP (SEQ ID No: 61), (wherein " / " indicates the cleavage site))

[0354] Domain-swapped mammalian expression constructs

[0355] To test the domain specificity of the antibodies, each domain from ITGA3 was replaced with the corresponding homologous domain from ITGA7 in the DNA sequence ( Figure 5 These were then inserted into a CMV-driven mammalian expression vector (pCMV6-Neo) for transient transfection.

[0356] SEQ ID NO: 62: Full-length human ITGA3 with exchanged ITGA7 Thigh-Genu:

[0357] MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYSVALHRQTER

[0358] QQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVA

[0359] SQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYL

[0360] ETGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQ

[0361] VGSFILHPKNITIVTGAPRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDG

[0362] WQDLLVGAPYYFERKEEVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQD

[0363] IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

[0364] LSDHIVLLRARPILHVSHEVSIAPRSIDLEQPNCAGGHSVCVDLRVCFSYIAVPSSYSPTVALDYVLD

[0365] ADTDRRLRGQVPRVTFLSRNLEEPKHQASGTVWLKHQHDRVCGDAMFQLQENVKDKLRAIVVTL

[0366] SYSLQTPRLRRQAPGQGLPPVAPILNAHQPSTQRAEIHFLKQGCGPDNKCESNLQMRAAFVSEQQQ

[0367] KLSRLQYSRDVRKLLLSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSVRPPGACQANETIFCELG

[0368] NPFKRNQRMELLIAFEVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLLVDYTLQTSLSMVNHRL

[0369] QSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLEWPYEVSNGKWLLYPTE

[0370] ITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGPPPVTLAAAKKAKSETV

[0371] LTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINM

[0372] ENKTTWFSVDIDSELVEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

[0373] SEQ ID NO:63: Full-length human ITGA3, with swapped ITGA7 Calf 1:

[0374] MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYSVALHRQTER

[0375] QQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVA

[0376] SQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYVRGNDLELDSSDWQTYHNEMCNSNTDYL

[0377] ETGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQ

[0378] VGSFILHPKNITIVTGAPRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDG

[0379] WQDLLVGAPYYFERKEEVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIDGDINQDGFQD

[0380] IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

[0381] LSDHIVLLRARPVINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEA

[0382] DRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLG

[0383] LRSLDAYPINLQAQALENHTEVQFQKECGPDNKCQSNLQLVRARFCTRVSDTEFQPLPMDVDGTT

[0384] ALFALSGQPVIGLELMVTNLPSDPAQPQADGDDAHEAQLLVMLPDSLHYSGVRALDPAEKPLCLS

[0385] NENASHVECELGNPMKRGAQVTFYLILLSTSGISIETTELEVELLLATISEQELHPVSARARVFIELLQT

[0386] SLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLEWPYEVSN

[0387] GKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGPPPVTLAA

[0388] AKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLF

[0389] LRTSIPTINMENKTTWFSVDIDSELVEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

[0390] SEQ ID NO:64: Full-length human ITGA3, Calf 2 replaced with ITGA7 Calf 2: <00008​​​​​​​​​​​​​​​​​

[0397] IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

[0398] LSDHIVLLRARPVINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEA

[0399] DRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLG

[0400] LRSLDAYPILNQAQALENHTEVQFQKECGPDNKCESNLQMRAAFVSEQQQKLSRLQYSRDVRKLL

[0401] LSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSVRPPGACQANETIFCELGNPFKRNQRMELLIAF

[0402] EVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLLVDYTPLSIAGMAIPQQLFFSGVVRGERAMQS

[0403] ERDVGSKVKYEVTVSNQGQSLRTLGSAFLNIMWPHEIANGKWLLYPMQVELEGGQGPGQKGLCS

[0404] PRPNILHLDVDSRDRRRRELEPPEQQEPGERQEPSMSWWPVSSAEKKKNITLDCARGTANCVVFSC

[0405] PLYSFDRAAVLHVWGRLWNSTFLEEYSAVKSLEVIVRANITVKSSIKNLMLRDASTVIPVMVYLDP

[0406] MEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

[0407] Cell line generation

[0408] K562 cells from ATCC were transfected with a linearized expression plasmid containing human integrin α3 via electroporation and maintained under 0.5 mg / mL G418 selection for 2 weeks. Cells expressing high integrin expression were enriched by fluorescence activated cell sorting (FACS) following a staining protocol using commercial antibody P1B5 (Millipore Sigma, Waltham, MA, USA) for anti-integrin α3 antibody staining.

