CN120202023A

CN120202023A - Compositions and methods involving integrin α3β1

Info

Publication number: CN120202023A
Application number: CN202380076446.XA
Authority: CN
Inventors: S·B·皮内达; D·洛佩斯-罗德里格斯; C·J·亚伯拉罕斯; A·J·巴尔博萨; V·古普塔
Original assignee: 149 Biological Co ltd; Rush University Medical Center
Current assignee: 149 Biological Co ltd; Rush University Medical Center
Priority date: 2022-10-31
Filing date: 2023-10-27
Publication date: 2025-06-24
Also published as: AU2023373741A1; EP4611808A1; WO2024097620A1

Abstract

Compositions and methods comprising antibodies that bind to integrin α3β1 are provided.

Description

Compositions and methods involving integrin alpha 3 beta 1

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/420,964 filed on 10/2022 at 31, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Background

Integrin α3β1 is a key integrin on the surface of cells including podocytes (cells that encapsulate glomerular capillaries within the bowman's capsule of the kidney). Integrin α3β1 is critical for podocyte attachment to the outside of blood vessels to form healthy glomeruli in the kidney. Allosteric agonist antibodies to integrin α3β1 can enhance integrin-dependent ligand binding and cell adhesion.

Disclosure of Invention

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

(1) Heavy chain complementarity determining region 1 (CDR H1) comprising a sequence of X ₁X₂SGX₃TFX₄X₅YX₆X₇X₈ (SEQ ID NO: 38) wherein X ₁ is A or K, X ₂ is A or T, X ₃ is F, G, or F, X ₄ is S or T, X ₅ is S or N, X ₆ is G, S, or A, X ₇ is M or I, and X ₈ is H, N, or S;

(2) CDR H2 comprising a sequence having up to two amino acid substitutions, or a sequence of WISAX ₁NGNX₂ N (SEQ ID NO: 39), relative to the sequence of GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), wherein X ₁ is Y or N, and X ₂ is T or S;

(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);

(4) A light chain complementarity determining region 1 (CDR L1) comprising the sequence of X ₁ASQX₂ISX₃ YLN (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 X ₁ is Q or A, X ₂ is D or Y, and X ₃ is N or S;

(5) CDR L2 comprising a sequence having at most one amino acid substitution relative to the sequence of YDASNLET (SEQ ID NO: 25), or a sequence of YX ₁X₂NX₃ RPS (SEQ ID NO: 41), wherein X ₁ is G or R, X ₂ is K or N, and X ₃ is N or Q, and

(6) CDR L3 comprising the sequence of X ₁QX₂YX₃X₄PX₅ T (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 X ₁ is L or Q, X ₂ is D or S, X ₃ is N, S, or R, X ₄ is Y or T, and X ₅ is L or P.

In some embodiments of this aspect, (1) CDR H1 comprises the sequence of any 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 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 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) CDR L1 comprises the sequence of any of QASQDISNYLN (SEQ ID NO: 21), RASQYISSYLN (SEQ ID NO: 22), QGDSLRSYYAS (SEQ ID NO: 23) and SGSSSNIGSNYVY (SEQ ID NO: 24), (5) CDR L2 comprises the sequence of any of YDASNLET (SEQ ID NO: 25), YGKNNRPS (SEQ ID NO: 26) and YRNNQRPS (SEQ ID NO: 27), and (6) L3 comprises the sequence of any of 95 (SEQ ID NO: 28), 69 (SEQ ID NO: 28), RASQYISSYLN (SEQ ID NO: 22), QGDSLRSYYAS (SEQ ID NO: 23) and SGSSSNIGSNYVY (SEQ ID NO: 24).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In some embodiments, 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. In some embodiments, 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.

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

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

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

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

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

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

In some embodiments of the antibodies described herein, the antibodies bind to cells expressing integrin α3β1 or a portion thereof. In certain embodiments, the cell is a podocyte, T cell, cancer cell, or neutrophil.

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 in the α3 moiety. In certain embodiments, the antibody binds to the sequence of SEQ ID NO. 44 or a sequence within the sequence of SEQ ID NO. 44. In some embodiments, the antibody binds to a specific conformation of α3. In some embodiments, the antibody binds to α3 and stabilizes it in a specific conformation.

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 ', F (ab') 2, fv, or single chain Fv (scFv) antibody. In some embodiments, the antibody is a bispecific antibody.

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

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

In another aspect, the present disclosure provides an isolated host cell comprising the vector described above.

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

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.

In some embodiments of the method, the kidney disease is a glomerular disease, such as nephritis, kidney disease (nephrotic disease), alport syndrome, or Focal Segmental Glomerulosclerosis (FSGS).

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

1) Removing antibodies that bind to the β1 chain of integrin α3β1 in the presence or absence of ligand mimetic peptides and/or antibodies;

2) Selecting antibodies that bind to integrin α3β1 from the antibodies remaining in step 1) in the presence or absence of β1 agonist antibodies;

3) Counter-selection of antibodies binding to integrin alpha 3 beta 1 against immobilized beta 1 agonist antibodies or ligand mimetic peptides alone, and

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

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 β1-containing integrin dimers other than α3β1 (e.g., α4β1 and α5β1). In some embodiments of the method, steps 1) and/or 2) are performed using human K562 cells that predominantly express human α5β1 integrin and that do not express α3β1.

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 agents (e.g., antibodies and ligands) that block the ligand binding site or domain of the integrin.

In some embodiments, integrin α3β1 is stabilized in a specific conformation by pre-complexing with an activator or inhibitor (e.g., activating antibody 9EG7 or TS 2/16). 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 an integrin dimer.

Brief Description of Drawings

FIGS. 1A-1D are representations of binding of integrin agonist antibodies by direct integrin ELISA. Bovine Serum Albumin (BSA), recombinant human integrin α3β1ECD, recombinant human integrin α4β1ECD, or recombinant mouse integrin α3β1ECD were coated on plates and incubated with human anti- α3ab or isoforms (n for each coated protein is 8, n for BSA is 4). Binding of (a) Ab74 a100, (B) Ab74a101, (C) Ab74 a102 and (D) Ab74 a104 was detected by incubation with anti-hIgG 1 Ab HRP conjugate and development of color by fluorogenic substrate, followed by reading the average fluorescence intensity on an enzyme-labeled instrument.

FIG. 2 epitope mapping of integrin agonist antibodies by direct integrin ELISA. Recombinant integrin α3β1 domain or Bovine Serum Albumin (BSA) was coated on plates and incubated with human anti- α3ab (red) or isotype (blue) (n is 4). Binding of Ab74 a101 was detected by incubation with anti-hIgG 1 Ab HRP conjugate and development of color via fluorogenic substrate, followed by reading the average fluorescence intensity on an enzyme-labeled instrument.

FIGS. 3A-3D show increased ligand binding in cells expressing mouse integrin alpha 3 beta 1 in the presence of agonist antibodies. The α3β1 expressing K562 cells were incubated with α3β1 ligand mimetic LXY 2-biotin conjugates and integrin agonist antibodies or isotype antibody controls. Cells were then stained with streptavidin-fluorophore conjugate and measured in a flow cytometer. The (a) Ab74 a100, (B) Ab74 a101, (C) Ab74 a102, (D) Ab74 a104 exhibited increased LXY2 binding when compared to the isotype control alone.

FIGS. 4A-4E show reduced cell migration in the presence of integrin agonist antibodies as determined by wound healing. SK-OV-3 cells expressing α3β1 were plated into ligand coated wells and allowed to adhere for 16 hours at 37 ℃. (A) Before addition of the treatments, scratch lesions were formed on the cell layer using a sterile pipette tip. (B) Isotype antibody control and (C) blocking of anti- α3 antibodies did not reduce cell migration, allowing cells to close the wound. (D) Control anti- β1 agonist antibody and (E) anti- α3 agonist antibody Ab74 a101 reduced wound closure after 16 hours.

FIG. 5 is a schematic representation of a domain exchanged mammalian expression construct.

FIGS. 6A-6C show staining of podocytes by antibodies with novel anti-integrin alpha 3 antibodies staining of podocyte expressed integrin alpha 3 beta 1. Kidney sections of C57B/L6 wild-type mice were immunofluorescent stained with various antibodies (5 μg/mL) and imaged using confocal microscopy. Representative images stained with ab74_a100 (a), 9EG7 (B), or human anti-mouse IgG1 isotype control antibody (C) are shown.

Detailed Description

I. Introduction to the invention

The present inventors have discovered novel 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 may enhance integrin-dependent functions such as ligand binding and cell adhesion. In particular, given that integrin α3β1 is a critical 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 nephritis, kidney disease, alport syndrome, or Focal Segmental Glomerulosclerosis (FSGS).

The inventors have also found that novel anti-integrin α3 allosteric antibodies induce intracellular signal transduction in the presence of external ligands. For example, when cells expressing integrin alpha 3 were incubated with the novel anti-alpha 3 integrin antibodies in the absence of integrin ligand, the level of phosphorylated focal adhesion kinase (pFAK) was unchanged. Co-incubation of cells with the novel antibodies and ligand laminin increased the relative levels of pFAK.