[0409] K562 mouse α3β1 and K562 cynomolgus monkey α3β1: K562 cells from ATCC were transfected with linearized expression plasmids containing mouse or cynomolgus monkey integrin α3 and a C-terminal FLAG tag via electroporation and maintained under 0.8 mg / mL puromycin selection for 2 weeks. Cells with high integrin expression were enriched by fluorescence-activated cell sorting (FACS) using an anti-FLAG antibody (Sino Biological, China).

[0410] Cell adhesion assay, fluorescent reporter system

[0411] K562 cells expressing human integrin α3β1 and target integrin were washed with TBS and 50,000 cells / well were transferred to ligand-coated wells of a high-binding clear 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) in a total of 90 μL of assay buffer (HEPES 20 mM / 2 mg / mL glucose / 140 mM NaCl containing 1 mM each of Ca 2+ and Mg 2+ , expressed as HEPES-CaMg). In the presence of antibodies, the plates were incubated at 37°C for 30 minutes. To induce detachment of non-adherent cells, the plates were gently inverted and kept in this position at room temperature for 45 minutes. The plates were placed upright and the wells were quickly aspirated using an automatic plate washer (Agilent Technologies, Santa Clara, CA, USA). Adherent cells were quantified using CyQuantNF (Invitrogen, Waltham, MA, USA). For minimum-maximum normalization, negative control assay buffer (HEPES buffer containing 10 mM EDTA, expressed as HEPES-EDTA) and positive control assay buffer (containing 1 mM Mn 2+ and 200 μM Ca 2+ of TBS, denoted as TBS-Mn) was included in the plate.

[0412] Cell adhesion assay, automated imaging

[0413] K562 cells expressing human integrin α3β1 and target integrin were washed with TBS and 50,000 cells / well were transferred to ligand-coated wells of a high-binding clear 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) in a total of 90 μL of assay buffer (HEPES 20 mM / 2 mg / mL glucose / 140 mM NaCl containing 1 mM each of Ca 2+ and Mg 2+ , expressed as HEPES-CaMg). In the presence of antibodies, the plate was incubated at 37 ° C for 30 minutes. In order to induce non-adherent cells to detach, the plate was gently inverted and kept in this position for 45 minutes at room temperature. The plate was upright, and at room temperature, the wells were fixed in an inverted position for 10 minutes using a final concentration of 2% paraformaldehyde stock solution. The plate was upright and quickly aspirated using an automatic plate washer (Agilent Technologies, Santa Clara, CA, USA). Using DAPI and an automatic imaging system with a cell nucleus segmentation algorithm, adherent cells were quantified. For minimum-maximum normalization, negative control assay buffer (TBS containing 10mM EDTA, expressed as TBS-EDTA) and positive control assay buffer (TBS containing 1mM Mn2+ and 200 μM Ca2+, expressed as TBS-Mn) were included in the plate.

[0414] Direct integrin ELISA

[0415] At 4 ° C, a high-binding black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated with 30 μL of TBS containing 3 μg / mL recombinant integrin. The plate was flicked to remove any liquid and blocked by adding 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) and incubated for 1 hour. After incubation, the plate was washed three times with 100 μL TBS using an automatic plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μ L assay buffer (TBS containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68, expressed as TBS-T) is added to each well. 1 μ L of test antibody storage solution is added to each corresponding well, and the plate is centrifuged at 1000 g for 1 minute, and incubated at room temperature for 1 hour. 100 μ L TBS wash plates are washed three times using an automatic washing machine. 30 μ L staining buffer (TBS-T, containing the anti-IgG HRP conjugate of 1:2000 dilution) (Invitrogen, Waltham, MA, USA) is added to each well and incubated for 30 minutes. 100 μ L wash plates are washed three times in an automatic plate washer. 30 μ L substrate buffer (TBS containing 100 μ M Amplex Red and 4 mM hydrogen peroxide) (Biotium, Fremont, CA, USA) is added and incubated at room temperature for 30 minutes. The plates were analyzed at 563 / 587 nm in a fluorescence microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0416] Integrin sandwich ELISA