II. Definition of

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 of disulfide bond in the hinge region to produce F (ab)' 2 (dimer of Fab, which is itself a light chain linked to V _H-C_H 1 by disulfide bond). F (ab) '2 can be reduced under mild conditions to break disulfide bonds in the hinge region, thereby converting the (Fab ') 2 dimer to Fab ' monomer. The Fab' monomer is essentially a Fab having a portion of the hinge region (see Fundamental Immunology, w.e.Paul et al, RAVEN PRESS, N.Y. (1993)) for a more detailed description of other antibody fragments. Although various antibody fragments are defined in terms of digestion of intact antibodies, one skilled in the art will appreciate that fragments may be synthesized de novo, either chemically or by using recombinant DNA methods. Thus, as used herein, the term antibody also includes antibody fragments produced by modification of whole antibodies or antibody fragments synthesized using recombinant DNA methods.

Antibodies as described herein may consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Confirmed immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, igM, igA, igD and IgE, respectively. In some embodiments, the antibody is IgG (e.g., igG1, igG2, igG3, igG 4), igM, igA, igD, or IgE.

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

In antibodies, substitution variants remove at least one amino acid residue and have different residues inserted in their positions. The most interesting sites for substitution mutagenesis include hypervariable regions, but framework changes are also contemplated. Examples of conservative substitutions are described above.

Basic modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in terms of maintaining the structure of the polypeptide backbone in the substitution region, e.g., as a β -sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following classes:

(1) Nonpolar norleucine Met, ala, val, leu, ile;

(2) A polar uncharged Cys, ser, thr, asn, gln;

(3) Acid (negatively charged): asp, glu;

(4) Basic (positively charged) Lys, arg;

(5) Residues affecting chain orientation, gly, pro, and

(6) Aromatic Trp, tyr, phe, his.

Non-conservative substitutions are made by exchanging members of one of these classes for another class.

One type of substitution that may be made is to change one or more cysteines in the antibody that may be chemically reactive to another residue, such as, but not limited to, alanine or serine. For example, there may be substitutions of non-classical cysteines. Substitutions may be made in the CDRs or framework regions of the variable domains or in the constant regions of the antibodies. In some embodiments, the cysteine is classical (e.g., involved in disulfide bond formation). Any cysteine residue that does not participate in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine linkages may be added to the antibody to increase its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

Antibodies include V _H-V_L dimers, including single chain antibodies (antibodies that exist as a single polypeptide chain), such as single chain Fv antibodies (sFv or scFv), in which variable heavy and variable light chain regions are joined together (either directly or through a peptide linker) to form a continuous polypeptide. Single chain Fv antibodies are covalently linked V _H-V_L, which can be expressed from a nucleic acid comprising V _H -and V _L -coding sequences linked directly or through a peptide-encoding linker (e.g., huston et al, proc. Nat. Acad. Sci. USA,85:5879-5883,1988). When V _H and V _L are linked to each other as a single polypeptide chain, the V _H and V _L domains associate non-covalently. Or the antibody may be another fragment. Other fragments may also be generated, for example using recombinant techniques, as soluble proteins or as fragments obtained from display methods. Antibodies may also include diabodies and minibodies. Antibodies of the present disclosure also include heavy chain dimers, such as antibodies from camels. In some embodiments, the antibody is dimeric. In other embodiments, the antibody may be in monomeric form with an active isotype. In some embodiments, the antibody is in a multivalent form, e.g., a trivalent or tetravalent form.

As used herein, the terms "variable region" and "variable domain" refer to the portion of the light and heavy chains of an antibody that includes the amino acid sequences of complementarity determining regions (CDRs, e.g., HCDR1, HCDR2, HCR3, LCDR1, LCDR2, and LCDR 3) and Framework Regions (FR). The variable regions of the heavy and light chains are commonly designated V _H and V _L, respectively. The variable regions are included on the Fab, F (ab') ₂, fv, and scFv antibody fragments described herein and are involved in specific antigen recognition.

As used herein, "Complementarity Determining Regions (CDRs)" refer to three hypervariable regions in each chain, interrupted by four framework regions established by light and heavy chain variable regions. CDRs are mainly responsible for binding to epitopes of antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially 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 CDR1 from the variable domain of the antibody light chain in which it is present.

The sequences of the framework regions of the different light or heavy chains are relatively conserved within a species. The framework regions of antibodies (i.e., the combined framework regions of the constitutive light and heavy chains) are used to position and align the CDRs in three-dimensional space.

The amino acid sequences of the CDRs and framework regions may be determined using a variety of well known definitions in the art, such as Kabat, north methods (see, e.g., north et al, J Mol biol 406 (2): 228-256, 2011), chothia, 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., 1989,Conformations of immunoglobulin hypervariable regions.Nature 342,877-883; chothia C. Et al, 1992,structural repertoire of the human V _H segments J.mol. Biol.227,799-817; al-Lazikani et al, J.mol. Biol1997,273 (4)). The definition of antigen combining sites is also described In 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 M.J.E. (eds.), protein Structure predictionOxford University Press, oxford,141-172 1996.

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, a 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 its natural ligand binding.

As used herein, "chimeric antibody" refers to an immunoglobulin molecule in which (a) the constant region or a portion thereof is altered, substituted or exchanged such 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 that confers novel properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc., or (b) the variable region or a portion thereof is altered, substituted or exchanged by a variable region or a portion thereof having different or altered antigen specificity, or by a corresponding sequence from another species or from another antibody class or subclass.

As used herein, "humanized antibody" refers to immunoglobulin molecules in CDRs from a donor antibody that are grafted onto a human framework sequence. Humanized antibodies may also contain residues of donor origin in the framework sequences. The humanized antibody may further comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also comprise residues that are present in neither the recipient antibody nor the imported CDR or framework sequences. Humanization (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) may be performed using methods known in the art, including techniques such as "super-humanizing" antibodies (Tan et al, J. Immunol.169:1119,2002) and "surface remodeling (resurfacing)" (e.g., staelens et al, mol. Immunol.43:1243,2006; and Roguska et al, proc. Natl. Acad. Sci USA 91:969, 1994).

The term "recombinant" when used in reference to, for example, a cell, or 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 a change in a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

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 term may refer to any molecule specifically recognized by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combination thereof (e.g., phosphorylated or glycosylated polypeptide, etc.). Those skilled in the art will appreciate that the term does not indicate that the molecule is immunogenic in each case, but simply that it can be targeted by an antibody.

Antibodies bind to an "epitope" on an antigen. An epitope is a localized site on an antigen that is recognized and bound by an antibody. An epitope may comprise several amino acids or portions of several amino acids, e.g. 5 or 6 or more, e.g. 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes a non-protein component, such as from a carbohydrate, a nucleic acid, or a lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope may consist of contiguous amino acids, or of amino acids from different parts of the protein that are accessed by protein folding (e.g., a discontinuous epitope). The same is true for other types of target molecules that form three-dimensional structures. Epitopes typically comprise at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., epitope Mapping Protocols, in Methods in Molecular Biology, volume 66, glenn E.Morris, eds. (1996).

The terms "specific for," "specifically binds," and like terms refer to a molecule (e.g., an antibody or antibody fragment) that binds a target with an affinity that is at least 2-fold greater than a non-target compound, e.g., any of an affinity that is at least 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 a target typically binds the target with at least 2-fold greater affinity than 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 descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

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

A "control" sample or value refers to a sample that serves as a reference (typically a known reference) for comparison with a test sample. For example, a test sample may be obtained from a test condition (e.g., in the presence of a test compound) and compared to 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 may also represent an average or range collected from multiple tests or results. Those skilled in the art will recognize that controls may be designed to evaluate any number of parameters. For example, controls 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). The control may be designed for in vitro applications. Those skilled in the art will understand which controls are valuable in a given situation and can analyze the data based on comparison with the control values. The control is also valuable for determining the significance of the data. For example, if the value of a given parameter varies greatly in the control, the variation in the test sample will not be considered significant.

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

For sequence comparison, typically one sequence serves as a reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are entered into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes reference to a segment selected from any one of a number of consecutive positions from 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150, wherein after optimal alignment of two sequences, the sequences can be compared to a reference sequence of the same number of consecutive positions. Sequence alignment methods for comparison are well known in the art.

Algorithms suitable for determining percent sequence identity and percent sequence similarity are the 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 the percent sequence identity of the nucleic acids and proteins of the present disclosure. Software for performing BLAST analysis is publicly available through the national center for Biotechnology information (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 either match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as a neighborhood word score threshold (neighborhood word score threshold) (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits extend in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score was calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The word hits are stopped from extending in each direction by the amount X of the reduction in cumulative alignment score from its maximum realized value, by the cumulative score becoming zero or lower due to the accumulation of one or more negative scoring residue alignments, or by reaching the end of either sequence. BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to 11 words long (W), 10 expectations (E), m=5, n= -4, and comparison of the two strands. For amino acid sequences, the BLASTP program defaults to use a word length of 3 and an expected value (E) of 10, and a BLOSUM62 scoring matrix of 50 (see Henikoff & Henikoff, proc. Natl. Acad. Sci. Usa 89:10915 (1989)) to compare (B), expected values (E) of 10, m=5, n= -4, and comparison of the two strands.

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

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 the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 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. Probes8:91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term encompasses amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, 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 hydrogen, a carboxyl group, an amino group, and an R group), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. 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 refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to naturally occurring amino acids.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee (IUPAC-IUB Biochemical Nomenclature Commission). Also, nucleotides may be referred to by their commonly accepted single letter codes.