[0417] At 4 ℃, high binding black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) was coated with 30 μL of TBS containing 4 μg / mL anti-integrin antibody overnight. Before the following steps, the assay buffer (TBS containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), denoted as TBS-T) was prepared. The plate was flicked to remove any liquid and blocked by adding 90 μL of TBS-T containing 5% bovine serum albumin (w / v) (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 hour. After incubation, the plate was washed three times with 100 μL TBS using an automatic plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL assay buffer (TBS-T containing 4 μg / mL labeled 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 TBS using an automatic washing machine. 30 μL of staining buffer (TBS-T containing 1:2000 diluted anti-tag antibody HRP conjugate) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. 100 μL of the plate was washed three times in an automatic 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 developed at room temperature for 30 minutes. The plate was analyzed at 563 / 587 nm in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0418] Recombinant Integrin Functional Assay (SoLISA), Integrin Detection

[0419] At 4 ℃, high binding black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) is coated overnight with 30 μ L of TBS containing 8 μ g / mL part. Before following steps, prepare assay buffer (TBS (Sigma-Aldrich, St. Louis, MO, USA) containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68, is expressed as TBS-T). Flick the plate to remove any liquid, and by adding 90 μ L of TBS-T (Sigma-Aldrich, St. Louis, MO, USA) containing 5% bovine serum albumin (w / v), block and incubate 1 hour. After incubation, use automatic plate washer (Agilent Technologies, Santa Clara, CA, USA) to wash the plate three times with 100 μ L TBS. Add 30 μL of assay buffer (TBS-T containing 4 μg / mL labeled recombinant integrin and 1 mM Ca) to each well. 2+ / 1mMMg 2+ 、1mM Mn 2+ / 200μM Ca 2+ , or one of 10mM EDTA). 1 μL agonist antibody (or isotype control) stock solution was added to each well at an appropriate concentration. The plate was centrifuged at 1000g for 1 minute and incubated at room temperature for 3 hours. An automatic washing machine was used to wash the plate three times with 100 μL TBS. 30 μL of staining buffer (TBS-T, containing an anti-tag antibody HRP conjugate diluted 1:2000) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. 100 μL of the plate was washed three times in an automatic plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and developed at room temperature for 30 minutes. The plate was analyzed at 563 / 587nm in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0420] Recombinant integrin functional assay (SoLISA), ligand detection

[0421] At 4 ℃, high binding black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) is coated with 30 μL of TBS containing 4 μg / mL anti-tag antibody overnight. Before the following steps, assay buffer (TBS containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68 (Sigma-Aldrich, St. Louis, MO, USA), expressed as TBS-T) is prepared. The plate is flicked to remove any liquid and blocked by adding 90 μL of TBS-T containing 5% bovine serum albumin (w / v), and then incubated for 1 hour. After incubation, the plate is washed three times with 100 μL TBS using an automatic plate washer (Agilent Technologies, Santa Clara, CA, USA). 30 μL capture buffer (TBS-T containing 4 μg / mL labeled recombinant integrin) is added to each well. Add 30 μL of assay buffer (TBS-T containing 8 μg / mL labeled recombinant ligand and 1 mM Ca) to each well. 2+ / 1mM Mg 2+ 、1mM Mn 2+ / 200μM Ca 2+ , or one of 10mM EDTA). 1 μL agonist antibody (or isotype control) stock solution was added to each well at an appropriate concentration. The plate was centrifuged at 1000g for 1 minute and incubated at room temperature for 3 hours. The plate was washed three times with 100 μL TBS using an automatic washing machine. 30 μL of staining buffer (TBS-T, containing a 1:2000 diluted anti-ligand-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 in an automatic plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and developed at room temperature for 30 minutes. The plate was analyzed at 563 / 587 nm in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0422] Recombinant Integrin Functional Assay (SoLISA), Antibody Detection