Antibodies that bind integrin alpha 3 beta 1 or a portion thereof

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 the α3 and β1 moieties. Integrin α3β1 is highly expressed on the surface of kidney podocytes and is critical for podocyte attachment to the outside of blood vessels to form healthy glomeruli in the kidney. The integrins are also expressed on other cells, such as T cells (Park et al ,Integrinα3promotes TH17 cellpolarization and extravasation during autoimmune neuroinflammation,Science Immunology,, volume 8 (88), 2023), cancer cells (Ke et al ,Novel monoclonal antibody against integrinα3shows therapeutic potential for ovarian cancer,Cancer Sci.,111(10),, page 3478, 2020) and neutrophils (Lerman et al ,Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrinα3β1-dependent,Blood,2014, 12, 4; 124 (24): 3515-23) and keratinocytes (Has et al INTEGRIN A, mutations WITH KIDNEY, lung, and skin disease.N Engl J Med 366:1508-1514,2012), and can affect the function of the cells expressing the integrins. 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 loss of kidney function. These antibodies can also reduce T cell migration and infiltration to reduce autoimmune disease, reduce cancer cell migration to reduce tumor growth and metastasis, and reduce pro-inflammatory neutrophil activation and tissue recruitment.

In some embodiments, the anti- α3β1 antibody is isolated (e.g., isolated from a component of its natural environment (e.g., animal, biological sample). In some embodiments, the anti- α3β1 antibody is a humanized antibody, or antigen-binding fragment thereof. In some embodiments, the anti- α3β1 antibody is a derivative of a humanized antibody that binds 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, to α3β1 in a flow cytometry assay, 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 cell (e.g., podocyte) of the subject).

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 the following sequence:

the thigh-genu region is in bold in SEQ ID NO. 45.

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 a sequence of ：MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK(SEQ ID NO:46).

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 in the α3 moiety. In certain embodiments, the antibody binds to the sequence of SEQ ID NO. 44 or a portion within the sequence of SEQ ID NO. 44.

VINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFA

GSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQKEC(SEQ ID NO:44)。

Generally, an anti- α3β1 antibody provided herein comprises at least one immunoglobulin heavy chain variable region and at least one immunoglobulin light chain variable region. In some embodiments, an anti- α3β1 antibody described herein comprises two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions. Typically, each immunoglobulin heavy chain variable region of an anti- α3β1 antibody comprises first, second, and third heavy chain complementarity determining regions (CDRs; HCDR1, HCDR2, and HCDR 3), and each immunoglobulin light chain variable region of an anti- α3β1 antibody comprises first, second, and third light chain CDRs (LCDR 1, LCDR2, and LCDR 3).

In some embodiments, the antibody is an antibody fragment, such as Fab, F (ab') ₂, fv, or scFv. Antibody fragments may be generated using any means known in the art, including chemical digestion (e.g., papain or pepsin) and recombinant methods. Methods for isolation and preparation of recombinant nucleic acids are known to those skilled in the art (see Sambrook et al, molecular cloning. A Laboratory Manual (2 nd edition, 1989); ausubel et al, current Protocols in Molecular Biology (1995)). Antibodies can be expressed in a variety of host cells, including E.coli (E.coli), other bacterial hosts, yeast, and various higher eukaryotic cells (e.g., COS, CHO, and HeLa cell lines, and myeloma cell lines).

In some embodiments, an antibody of the disclosure may comprise the sequences of heavy chain complementarity determining region 1 (HCDR 1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR 1), LCDR2, LCDR3, heavy chain variable region (V _H), and/or light chain variable region (V _L) as set forth in table 1.

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) may comprise:

(1) Heavy chain complementarity determining region 1 (HCDR 1) comprising a sequence of X ₁X₂SGX₃TFX₄X₅YX₆X₇X₈ (SEQ ID NO: 38) wherein X ₁ is A or K, X ₂ is A or T, X ₃ is F, G, or F, X ₄ is S or T, X ₅ is S or N, X ₆ is G, S, or A, X ₇ is M or I, and X ₈ is H, N, or S;

(2) HCDR2 comprising a sequence having up to two amino acid substitutions, or a sequence of WISAX ₁NGNX₂ N (SEQ ID NO: 39), relative to the sequence of GISGSADTTY (SEQ ID NO: 6), SISSSSSYIY (SEQ ID NO: 9), or GIIPIFGTAN (SEQ ID NO: 10), wherein X ₁ is Y or N, and X ₂ is T or S;

(3) HCDR3 comprising 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 (LCDR 1) comprising the sequence of X ₁ASQX₂ISX₃ YLN (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 X ₁ is Q or A, X ₂ is D or Y, and X ₃ is N or S;

(5) LCDR2 comprising a sequence having at most one amino acid substitution relative to the sequence of YDASNLET (SEQ ID NO: 25), or a sequence of YX ₁X₂NX₃ RPS (SEQ ID NO: 41), wherein X ₁ is G or R, X ₂ is K or N, and X ₃ is N or Q, and

(6) LCDR3 comprising the sequence of X ₁QX₂YX₃X₄PX₅ T (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 X ₁ is L or Q, X ₂ is D or S, X ₃ is N, S, or R, X ₄ is Y or T, and X ₅ is L or P.

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

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

HCDR1-3 and V _H

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

Antibodies of the disclosure may comprise heavy chain variable regions (V _H) having HCDR1, HCDR2, and HCDR3 as described herein. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any of SEQ ID NOS: 16-20. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 1,6, and 11, 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. 16. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 2, 7, and 12, 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 17. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 3, 8, and 13, 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: 18. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 4, 9, and 14, 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 19. In certain embodiments, antibodies of the disclosure may comprise a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 5, 10, and 15, 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: 20.

LCDR1-3 and V _L

In some embodiments, an antibody of the present disclosure may comprise LCDR1 having the 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, LCDR2 having the 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 LCDR3 having the 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 LCDR1 having the 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, LCDR2 having the 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 LCDR3 having the 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 LCDR1 having the 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, LCDR2 having the 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 LCDR3 having the 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 LCDR1 having the 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, LCDR2 having the 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 LCDR3 having the 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 LCDR1 having the sequence of SEQ ID No. 24 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID No. 24, LCDR2 having the sequence of SEQ ID No. 27 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID No. 27, and LCDR3 having the sequence of SEQ ID No. 32 or a variant thereof having a sequence with one amino acid substitution relative to the sequence of SEQ ID No. 32.

Antibodies of the disclosure may comprise a light chain variable region (V _L) having LCDR1, LCDR2, and LCDR3 as described herein. In certain embodiments, antibodies of the disclosure may comprise a light chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of any of SEQ ID NOs 33-37. In certain embodiments, antibodies of the disclosure may comprise a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 21, 25, and 28, 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 33. In certain embodiments, antibodies of the 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, antibodies of the 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, antibodies of the 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, antibodies of the 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.

A100

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

In some embodiments, an antibody may comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 1,6, and 11, 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. 16, and (2) a light chain variable region having LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 21, 25, and 28, 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. 33.

In certain embodiments, the antibody comprises a heavy chain 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. 47:

EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWLSGISGSADTTYYADSVK

GRFTISRDNSKNTLYLQMTSLRAEDTAVYYCVRDDIQLRDWGQGTLVTVSSASTKGPSVFPLAPSS

KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI

CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

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 sequence of SEQ ID NO: 48:

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

GTDFALTISSLQPEDFATYYCLQDYNYPLTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCL

LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

In certain embodiments, an antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO. 16 and a sequence 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. 47, and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO. 33 and a sequence 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. 48.

A101

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

In some embodiments, an antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 2, 7, and 12, 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 17, and (2) 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, the antibody comprises a heavy chain 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. 49:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQK

LQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYSGSWYPSNGPALDYWGQGTMVTVSSAS

TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, 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 sequence of SEQ ID NO: 50:

DIQMTQSPSSLSASVGDRVTITCRASQYISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

GTDFTFTISSLQPEDIATYYCLQDYSYPLTFGGGIKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

In certain embodiments, an antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO. 17 and a sequence 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. 49, and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO. 34 and a sequence 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. 50.

A102

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

In some embodiments, an antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 3, 8, and 13, 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:18, and (2) 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, the antibody comprises a heavy chain 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: 51:

EVQLVQSGAEVKKPGASVKVSCKTSGFTFTNYGISWVRQAPGQGLEWMGWISANNGNSNYAQD

HQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAREFPGWYFDYWGQGTLVTVSSASTKGPSVFP

LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

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 sequence of SEQ ID NO: 52:

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS

GTDFTFTISSLQPDDFATYYCQQSYRTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

In certain embodiments, an antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO. 18 and a sequence 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. 51, and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO. 35 and a sequence 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. 52.

A103

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

In some embodiments, an antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 4, 9, and 14, 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 19, and (2) 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, the antibody comprises a heavy chain 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: 53:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKG

RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAREYYDFWSGYPSGYAFDIWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, 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 sequence of SEQ ID NO: 54:

QSALTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSG

NTASLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

In certain embodiments, an antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO. 19 and a sequence 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. 53, and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO. 36 and a sequence 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. 54.

A104

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

In some embodiments, an antibody can comprise (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 5, 10, and 15, 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:20, and (2) 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.

In certain embodiments, the antibody comprises a heavy chain 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: 55:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGTANYAQKFQ

GRVTITADKSTSTAYMELSSLRSEDTAVYYCARGVPSGSGYYLGLDYWGQGTMVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

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 sequence of SEQ ID NO: 56:

QSELTQPPSASGAPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSK

SGTSASLAISGLRSEDEADYYCAAWDDSLSGPVFSGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTAS

VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

In certain embodiments, an antibody comprises (i) a heavy chain comprising a heavy chain variable region having the sequence of SEQ ID NO. 20 and a sequence 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. 55, and (ii) a light chain comprising a light chain variable region having the sequence of SEQ ID NO. 37 and a sequence 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. 56.

Fc polypeptides

The anti- α3β1 antibodies provided herein may comprise a crystallizable fragment region (Fc region), also referred to herein as an Fc polypeptide. Fc polypeptides are part 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. Fc polypeptides typically include a CH2 domain and a CH3 domain, which are immunoglobulin constant region domain polypeptides. In some embodiments, the Fc polypeptide in an antibody described herein can be a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide. In certain embodiments, an antibody described herein may comprise a wild-type Fc polypeptide having the sequence of SEQ ID NO. 43:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN

QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

In other embodiments, antibodies described herein can comprise a variant of a wild-type Fc polypeptide that has 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 at least one amino acid substitution relative to the sequence of the wild-type Fc polypeptide (e.g., SEQ ID NO: 43).

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 altered phenotypes relative to antibodies comprising wild-type Fc polypeptides. 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 an NK-dependent or macrophage-dependent assay).

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, such as at positions 4 and 5 (numbering relative to the sequence of SEQ ID NO: 43) (see, e.g., lund et al, JImmunol.147 (8): 2657-62, 1991). For example, in some embodiments, one or both Fc polypeptides in an antibody described herein may comprise L4A and L5A substitutions.

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

In some embodiments, the Fc polypeptide comprises one or more modifications that alter the ratio of affinity of the modified Fc polypeptide for an active fcγr (e.g., fcγriia or fcγriiia) relative to inhibiting an fcγr (e.g., fcγriib):

Where the modified Fc polypeptide has an affinity ratio of 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 symptoms of a disease, disorder, or infection, wherein enhanced efficacy of effector cell function (e.g., ADCC) mediated by fcγr, e.g., cancer or infectious disease, is desired. Where the modified Fc region has an affinity ratio of 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 symptoms of a disease or disorder, wherein reduced efficacy of effector cell function mediated by fcγr, e.g., autoimmune or inflammatory disorders, is desired. Examples of single, two, three, four and five amino acid substitutions in Fc polypeptides providing an affinity ratio of greater than 1 or less than 1 are listed in table 2 (see, e.g., PCT publication nos. WO 04/063251; WO 06/088494; WO 07/024449; WO 06/113665; WO 07/021841; WO 07/106707; WO 2008/140603). Amino acid positions are numbered according to the EU numbering scheme.

TABLE 2

Antibodies that competitively bind to anti-alpha 3 beta 1 antibodies

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

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

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

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 competitively bind to the same epitope (e.g., the same peptide (linear epitope) or the same surface amino acid (conformational epitope)) on α3β1 as one or more anti- α3β1 antibodies described herein. Such antibodies that bind the same epitope may be referred to as epitope competitors.

VI polyclonal and monoclonal antibodies

Polyclonal antibodies can be raised in animals (vertebrates or invertebrates, including mammals, birds, and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen with a protein or other carrier that is immunogenic in the species to be immunized (e.g., key Kongxie blue protein, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent, such as maleimidobenzenesuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂, or r1n=c=nr (where R and R1 are different alkyl groups). Non-protein carriers (e.g., colloidal gold) may also be used for antibody production.

Animals may be immunized against an antigen, immunogenic conjugate, or derivative by, for example, mixing 100 μg or 5 μg (for rabbit or mouse, respectively) of protein or conjugate with three volumes of freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, animals were bled and serum antibody titers were determined. The booster immunization of the animals was performed until the titers were stable. Typically, animals are boosted with conjugates of the same antigen but conjugated to different proteins and/or by different crosslinking agents. Conjugates can also be prepared as protein fusions in recombinant cell cultures. Furthermore, coagulants (e.g., alum) are suitable for enhancing immune responses.

Monoclonal antibodies can be made using hybridomas (e.g., the hybridoma method described for the first time by Kohler et al, nature,256:495 (1975)), or can be made by other methods such as recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal (e.g., hamster or cynomolgus monkey) is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Or can be an in vitro immune lymphocyte. Lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (see, e.g., goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pages 59-103 (ACADEMIC PRESS, 1986)).

The hybridoma cells so produced are inoculated into a suitable medium and grown, which may contain one or more substances that inhibit the growth or survival of the non-fused parent myeloma cells. For example, if the parent myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine, which inhibit the growth of HGPRT-deficient cells (HAT medium). Preferred myeloma cells are those cells that have high fusion efficiency, support stable and high level production of antibodies by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma cell lines, such as SP-2 or X63-Ag8-653 cells (available from the American type culture Collection (AMERICAN TYPE Culture Collection), rockville, md. USA). Human myeloma and mouse-human heterologous myeloma cell lines for the production of human monoclonal antibodies are also described (Kozbor, J.Immunol.,133:3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (MARCEL DEKKER, inc., new York, 1987)).

The medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation, in vitro binding assays such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA), or flow cytometry analysis of cells expressing membrane antigens. For example, the binding affinity of a monoclonal antibody can be determined by Scatchard analysis by Munson et al, anal. Biochem.,107:220 (1980).

After hybridoma cells have been identified to produce 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, pages 59-103 (ACADEMIC PRESS, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in animals as ascites tumors. 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, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). Alternatively, cDNA may be prepared from mRNA and then DNA sequenced. Hybridoma cells serve as a preferred source of such genomic DNA or RNA from which cDNA is prepared. Once isolated, the DNA can be placed into an expression vector well known in the art, which is then transfected into a host cell (e.g., an escherichia coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or hybridoma cell) that does not otherwise produce immunoglobulins, thereby obtaining synthetic monoclonal antibodies in the recombinant host cell.

VII humanized and amino acid variants

General methods for humanization of antibodies are described, for example, in U.S. patent nos. 5861155、6479284、6407213、6639055、6500931、5530101、5585089、5693761、5693762、6180370、5714350、6350861、5777085、5834597、5882644、5932448、6013256、6129914、6210671、6329511、5225539、6548640 and 5624821. 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 antibodies.

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. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterpart. 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 described, e.g., in U.S. patent No. 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) Nature321:522, 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, polynucleotides comprising a first sequence encoding a humanized immunoglobulin framework region and a second sequence set encoding a desired immunoglobulin complementarity determining region may be synthetically produced or produced by combining appropriate cDNA and genomic DNA segments. The human constant region DNA sequence can be isolated from a variety of human cells according to well known procedures. CDRs used to produce immunoglobulins of the present disclosure may similarly be derived from monoclonal antibodies capable of specifically binding to α3β1.

Amino acid sequence variants of the anti- α3β1 antibodies can be prepared by introducing appropriate nucleotide changes into the DNA of the anti- α3β1 antibody or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti- α3β1 antibodies of the examples herein. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics. Amino acid changes may also alter post-translational processing of humanized or variant anti- α3β1 antibodies, such as altering the number or position of glycosylation sites.

One method for identifying specific residues or regions in an anti- α3β1 antibody that are preferred mutagenesis sites is referred to as "alanine scanning mutagenesis" as described, for example, by Cunningham and Wells, science,244:1081-1085 (1989). Here, a residue or set of residues of interest (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 polyAla) to affect the interaction of the amino acids with the α3β1 antigen (e.g., sequence within the thigh-genu region of integrin α3β1). Those amino acid positions that exhibit functional sensitivity to substitution are then refined by introducing further or other variants at or against the substitution site. Thus, although the site at which the amino acid sequence variation is introduced 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 carboxy-terminal fusions ranging in length from one residue to polypeptides containing 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 insertional variants include enzymes or polypeptides fused to the N-or C-terminus of an antibody that increase the serum half-life of the antibody.

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 have different residues inserted in their positions. The most interesting sites for substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are preferred, but more substantial changes may be introduced and the product may be screened. Examples of substitutions are listed below:

Ala(A):Val;Leu;Ile;Val

Arg(R):Lys;Gln;Asn;Lys

Asn(N):Gln;His;Asp、Lys;Gln;Arg

Asp(D):Glu;Asn

Cys(C):Ser;Ala

Gln(Q):Asn;Glu

Glu(E):Asp;Gln

Gly(G):Ala

His(H):Asn;Gln;Lys;Arg

Ile (I) Leu, val, met, ala, leu, phe, norleucine

Leu (L) is norleucine, ile, val, ile, met, ala, phe

Lys(K):Arg;Gln;Asn

Met(M):Leu;Phe;Ile

Phe(F):Leu;Val;Ile;Ala;Tyr

Pro(P):Ala

Ser(S):Thr

Thr(T):Ser

Trp(W):Tyr;Phe

Tyr(Y):Trp;Phe;Thr;Ser

Val (V) is Ile, leu, met, phe, ala, norleucine

Basic modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in terms of maintaining the structure of the polypeptide backbone in the substitution region, e.g., as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following classes:

(1) Hydrophobicity, norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilicity Cys, ser, thr;

(3) Acid, asp, glu;

(4) Basicity Asn, gln, his, lys, arg;

(5) Residues affecting chain orientation, gly, pro, and

(6) Aromatic Trp, tyr, phe

Non-conservative substitutions require the exchange of members of one of the above classes for another class.