[0423] At 4 ℃, high binding black 384-well microplate (Corning Incorporated, One Riverfront Plaza, NY, USA) is coated overnight with 30 μ L of TBS containing 8 μ g / mL part. Before following steps, prepare assay buffer (TBS (Sigma-Aldrich, St. Louis, MO, USA) containing 0.05% Triton X-100 (v / v) and 0.025% (v / v) Pluronic F68, is expressed as TBS-T). Flick the plate to remove any liquid, and by adding 90 μ L of TBS-T (Sigma-Aldrich, St. Louis, MO, USA) containing 5% bovine serum albumin (w / v), then hatch 1 hour. After hatching, use automatic plate washer (Agilent Technologies, Santa Clara, CA, USA) to wash the plate three times with 100 μ L TBS. Add 30 μL of assay buffer (TBS-T containing 4 μg / mL labeled recombinant integrin and 1 mM Ca) to each well. 2+ / 1mM Mg 2+ 、1mM Mn 2+ / 200μM Ca 2+ , or one of 10mM EDTA). 1 μL agonist antibody (or isotype control) storage solution was added to each well at an appropriate concentration. The plate was centrifuged at 1000g for 1 minute and incubated at room temperature for 3 hours. An automatic washing machine was used to wash the plate three times with 100 μL TBS. 30 μL of staining buffer (TBS-T, containing an anti-IgG antibody HRP conjugate diluted at 1:2000) (Invitrogen, Waltham, MA, USA) was added to each well and incubated at room temperature for 30 minutes. 100 μL of the plate was washed three times in an automatic plate washer. 30 μL of substrate buffer (TBS containing 100 μM Amplex Red and 4mM hydrogen peroxide) (Biotium, Fremont, CA, USA) was added to each well and developed at room temperature for 30 minutes. Plates were analyzed at 563 / 587nm in a fluorescent microplate reader (Agilent Technologies, Santa Clara, CA, USA).

[0424] Soluble ligand (laminin 511) binding assay by flow cytometry

[0425] The day before the assay, K562 cells expressing integrins in antibiotic-containing culture medium were counted, washed with 10 mL of PBS, centrifuged and resuspended in complete culture medium without positive selection antibiotics. These cells were cultured overnight at 37 ° C and 5% CO2. Laminin-511E8 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 1: 1.5 molar ratio (5 μg laminin-511E8 Fc per test group) and incubated at room temperature in the dark for 30 minutes. The cells were centrifuged and resuspended in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, Hampton, NH, USA) at 10 million cells / mL. Human Fc blocker 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, FACS buffer was added to dilute the concentration to 1 million cells / mL. Once the cells were transferred to a V-bottom 96-well plate [Cat. No. 290-8116-01V], 40 μL of 1 mM CaCl2 was added to each corresponding well. 2+ / 1mM Mg 2+ or 1 mM Mn 2+ / 200μM Ca 2+ Or one of 10mM EDTA assay buffer, subsequently add agonist antibody or isotype, and incubate at room temperature for 5 minutes.Next, add the laminin 511E8 Fc / Ab-AF647 solution of 5 μ L to each well, and incubate at room temperature for 25 minutes.With 200 μ L assay buffer wash plate.In order to detect the combination of agonist antibody, the cell is resuspended in 100 μ L assay buffer (the assay buffer containing the anti-IgG of 2.5-5.0 μ g / mL) (BD Pharmigen) and incubate at room temperature for 30 minutes in the dark.With 200 μ L assay buffer wash plate, and the precipitation is resuspended in 100 μ L freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [catalog number AA47377-9M] and incubate on ice for 10 minutes.Finally, the cell is resuspended in PBS, and on CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA) analyzes cell.