Any cysteine residue that does not participate 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 linkages may be added to the antibody to increase its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

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

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By "altering" is meant 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. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are the most common recognition sequences for enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The addition of glycosylation sites to antibodies can be achieved by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Alterations (for O-linked glycosylation sites) may also be made by adding or substituting one or more serine or threonine residues in the sequence of the original antibody.

VIII other modifications

Other modifications of the 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 fragment thereof) or a radioisotope (e.g., a radio conjugate) 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-pyridinedithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide compounds (such as bis- (p-azidobenzoyl) hexamethylenediamine), diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and di-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene).

The anti- α3β1 antibodies (e.g., anti- α3β1 antibodies) disclosed herein can be formulated as immunoliposomes. Antibodies containing liposomes are prepared by methods known in the art, such as those described in Epstein et al, proc.Natl. Acad.Sci.USA 82:3688 (1985), hwang et al, proc.Natl. Acad.Sci.USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes can be formed by reverse phase evaporation from a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter defining a pore size to produce liposomes having a desired diameter. Fab' fragments of the antibodies provided herein can be conjugated to liposomes via disulfide interchange reactions as described in Martin et al, J.biol.chem.257:286-288 (1982). The liposomes optionally contain another active ingredient therein.

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

In certain embodiments, it may be desirable to use antibody fragments rather than whole antibodies to increase penetration of, for example, target tissues and cells. In such cases, 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 in the antibody fragment (e.g., by mutating the appropriate region 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, e.g., by DNA or peptide synthesis; see, e.g., WO96/32478 published at 10/17 1996).

In some embodiments, modifications may optionally be introduced into the antibody (e.g., within the polypeptide chain or at either the N-terminus or the C-terminus) to, for example, extend in vivo half-life, 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, e.g., 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 linkers may be, for example, glu residues, or acyl residues containing thiol functionality for attachment 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 at a residue exposed to solvent (e.g., a substitution of an Arg or Lys residue). The Cys residue is then site-specifically attached to a PEG chain containing, for example, a maleimide functional group. Methods for incorporating PEG or PEG long chain polymers are known in the art (e.g., veronese, F.M. et al, drug disc. Today 10:1451-8 (2005); greenwald, R.B. et al, adv. Drug Deliv. Rev.55:217-50 (2003); roberts, M.J. et al, adv. Drug Deliv. Rev.,54:459-76 (2002), the contents of which are incorporated herein by reference).

Covalent modifications of anti- α3β1 antibodies are also included within the scope of this technology. For example, the modification may be by chemical synthesis or by enzymatic or chemical cleavage of an anti- α3β1 antibody. Other types of covalent modifications of antibodies are introduced into the molecule by reacting 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. Pat. No. 5,534,615, which is expressly incorporated herein by reference. One preferred type of covalent modification of an antibody comprises attaching the antibody to one of a plurality of non-protein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene) in a manner such as that set forth in U.S. Pat. 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

The 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. The nucleic acids herein may include one or more subsequences, each subsequence referred to as a polynucleotide.

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 an immunoglobulin heavy chain variable domain of an anti- α3β1 antibody provided herein. In some embodiments, the nucleic acid encodes an immunoglobulin light chain variable domain of an anti- α3β1 antibody provided herein. In some embodiments, the nucleic acid encodes an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of an 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.

For recombinant production of anti- α3β1 antibodies, the nucleic acid encoding the anti- α3β1 antibody may be isolated and inserted into a replication vector for further cloning (amplification of DNA) or expression. In some cases, the anti- α3β1 antibody may be produced by homologous recombination. DNA encoding the anti- α3β1 antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibodies). Many vectors are available. The vector component typically includes, but is not limited to, one or more of a signal sequence, and an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Suitable host cells for cloning or expressing the DNA in the vectors herein may be prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae (Enterobacteriaceae) such as Escherichia (ESCHERIACHIA) (e.g. E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (e.g. Salmonella typhimurium (Salmonella typhimurium)), serratia (e.g. Serratia marcescens (SERRATIA MARCESCANS)) and Shigella (Shigella), and Bacillus (Bacillus) (e.g. Bacillus subtilis (B. Subtilis) and Bacillus licheniformis (B. Licheniformis)), pseudomonas (Pseudomonas) (e.g. Pseudomonas aeruginosa) and Streptomyces (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.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the anti- α3β1 antibody encoding vectors. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. A number of other genera, species or strains are generally available and useful herein, such as Schizosaccharomyces (Schizosaccharomyces pombe), kluyveromyces (Kluyveromyces) hosts, e.g., kluyveromyces lactis (K.lactis), kluyveromyces fragilis (K.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), kluyveromyces wegenensis (K.wichemii) (ATCC 24,178), kluyveromyces krill (K.waii) (ATCC 56,500), kluyveromyces drosophila (K.drosophorae) (ATCC 36,906), kluyveromyces thermotolens (K.thermalis) and Kluyveromyces marxianus (K.marxianus), yarrowia (yarrowia) (EP 402,226), kluyveromyces (Pichia pastoris) (EP 183,070), trichoderma (Candida) Trichoderma (Schwanese) and Aspergillus (Schwannomia), aspergillus (Aspergillus kaki) and Aspergillus (Aspergillus kawachii) (Aspergillus kaki) such as Aspergillus kaki (K.drosophila) and Aspergillus kaki (Aspergillus kaki) and Aspergillus (Toxiconae.38.

Suitable host cells for expressing the anti- α3β1 antibodies may 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 (Spodoptera frugiperda) (armyworm), aedes aegypti (AEDES AEGYPTI) (mosquito), aedes albopictus (Aedes albopictus) (mosquito), drosophila melanogaster (Drosophila melanogaster) (drosophila) and Bombyx mori (silkworm moth). A variety of viral strains for transfection are publicly available, for example, the L-1 variant of the Spodoptera frugiperda (Autographa californica) NPV and the Bm-5 strain of the silkworm (Bombyx mori) NPV, and such viruses may be used herein in accordance with the present technology as viruses for use in particular in transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

Suitable host cells for expressing the anti- α3β1 antibodies may also include vertebrate cells (e.g., mammalian cells). Vertebrate cells can be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), the 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. USA77:4216 (1980)), mouse Sertoli cells (TM 4, mather, biol. Reprod.23:243-251 (1980)), african green monkey kidney cells (RO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo mouse liver cells (39L) A, urlaub et al, proc. Natl. Acad. Sci. USA77:4216 (1980)), mouse Sertoli cells (TM 4, mather, biol. Reprod.23:243-251 (1980)), african green monkey kidney cells (RO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDC 4, ATCC CCL 34), buffalo mouse liver cells (39L 4, mr. Lev. SCL 4, mr. 3.F. 3, mr. 4, and human Mr. 3.F.3.3.F.6, mr. 3.

The host cells may be transformed with the expression or cloning vectors described above for antibody production and cultured in conventional nutrient media which are suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. Host cells for producing antibodies provided herein can be cultured in a variety of media. Commercially available media such as Ham F10 (Sigma), minimal essential media (MEM, sigma), RPMI-1640 (Sigma) and Dulbecco's modified eagle's medium ((DMEM, sigma) are suitable for culturing host cells. 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. No. 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 may also be used as the medium for the host cells. Any of these media may be supplemented as desired 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., GENTAMYCINTM), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included in suitable concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are conditions previously used to select host cells for expression and will be apparent to the ordinarily skilled artisan.

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

Antibody compositions produced from cells may be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being 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 may be recommended for all mouse isoforms and human gamma 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 useful. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, bakerbond abx.tm. Resin (j.t.baker, philips burg, n.j.) may be used for purification. Other protein purification techniques such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel column chromatography, chromatography on heparin SEPHAROSE ^TM, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also useful depending on the antibody to be recovered.

After any one or more preliminary purification steps, the mixture comprising the antibody of interest and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between, for example, about 2.5-4.5, and may be performed at low salt concentrations (e.g., about 0-0.2 m salt).

X. pharmaceutical formulations, administration and route of administration

The present disclosure provides anti- α3β1 antibodies and related compositions that are useful, for example, for eliminating α3β1-expressing pathogens from the body, and for, for example, identifying and quantifying the amount of α3β1-expressing pathogens in biological samples.

Anti- α3β1 antibodies can be formulated into pharmaceutical compositions that can be used for a variety of purposes, including the treatment of diseases or conditions. Pharmaceutical compositions comprising one or more anti- α3β1 antibodies may be administered to a patient in need thereof using a pharmaceutical device, and according to one embodiment of the technology, kits comprising such devices are provided. Such devices and kits can be designed for conventional administration, including self-administration, of the pharmaceutical compositions herein.

Antibody therapeutic formulations in the form of lyophilized formulations or aqueous solutions can be prepared for storage by mixing an agent or antibody of the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences th edition, osol, editions (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic 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, hexahydrocarbon quaternary ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, 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, sugar, or sorbitol, salt forming ions such as sodium, metal complexes such as sodium, or non-aqueous seaweed (e.g., ^TM、PLURONICS^TM), or non-surface active protein such as Zn-PEG or non-Protein (PEG) or non-equilibrium complex.

The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those having complementary activities without adversely affecting each other. Such molecules are suitably present in combination in an amount effective for the intended purpose.

Formulations for in vivo administration are typically sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

A slow release preparation may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent/antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate), or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-gamma ethyl glutamate, nondegradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron(Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of sustained release of molecules for more than 100 days, certain hydrogels release proteins in a shorter period of time. When encapsulated agents/antibodies remain in the body for a longer period of time, they may denature or aggregate as a result of exposure to moisture at 37 ℃, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanisms involved, reasonable stabilization strategies can be devised. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds by sulfhydryl-disulfide interchange (thio-disulfide interchange), stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymeric matrix compositions.

For therapeutic use, the anti- α3β1 antibodies provided herein are administered to a mammal, such as a human, in pharmaceutically acceptable dosage forms (such as those discussed above, including those that can be administered to a human by intravenous as a bolus or by continuous infusion over a period of time, or by intramuscular, intraperitoneal, cerebrospinal, subcutaneous, intra-articular, synovial, intrathecal, oral, topical, or inhalation routes). For the prevention or treatment of a disease, the appropriate dosage of the agent or antibody will depend on the type of disease to be treated, the severity and course of the disease, as defined above, the purpose for which the antibody is administered, previous therapies, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

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

In some embodiments, a composition comprising an anti- α3β1 antibody herein may be administered as monotherapy, and in some embodiments, a composition comprising an anti- α3β1 antibody may be administered as part of a combination therapy. In some cases, the effectiveness of an antibody in preventing or treating a disease may be improved by administering the antibody continuously or in combination with another agent that is effective for those purposes (e.g., a chemotherapeutic agent for treating cancer or a microbial infection). In other cases, anti- α3β1 antibodies can be used to enhance or sensitize cells to chemotherapy treatment, allowing efficacy to occur 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 further comprise delivering a second therapeutic regimen selected from the group consisting of a chemotherapeutic agent, radiation therapy, surgery, and combinations of any of the foregoing. Such other agents may be present in the composition to be administered, or may be administered separately. In addition, the anti- α3β1 antibodies may be suitably administered sequentially or in combination with other agents or forms (e.g., chemotherapeutic agents or radiation for treating cancer, infection, etc., or immunosuppressive drugs).

XI method

As described herein, integrin α3β1 is a key integrin on the surface of podocytes (cells that encapsulate glomerular capillaries within the kidney bowman's capsule). Integrin α3β1 is critical for podocyte attachment to the outside of blood vessels to form healthy glomeruli in the kidney. The antibodies described herein can act as allosteric agonist antibodies to integrin α3β1 and can enhance integrin-dependent ligand binding and cell adhesion, thereby preventing cell loss in urine and preventing loss of kidney function. Also provided herein are methods of treating a disease and/or condition associated with podocyte loss in a subject in need thereof by administering to the subject an anti- α3β1 antibody described herein that binds to 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 may be a disease and/or condition caused by podocyte loss (i.e., loss of cell number and/or loss of cell function). 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 kidney disease and/or condition.

In some embodiments of the method, the subject suffers from kidney disease associated with podocyte loss (i.e., loss of cell number and/or loss of cell function). The kidney disease may be a glomerular disease, such as nephritis, kidney disease, alport syndrome, or Focal Segmental Glomerulosclerosis (FSGS).

In some embodiments, the subject is receiving, has received, or is about to receive a transplant procedure. In some embodiments, the transplant procedure is a kidney transplant. In certain embodiments, the antibody is administered after the transplant procedure. In certain embodiments, antibodies are administered after kidney transplantation to protect, maintain, and/or improve kidney function and health.

In some embodiments, the disease or condition associated with podocyte loss in the 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 an anti-glomerular basement membrane (anti-GBM) disease. In some embodiments, the autoimmune disease is ANCA-related vasculitis and glomerulonephritis.

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

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 Membranous Proliferative Glomerulonephritis (MPGN), interstitial nephritis, igA nephropathy (Berger's disease), pyelonephritis, lupus nephritis, or Wegener's granulomatosis.

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

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 predominantly express human α5β1 integrin and that do not express α3β1.

We cleared β1 binders by clearing against "other" β1 integrins (either as recombinant proteins or using cell lines, such as α5β1 expressing K562, etc.).

We prepared the α3β1 complex by pre-complexing α3β1 (recombinant or expressed on cell lines) with a β1 activating antibody such that α3β1 is in a more "open" or "active" like conformation. This allows for easier identification of antibodies that bind to active α3β1 or activate α3β1. We further increased the opportunity to identify activated antibodies by adding a ligand or ligand mimetic to the complex. This prepared pre-complex was used to select for activated antibodies.

We further facilitated the identification of new antibodies directed against allosteric sites by blocking the ligand binding face of the integrin complex using ligand mimics or blocking antibodies.

Examples

Example 1 identification of antibody fragments Using phage display

To find variable short chain fragments (scfvs), phage display libraries of natural human scfvs were run through novel selection strategies to identify allosteric agonists. This strategy facilitates the identification of allosteric agonist conjugates, the identification of conjugates specific for one integrin chain relative to another integrin chain, and the identification of conjugates that increase ligand binding. In addition, this strategy relies on conformationally stabilized integrins (e.g., integrin α3β1 complexed with β1 activating antibodies to stabilize the integrin in an "active" conformation) to aid in the identification of conformationally sensitive conjugates. In addition, this strategy uses ligand blockers (e.g., integrin α3β1 complexed with ligand mimetic peptide LXY2 or ligand laminin to block highly antigenic ligand binding pockets and MIDAS sites, with or without β1 activating antibodies) to intentionally exclude binders directed against the ligand binding pockets. Here we used two selection methods (termed selection 1 and selection 2) to identify the binders. Selection 1 utilizes recombinant integrins, while selection 2 utilizes cell surface expressed integrins. For option 1, a round of screening uses three steps, pre-exhaustion, selection and counter-selection. The pre-depletion step is used to remove the binding of the β1 chain to the α3β1 dimer by using a negative selection, in which step phage binding to immobilized recombinant human integrin α4β1 is selected from the screening pool. In addition, in certain steps, both commercial β1 agonist antibodies (TS 2/16) and ligand mimetic peptides (LXY 2) are included with immobilized integrin α4β1 to further remove any phage conjugates of these agents. The non-binding phage is used in the next step, the selection step for positive selection, in which phage are 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 ligand mimetic peptide LXY 2. Unbound phage were removed and discarded. Bound phage are eluted and used in the final step, where any conjugate of β1 agonist antibody and LXY2 is removed using counter-selection against immobilized β1 agonist antibody and LXY2 alone. All unbound phages were considered to be enriched for the anti-integrin α3 allosteric binder. In addition, this process can be repeated for multiple rounds to further enrich the phage clones of interest.

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 cell surface expressed integrins (similar to the methods described above). Here, a round of screening consisted of 1) a pre-depletion step against K562 cells (expressing predominantly human α5β1 integrin and not expressing α3β1) in the presence or absence of the β1 agonist antibody and ligand mimetic peptide LXY2, 2) a positive selection against human K562 cells expressing integrin α3β1 in the presence or absence of the β1 agonist antibody TS2/16, and a counter selection against the immobilized β1 agonist antibody and LXY2 alone. Cell line production is described in this method.

The enriched phage library from selection 2 is optionally further amplified and plated using standard methods. 184 individual clones were identified and selected for the clonal expansion step. Individual clones were purified to produce Periplasmic Extract (PE) solutions containing soluble parent clone scFv. These extracts were tested via direct integrin ELISA (as described in the methods) for each of human α3β1, mouse α3β1, and human α4β1, with or without LXY 2. Extracts were then tested by flow cytometry for both K562 expressing human α3 and K562 not expressing α3.

After PE characterization, DNA of the variable domains of each scFv was sequenced. CDR sequences were assigned to each of 184 clones, and alignment and clustering were then performed to eliminate duplicates. The 184 clones isolated from the selection yielded 25 unique scFv CDR sequences. Sequencing data was combined with assay data from PE ELISA and FACS to enable selection of the best hits.

EXAMPLE 2 Generation of full-Length IgG antibodies

Using the dataset generated by the sequencing and characterization assays, the first five sequences were selected for reconstruction. The heavy chain variable region DNA sequence of each scFv was attached to full length human heavy chain constant IgG 1DNA (IGHC gene transcripts) via gene synthesis and cloning. The light chain human k-variable region and lambda variable region DNA sequences of each scFv were attached to full length human light chain constant IGKC1 and IGLC 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 mammalian expression vectors.

Each of the five paired constructs was transfected and expressed in 10mL mammalian cells, and then the antibodies were 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 SDS-PAGE gels and the SEC-HPLC peak determined that the samples were quite pure.

Example 3 validation of antibody binding Using ELISA

Five full length IgG antibodies are called Ab74 a100 to a104, or Ab74 for short. First, the binding of five abs 74 to ECD was characterized by direct integrin ELISA. Briefly, BSA, recombinant human integrin α3β1ECD, recombinant human integrin α4β1ECD, or recombinant mouse integrin α3β1ECD were coated on the plates overnight and then incubated with each of the five abs 74 or with isotype human IgG1 antibody negative control or commercial anti-human α3 antibody positive control, respectively. Binding was detected by incubation with an anti-human IgG1 antibody horseradish peroxidase (HRP) conjugate, followed by treatment with a fluorogenic substrate, development of the reaction, and reading of the average fluorescence intensity using an enzyme-labeled instrument.