[0426] Soluble ligand (ligand mimetic) binding assay by flow cytometry

[0427] The day before the assay, K562 cells expressing integrins in antibiotic-containing medium were counted, washed with 10 mL of PBS, centrifuged and resuspended in complete medium without positive selection antibiotics. The cells were cultured overnight at 37°C and 5% CO2. The cells were centrifuged and resuspended in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, Hampton, NH, USA) at 10 million cells / mL. Human Fc blocker was added to the cells (BD Biosciences, Franklin Lakes, NJ, USA) to a final 25 μg / mL and incubated on ice for 15 minutes. After incubation, FACS buffer was added to dilute the concentration to 1 million cells / mL. Once the cells were transferred to a V-bottom 96-well plate [Catalog No. 290-8116-01V], 40 μL of 1 mM Ca was added to each corresponding well. 2+ / 1mM Mg 2+ or 1 mM Mn 2+ / 200μM Ca 2+ Or one of 10mM EDTA assay buffer, followed by addition of agonist antibodies or isotypes, and incubation at room temperature for 5 minutes. Next, 5 μL of 10X biotinylated ligand mimics were added to each well and incubated at room temperature for 25 minutes. The plates were washed with 200 μL of assay buffer. In order to detect the binding of the agonist antibodies, the cells were resuspended in 100 μL of assay buffer (containing 2.5-5.0 μg / mL of anti-IgG assay buffer) (BD Pharmigen). Biotinylated ligand mimics were detected by fluorescently labeled streptavidin and incubated at room temperature for 30 minutes in the dark. The plates were washed with 200 μL of assay buffer, and the precipitate was resuspended in 100 μL of freshly prepared fixative buffer (PBS containing 4% paraformaldehyde) [Catalog No. AA47377-9M] and incubated on ice for 10 minutes. Finally, cells were resuspended in PBS and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA).

[0428] Wound healing / scratch assay

[0429] The day before, a tissue culture treated flat-bottom 96-well plate was coated with 100 μL of 2.0 μg / mL laminin-511 (iMatrix) prepared in 1X PBS under sterile conditions and placed at 4°C overnight. The next day, the coating solution was aspirated and the plate was blocked with 100 μL of sterile 2% FBS for 1 hour at room temperature. To detach cells for seeding for the scratch assay, the SKOV3 cell layer was treated with 0.25% trypsin-EDTA and 30,000 cells were seeded into 100 μL of warm serum-free medium per well. The plate was centrifuged at 500 g for 5 minutes to pellet the cells and incubated in a cell culture incubator overnight. The next day, a vertical wound / scratch was created in the middle of the well using a sterile p200 pipette tip. Agonist or isotype antibody treatments were prepared in warm complete medium and 100 μL of treatment was added to each well. Finally, Ca was added to each well. 2+ Mg 2+ or Mn 2+ To a final concentration of 0.5 mM in 10 μL. Wound healing was observed at 24 hours to determine wound closure. When wounds were nearly closed after treatment with complete medium alone, the medium was aspirated, and 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 for 30 minutes at room temperature.

[0430] Transient transfection of 293HEK cells with integrin α subunit chimeric DNA constructs

[0431] 293HEK cells were plated into 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 800 μL of Opti-MEM was gently added to the cells. The transfection reagent was prepared with 2.5 μg of DNA and 3 μL of lipofectamine 2000 in 250 μL of Opti-MEM, followed by incubation at room temperature for 5 minutes. The solution was dispensed dropwise into the wells, incubated overnight, and then the complete medium was replaced. The cells were analyzed for Ab74 binding by flow cytometry and detected by anti-human IgG1 antibody conjugated to a fluorophore.