ELISA results showed that five Ab74 preferentially bound human α3β1ECD, rather than all other antigens coated on the plate (FIGS. 1A-1D). Isotype negative control and anti- α3 positive control gave the expected negative and positive results, confirming the low background of the assay and positive signal to human α3, respectively. Two of the five Ab74 antibodies exhibited low binding to mouse α3, all five Ab74 showed only background signals to coated BSA and human α4β1ECD, confirming that Ab74 binds human α3ECD rather than human β1ECD by ELISA.

To further refine the binding site, the individual human α3 domains are expressed recombinantly in mammalian cells and purified. Ab74 was tested against BSA, soluble human α3thigh-Genu, human α Calf1-Calf2, or human α3β1ECD by direct integrin ELISA. The data indicate that Ab74 a101 binds to the th-Genu region in the sequence of the expressed construct (fig. 2).

Example 4 validation of antibody binding Using flow cytometry-based assays

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

After treatment of K562 cells over-expressing human or mouse integrin α3, all full-length human IgG antibodies were detected by flow cytometry after treatment with anti-human IgG1 antibody conjugated to fluorophores and secondary antibody staining. The results in table 3 show that Ab74 detection was higher in both human and mouse integrin α3 expressing K562 cells when compared to the negative control isotype antibody.

TABLE 3 characterization of integrin agonist antibodies. Ab74 clones bound both human and mouse integrins α3β1 (as measured by ELISA and flow cytometry). The α3β1 domain localization was performed by binding ELISA and flow cytometry using recombinant protein domain and domain exchanged integrin expressing cell lines, respectively.

Example 5 increased ligand binding by cells expressing human integrin α3β1 in the presence of agonist antibodies

To explore ligand binding agonism, K562 expressing human α3 was treated with anti- α3ab74 or negative control isotype antibody or positive control commercial β1 agonist antibody TS2/16 in the presence of biotinylated ligand mimetic peptide LXY2 in low affinity ca2+/mg2+ buffer. LXY2 binding was detected by treating cells with streptavidin fluorophore conjugate and reading the cells in a flow cytometer. Negative and positive controls showed correctly low to no and high ligand binding in low affinity buffers, respectively. The results in table 4 show that Ab74 increased LXY2 binding in low affinity buffers compared to isotype antibodies.

TABLE 4 increased ligand binding in cells expressing human integrin alpha 3 beta 1 in the presence of agonist antibodies. The α3β1 expressing K562 cells were incubated with α3β1 ligand mimetic LXY 2-biotin conjugates, and integrin agonist antibodies or isotype controls. Cells were then stained with streptavidin-fluorophore conjugate and measured by flow cytometry.

Example 6 increased ligand binding by cells expressing mouse integrin alpha 3 beta 1 in the Presence of agonist antibodies

Cross-reactivity with mouse α3 was characterized by repeating the ligand experiments on K562 expressing mouse α3. Briefly, cells were treated with anti- α3ab74 or negative control isotype antibody or positive control commercial β1 agonist antibody 9EG7 in the presence of biotinylated ligand mimetic peptide LXY2 in low affinity ca2+/mg2+ buffer. LXY2 binding was detected by treating cells with streptavidin fluorophore conjugate and reading the cells in a flow cytometer. Negative and positive controls showed correctly low to no and high ligand binding in low affinity buffers, respectively. The results in figures 3A-3D also show that Ab74 increases LXY2 binding in low affinity buffers.

Example 7 reduction of cell migration in the Presence of integrin agonist antibodies

The adherent human ovarian cancer cell line SK-OV-3 expresses integrin alpha 3 beta 1 at high levels and mediates ligand binding to laminin-511. Tissue culture treated 96-well plates were coated with integrin α3 ligand laminin-511 and incubated overnight. The following day, wells were plated with SK-OV-3 cells in serum-free medium, allowing cells to adhere. After 16 hours, scratches (lesions) were made with sterile plastic P200 pipette tips before adding anti- α3ab74 or negative control isotype antibody or positive control β1 agonist antibody to the complete medium. Changing from serum-free medium to Fetal Bovine Serum (FBS) -containing medium promotes cell migration and thus wound closure via cell movement and migration. After 48 hours, the medium was removed, the cells were fixed with 4% paraformaldehyde and then stained with 0.2% crystal violet. The results in figures 4A-4E demonstrate that wound closure is inhibited in wells treated with all Ab74 antibodies or positive control anti- β1 agonist antibodies compared to negative control or blocking anti- α3 treated with isoforms that produce wound closure.

Example 8 integrin agonist antibody targeting thigh-genu domain

To further refine the binding epitope of the antibody, several α3 integrin domains are "swapped" (as described in the methods) with their homologous counterparts in similar protein human integrin α7 (also containing the Thigh-Genu, calf 1 and Calf domains).

To this end, integrin DNA constructs were created in which the Thigh-genu, calf 1 and Calf domains on integrin alpha 7 replaced equivalent domains on integrin alpha 3 in mammalian expression plasmids. These three constructs were separately transfected into HEK-293 cells using Lipofectamine and allowed to expand under culture conditions for 48 hours. These cells were then treated with Ab74 or negative control isotype antibody or positive control commercial α3 antibody P1B5 prior to staining with fluorophore conjugated anti-human IgG1 for detection in a flow cytometer.

The results in table 5 show that Ab74 was detected in cells transfected with full-length integrin α3DNA and constructs with integrin α3 containing calf-2 of integrin α7 and constructs with integrin α3 containing calf-1 of integrin α7, while little to no binding occurred in integrin α3 constructs substituted with the Thigh-Genu domain of integrin α7, indicating that the five Ab74 antibodies recognized epitopes in the Thigh-Genu region of α3.

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 are ITGA7 th-genu, calf 1 and Calf 2 inserted into alpha 3, respectively, to replace the wild-type sequence. These DNA constructs were then cloned into mammalian expression vectors and transfected into mammalian cells within 48 hours, respectively, and then incubated with activated human anti- α3ab. Antibody binding was detected by staining cells with anti-hIgG 1 antibody APC conjugate and reading on a flow cytometer.

TABLE 5 epitope domain localization of integrin agonist antibodies by flow cytometry.

Example 9 method

Cell culture and transient protein expression in HEK293 cell lines

HEK293 cells were cultured in serum-free CD medium (Sino Biological catalog No. SMM 293-TI) until they reached optimal cell density. Expression vectors were added to cells in the presence of TF1 transfection reagent and serum-free feeding solution (Sino Biological cat. No. M293-SUPI-100) was added to the culture at days 1, 3 and 5 post-transfection. Cells were harvested on day 7 of culture and protein purification was performed.

Purification of proteins from HEK293 cells

Cells were removed via centrifugation and culture supernatants were collected for protein purification.

Affinity purification by equilibrating the column with a loading buffer and loading the culture supernatant onto the column. The column was re-equilibrated and the target protein eluted by a buffer gradient containing imidazole (Ni-affinity), or glycine and NaCl (protein a affinity or FLAG affinity). The protein is subjected to further buffer exchange to remove excess imidazole or other salts. The protein solution was concentrated and the protein concentration and purity were determined by the corresponding methods. Protein concentration was determined by UV, while its purity was determined by SDS-PAGE and Western blotting.

Cloning in expression vectors

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

Signal peptides

N-terminal-MGWSCIILFLVATATGVHS- (SEQ ID NO: 57)

Protein tags and features

Some constructs have an N-terminal FLAG, a C-terminal 6XHIS, and 3 Gly4Ser linkers, and a 3C protease cleavage site.

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

C-terminal 6 XHis tag HHHHHH (SEQ ID NO: 59)

(Gly 4 Ser) 3 linkers GGGGSGGGGSGGGGS (SEQ ID NO: 60)

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

Domain exchanged mammalian expression constructs

To test the domain specificity of antibodies, each domain from ITGA3 was replaced in the DNA sequence with the corresponding cognate domain from ITGA7 (fig. 5). These were then transiently transfected into a CMV driven mammalian expression vector (pCMV 6-Neo).