[0432] Additional Sequence

[0433] Human recombinant integrin α3β1:

[0434] ITGA3 sp|P26006|33-991; protein sequence: 1034aa

[0435] MGWSCIILFLVATATGVHSFNLDTRFLVVKEAGNPGSLFGYSVALHRQTERQQRYLLLAGAPRELAV

[0436] PDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVASQGPAGRVLVCAHRYT

[0437] QVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYLETGMCQLGTSGGFTQ

[0438] NTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQVGSFILHPKNITIVTGA

[0439] PRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDGWQDLLVGAPYYFERKE

[0440] EVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQDIAVGAPFEGLGKVYIYH

[0441] SSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGSLSDHIVLLRARPVINIVH

[0442] KTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESA

[0443] VFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALE

[0444] NHTEVQFQKECGPDNKCESNLQMRAAFVSEQQQKLSRLQYSRDVRKLLLSINVTNTRTSERSGED

[0445] AHEALLTLVVPPALLLSSVRPPGACQANETIFCELGNPFKRNQRMELLIAFEVIGVTLHTRDLQVQL

[0446] QLSTSSHQDNLWPMILTLLVDYTLQTSLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQV

[0447] GPMGEGLVGLGTLVLGLEWPYEVSNGKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRP

[0448] SSPQRRRRQLDPGGGQGPPPVTLAAAKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKAR

[0449] VWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENAQLEKELQALEKENAQLEWELQALEKELAQGGDYKDDDDK(SEQ ID NO:65).

[0450] ITGB1 sp|P05556|21 - 728; Protein sequence: 781aa

[0451] MGWSCIILFLVATATGVHSQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDD

[0452] LEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTL

[0453] KFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTT

[0454] PAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIG

[0455] WRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNI

[0456] QTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYC

[0457] KNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEG

[0458] IPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQ

[0459] CVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCE

[0460] ASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQE

[0461] CSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDDTSGLLEVLFQGPGKNAQLKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHHHH(SEQ ID NO:66).

[0462] Human recombinant integrin α3β1 domains Calf1-Calf2:

[0463] ESNLQMRAAFVSEQQQKLSRLQYSRDVRKLLLSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSV

[0464] RPPGACQANETIFCELGNPFKRNQRMELLIAFEVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLL

[0465] VDYTLQTSLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLE

[0466] WPYEVSNGKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGP

[0467] PPVTLAAAKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENHHHHHH(SEQ ID NO:67)。

[0468] Human recombinant integrin α3β1 domain Thigh:

[0469] MGWSCIILFLVATATGVHSMDYKDDDDKGGGGSGGGGSGGGGSLEVLFQGPLRARPVINIVHKTL

[0470] VPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESAVFH

[0471] GFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQLEVLFQGPGGGGSGGGGSGGGGSHHHHHH(SEQ ID NO:68)。

[0472] Mouse recombinant integrin α3β1:

[0473] Mouse α3ECD sequence; Protein sequence: 1036aa

[0474] MGWSCIILFLVATATGVHSFNLDTRFLVVKEAVNPGSLFGYSVALHRQTERQQRYLLLAGAPRDLAV

[0475] GDDYTNRTGAVYLCPLTAHKDDCERMDISEKSDPDHHIIEDMWLGVTVASQGPAGRVLVCAHRYT

[0476] KVLWSGLEDQRRMVGKCYVRGNDLQLDPGDDWQTYHNEMCNSNTDYLQTGMCQLGTSGGFTQ

[0477] NTVYFGAPGAYNWKGNSYMIQRKDWDLSEYSYRGSEEQGNLYIGYTVQVGNAILHPTDIITVVTG

[0478] APRHQHMGAVFLLKQESGGDLQRKQVLKGTQVGAYFGSAIALADLNNDGWQDLLVGAPYYFER

[0479] KEEVGGAVYVFMNQAGASFPDQPSLLLHGPSRSAFGISIASIGDINQDGFQDIAVGAPFEGLGKVYI

[0480] YHSSSGGLLRQPQQIIHGEKLGLPGLATFGYSLSGKMDVDENLYPDLLVGSLSDHIVLLRARPVINIL

[0481] HRTLVARPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFARSQS

[0482] SVFHGFFSMPETHCQTLELLLMDNVRDKLRPIVIAMNYSLPLRMPDRLKLGLRSLDAYPVLNQAQ

[0483] AMENHTEVHFQKECGPDNKCDSNLQMRAAFLSEQLQPLSRLQYSRDTKKLFLSINVTNSPSSQRA

[0484] GEDAHEALLTLEVPSALLLSSVRPSGTCQANNETILCELGNPFKRNQRMELLIAFEVIGVTLHTRDL

[0485] PVLLQLSTSSHQDNLQPVLLTLQVDYTLQASLSLMNHRLQSFFGGTVMGEAAMKTAEDVGSPLKY

[0486] EFQVSPVGDGLAALGTLVLGLEWPYEVTNGKWLLYPTEITIHSNGSWPCQPSGNLVNPLNLTLSDP

[0487] GVTPLSPQRRRRQLDPGGDQSSPPVTLAAAKKAKSETVLTCSNGRARCVWLECPLPDTSNITNVTV

[0488] KARVWNSTFIEDYKDFDRVRVDGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENAQLEKELQALEKENAQLEWELQALEKELAQGGDYKDDDDK(SEQ ID NO:69).

[0489] Mouse β1ECD sequence; Protein sequence: 781aa

[0490] MGWSCIILFLVATATGVHSQTDKNRCLKANAKSCGECIQAGPNCGWCTNTTFLQEGMPTSARCDD

[0491] LEALKKKGCQPSDIENPRGSQTIKKNKNVTNRSKGMAEKLRPEDITQIQPQQLLLKLRSGEPQKFTL

[0492] KFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTT

[0493] PAKLRNPCTSEQNCTSPFSYKNVLSLTDRGEFFNELVGQQRISGNLDSPEGGFDAIMQVAVCGSLIG

[0494] WRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNVYTMSHYYDYPSIAHLVQKLSENNI

[0495] QTIFAVTEEFQPVYKELKNLIPKSAVGTLSGNSSNVIQLIIDAYNSLSSEVILENSKLPDGVTINYKSY

[0496] CKNGVNGTGENGRKCSNISIGDEVQFEISITANKCPNKESETIKIKPLGFTEEVEVVLQFICKCNCQS

[0497] HGIPASPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVC

[0498] GQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCRCRVCECYPNYTGSACDCSLDTGP

[0499] CLASNGQICNGRGICECGACKCTDPKFQGPTCETCQTCLGVCAEHKECVQCRAFNKGEKKDTCAQ

[0500] ECSHFNLTKVESREKLPQPVQVDPVTHCKEKDIDDCWFYFTYSVNGNNEAIVHVVETPDCPTGPDDTSGLLEVLFQGPGKNAQLKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHHHH(SEQ ID NO:70).

[0501] Mouse recombinant integrin α3β1 domains Calf1 - Calf2:

[0502] MGWSCIILFLVATATGVHSDSNLQMRAAFLSEQLQPLSRLQYSRDTKKLFLSINVTNSPSSQRAGED

[0503] AHEALLTLEVPSALLLSSVRPSGTCQANNETILCELGNPFKRNQRMELLIAFEVIGVTLHTRDLPVL

[0504] LQLSTSSHQDNLQPVLLTLQVDYTLQASLSLMNHRLQSFFGGTVMGEAAMKTAEDVGSPLKYEFQ

[0505] VSPVGDGLAALGTLVLGLEWPYEVTNGKWLLYPTEITIHSNGSWPCQPSGNLVNPLNLTLSDPGVT

[0506] PLSPQRRRRQLDPGGDQSSPPVTLAAAKKAKSETVLTCSNGRARCVWLECPLPDTSNITNVTVKAR

[0507] [[ID=2,7]]VWNSTFIEDYKDFDRVRVDGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGENHHHHHH(SEQ ID NO:71).

[0508] The above examples are provided to illustrate the present disclosure and not to limit its scope. Other variations of the present disclosure will be apparent to those skilled in the art and are encompassed by the appended claims. All publications, databases, internet resources, patents, patent applications, and registration numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. An isolated antibody that binds to integrin α3β1 or a portion thereof, comprising: (1) Heavy chain complementarity determining region 1 (CDR H1) that comprises the sequence X1X2SGX3TFX4X5YX6X7X8 (SEQ ID NO: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) CDR H2 that comprises a sequence having at most two amino acid substitutions relative to the sequence of GISGSADTTY (SEQ ID NO:6), SISSSSSYIY (SEQ ID NO:9), or GIIPIFGTAN (SEQ ID NO:10), or the sequence of WISAX1NGNX2N (SEQ ID NO:39), wherein X1 is Y or N; and X2 is T or S; (3) CDR H3 that comprises a sequence having at most two amino acid substitutions relative to the sequence of VRDDIQLRD (SEQ ID NO:11) or AREFPGWYFDY (SEQ ID NO:13), or a sequence having at most four amino acid substitutions relative to the sequence of ARDYSGSWYPSNGPALDY (SEQ ID NO:12), AREYYDFWSGYPSGYAFDI (SEQ ID NO:14), or ARGVPSGSGYYLGLDY (SEQ ID NO:15); (4) Light chain complementarity determining region 1 (CDR L1) that comprises the sequence X1ASQX2ISX3YLN (SEQ ID NO:40), or a sequence having at most three amino acid substitutions relative to the sequence of QGDSLRSYYAS (SEQ ID NO:23) or SGSSSNIGSNYVY (SEQ ID NO:24), wherein X1 is Q or A; X2 is D or Y; and X3 is N or S; (5) CDR L2 that comprises a sequence having at most one amino acid substitution relative 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 that comprises the sequence X1QX2YX3X4PX5T (SEQ ID NO:42), or a sequence having at most two amino acid substitutions relative to the sequence of NSRDSSGNHWV (SEQ ID NO:31) or AAWDDSLSGPV (SEQ ID NO:32), wherein 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.

2. The isolated antibody according to claim 1, wherein: (1) The CDR H1 comprises the sequence of any one of 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) The CDR H2 comprises the sequence of any one of 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) The CDR H3 comprises the sequence of any one of 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) The CDR L1 comprises the sequence of any one of 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 comprises the sequence of any one of YDASNLET (SEQ ID NO:25), YGKNNRPS (SEQ ID NO:26), and YRNNQRPS (SEQ ID NO:27); and (6) The CDR L3 comprises the sequence of any one of 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).

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

11.

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

12.

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

13.

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

14.

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

15.

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

28.

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

29.

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

30.

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

31.

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

32.

13. An isolated antibody according to any one of claims 1 to 12, wherein the antibody comprises a heavy chain variable region having at least 90% identity to the sequence of any one of SEQ ID NOs: 16 - 20.

14. An isolated antibody according to any one of claims 1 to 13, wherein the antibody comprises a light chain variable region having at least 90% identity to the sequence of any one of SEQ ID NOs: 33 - 37.

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

28.

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

16.

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

33.

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

29.

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

17.

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

34.

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

30.

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

18.

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

35.

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

31.

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

19.

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

36.

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

32.

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

20.

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

37.

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

43.

31. The isolated antibody according to any one of claims 1 to 30, wherein the antibody binds to a cell expressing integrin α3β1 or a portion thereof.

32. The isolated antibody according to claim 31, wherein the cell is a podocyte or a neutrophil.

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

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

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

44.

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

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

38. An 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 single-chain Fv (scFv) antibody.

39. An 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 39.

41. An expression vector comprising the nucleic acid according to claim 40.

42. An isolated host cell comprising 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 in need thereof, 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 transplantation 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 a nephritic disease, a renal disease, Alport syndrome, or focal segmental glomerulosclerosis (FSGS).

49. A method for identifying an antibody that binds to integrin α3β1 or a portion thereof, comprising: 1) removing antibodies that bind to the β1 chain of the integrin α3β1 in the presence or absence of a ligand mimetic peptide and / or an antibody; 2) selecting antibodies that bind to the integrin α3β1 from the antibodies remaining in step 1) in the presence or absence of a β1 agonist antibody; 3) counterselecting the antibodies that bind to the integrin α3β1 against an immobilized β1 agonist antibody or ligand mimetic peptide, separately; and 4) repeating steps 1), 2), and 3) above to enrich for antibodies that are allosteric agonists of integrin α3 in the presence of integrin α3β1 expressed on the cell surface.

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

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

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