SEQ ID NO. 62 full-length human ITGA3 with exchanged ITGA7 Thigh-Genu:

MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYSVALHRQTER

QQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVA

SQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYL

ETGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQ

VGSFILHPKNITIVTGAPRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDG

WQDLLVGAPYYFERKEEVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQD

IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

LSDHIVLLRARPILHVSHEVSIAPRSIDLEQPNCAGGHSVCVDLRVCFSYIAVPSSYSPTVALDYVLD

ADTDRRLRGQVPRVTFLSRNLEEPKHQASGTVWLKHQHDRVCGDAMFQLQENVKDKLRAIVVTL

SYSLQTPRLRRQAPGQGLPPVAPILNAHQPSTQRAEIHFLKQGCGPDNKCESNLQMRAAFVSEQQQ

KLSRLQYSRDVRKLLLSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSVRPPGACQANETIFCELG

NPFKRNQRMELLIAFEVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLLVDYTLQTSLSMVNHRL

QSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLEWPYEVSNGKWLLYPTE

ITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGPPPVTLAAAKKAKSETV

LTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINM

ENKTTWFSVDIDSELVEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

SEQ ID NO. 63 full-length human ITGA3, with exchanged ITGA7 Calf 1:

MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYSVALHRQTER

QQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVA

SQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYL

ETGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQ

VGSFILHPKNITIVTGAPRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDG

WQDLLVGAPYYFERKEEVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQD

IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

LSDHIVLLRARPVINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEA

DRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLG

LRSLDAYPILNQAQALENHTEVQFQKECGPDNKCQSNLQLVRARFCTRVSDTEFQPLPMDVDGTT

ALFALSGQPVIGLELMVTNLPSDPAQPQADGDDAHEAQLLVMLPDSLHYSGVRALDPAEKPLCLS

NENASHVECELGNPMKRGAQVTFYLILSTSGISIETTELEVELLLATISEQELHPVSARARVFIELLQT

SLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLEWPYEVSN

GKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGPPPVTLAA

AKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLF

LRTSIPTINMENKTTWFSVDIDSELVEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

SEQ ID NO. 64 full-length human ITGA3, calf 2 replaced with ITGA7 Calf 2:

MGPGPSRAPRAPRLMLCALALMVAAGGCVVSAFNLDTRFLVVKEAGNPGSLFGYSVALHRQTER

QQRYLLLAGAPRELAVPDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVA

SQGPAGRVLVCAHRYTQVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYL

ETGMCQLGTSGGFTQNTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQ

VGSFILHPKNITIVTGAPRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDG

WQDLLVGAPYYFERKEEVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQD

IAVGAPFEGLGKVYIYHSSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGS

LSDHIVLLRARPVINIVHKTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEA

DRDRRPPRLRFAGSESAVFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLG

LRSLDAYPILNQAQALENHTEVQFQKECGPDNKCESNLQMRAAFVSEQQQKLSRLQYSRDVRKLL

LSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSVRPPGACQANETIFCELGNPFKRNQRMELLIAF

EVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLLVDYTPLSIAGMAIPQQLFFSGVVRGERAMQS

ERDVGSKVKYEVTVSNQGQSLRTLGSAFLNIMWPHEIANGKWLLYPMQVELEGGQGPGQKGLCS

PRPNILHLDVDSRDRRRRELEPPEQQEPGERQEPSMSWWPVSSAEKKKNITLDCARGTANCVVFSC

PLYSFDRAAVLHVWGRLWNSTFLEEYSAVKSLEVIVRANITVKSSIKNLMLRDASTVIPVMVYLDP

MEELPAEIELWLVLVAVGAGLLLLGLIILLLWKCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY*

Cell line generation

K562 cells from ATCC were transfected via electroporation with linearized expression plasmid containing human integrin alpha 3 and maintained for 2 weeks under G418 selection at 0.5 mg/mL. Cells highly expressing integrin were enriched by Fluorescence Activated Cell Sorting (FACS) following the staining protocol with commercial antibody P1B5 (Millipore Sigma, MA WALTHAM, MA, USA) for anti-integrin α3 antibody staining.

K562 mice α3β1 and K562 cynomolgus α3β1 with linearized expression plasmids containing mouse or cynomolgus integrin α3 and containing a C-terminal FLAG tag, K562 cells from ATCC were transfected via electroporation and maintained for 2 weeks under selection of puromycin at 0.8 mg/mL. Cells highly expressing integrin were enriched by Fluorescence Activated Cell Sorting (FACS) following staining protocol against FLAG antibody (Sino Biological, china).

Cell wall-attachment measurement and fluorescence reporting system

Target integrin-expressing K562 cells expressing human integrin α3β1 were washed with TBS and 50,000 cells/well transferred into a total of 90. Mu.L assay buffer (HEPES 20mM/2Mg/mL glucose/140 mM NaCl, containing 1mM each of Ca ₂₊ and Mg ₂₊, denoted HEPES-CaMg) in ligand-coated wells of a high binding transparent 384 well microplate (Corning Incorporated, one Riverfront Plaza, NY, USA). Plates were incubated at 37 ℃ for 30 minutes in the presence of antibodies. To induce detachment of non-adherent cells, the plates were gently inverted and held in this position for 45 minutes at room temperature. The plate was set up and the wells were quickly aspirated with an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). Adherent cells were quantified using CyQuantNF (Invitrogen, waltham, mass., USA). For min-max normalization, negative control assay buffer (HEPES buffer containing 10mM EDTA, denoted HEPES-EDTA) and positive control assay buffer (TBS containing 1mM Mn ₂₊ and 200uM Ca ₂₊, denoted TBS-Mn) are included in the plates.

Cell attachment assay, automated imaging

Target integrin-expressing K562 cells expressing human integrin α3β1 were washed with TBS and 50,000 cells/well transferred into a total of 90. Mu.L assay buffer (HEPES 20mM/2Mg/mL glucose/140 mM NaCl, containing 1mM each of Ca ₂₊ and Mg ₂₊, denoted HEPES-CaMg) in ligand-coated wells of a high binding transparent 384 well microplate (Corning Incorporated, one Riverfront Plaza, NY, USA). Plates were incubated at 37 ℃ for 30 minutes in the presence of antibodies. To induce detachment of non-adherent cells, the plates were gently inverted and held in this position for 45 minutes at room temperature. The plates were set up and wells were fixed in an inverted position for 10 minutes at room temperature using a final concentration of 2% paraformaldehyde stock. The panels were set up and rapidly aspirated using an automatic board wash machine (Agilent Technologies, SANTA CLARA, CA, USA). Adherent cells were quantified using DAPI and an automated imaging system with a nuclear segmentation algorithm. For min-max normalization, negative control assay buffer (TBS containing 10mM EDTA, indicated as TBS-EDTA) and positive control assay buffer (TBS containing 1mM Mn2+ and 200. Mu.M Ca2+, indicated as TBS-Mn) are included in the plates.

Direct integrin ELISA

High binding black 384 well microplates (Corning Incorporated, one Riverfront Plaza, NY, USA) were coated overnight at 4℃with 30. Mu.L of TBS containing 3. Mu.g/mL recombinant integrin. The plates were flicked to remove any liquid and blocked and incubated for 1 hour by adding 90. Mu.L of TBS (Sigma-Aldrich, st.Louis, MO, USA) containing 5% bovine serum albumin (w/v), 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68. After incubation, the plates were washed three times with 100 μl TBS using an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). To each well was added 30. Mu.L of assay buffer (TBS containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, denoted TBS-T). To each corresponding well 1 μl of test antibody stock was added and the plate was centrifuged at 1000g for 1 min and incubated for 1 hr at room temperature. Plates were washed three times with 100 μl TBS using an automatic washer. To each well was added 30. Mu.L of staining buffer (TBS-T containing anti-IgG HRP conjugate diluted 1:2000) (Invitrogen, waltham, mass., USA) and incubated for 30 minutes. The plate was washed three times with 100. Mu.L in an automatic plate washer. mu.L of substrate buffer (TBS containing 100. Mu.M Amplex Red and 4mM hydrogen peroxide) (Biotium, fremont, calif., USA) was added and incubated for 30 minutes at room temperature. Plates were analysed at 563/587nm in a fluorescent microplate reader (Agilent Technologies, SANTA CLARA, CA, USA).

Integrin sandwich ELISA

High binding black 384 well microplates (Corning Incorporated, one Riverfront Plaza, NY, USA) were coated overnight at 4 ℃ with 30 μl of TBS containing 4 μg/mL anti-integrin antibody. Assay buffer (TBS (Sigma-Aldrich, st.Louis, MO, USA) containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, indicated as TBS-T) was prepared prior to the following steps. The plates were flicked to remove any liquid and blocked by adding 90. Mu.L of TBS-T (Sigma-Aldrich, st.Louis, MO, USA) containing 5% bovine serum albumin (w/v) and incubated for 1 hour. After incubation, the plates were washed three times with 100 μl TBS using an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). To each well was added 30. Mu.L of assay buffer (TBS-T containing 4. Mu.g/mL labeled recombinant integrin). Plates were centrifuged at 1000g for 1 min and incubated for 1 hr at room temperature. Plates were washed three times with 100 μl TBS using an automatic washer. To each well was added 30. Mu.L of staining buffer (TBS-T containing anti-tag antibody HRP conjugate diluted 1:2000) (Invitrogen, waltham, mass., USA) and incubated for 30 minutes at room temperature. The plate was washed three times with 100. Mu.L in an automatic plate washer. To each well was added 30. Mu.L of substrate buffer (TBS containing 100. Mu.M Amplex Red and 4mM hydrogen peroxide) (Biotium, fremont, calif., USA) and developed for 30 minutes at room temperature. Plates were analysed at 563/587nm in a fluorescent microplate reader (Agilent Technologies, SANTA CLARA, CA, USA).

Recombinant integrin function assay (SoLISA), integrin assay

High binding black 384 well microplates (Corning Incorporated, one Riverfront Plaza, NY, USA) were coated overnight with 30 μl of TBS containing 8 μg/mL ligand at 4 ℃. Assay buffer (TBS (Sigma-Aldrich, st.Louis, MO, USA) containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, indicated as TBS-T) was prepared prior to the following steps. The plates were flicked to remove any liquid and blocked by adding 90. Mu.L of TBS-T (Sigma-Aldrich, st.Louis, MO, USA) containing 5% bovine serum albumin (w/v) and incubated for 1 hour. After incubation, the plates were washed three times with 100 μl TBS using an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). To each well was added 30. Mu.L of assay buffer (TBS-T containing 4. Mu.g/mL labeled recombinant integrin and one of 1mM Ca ₂₊/1mM Mg₂₊、1mM Mn₂₊/200μM Ca₂₊, or 10mM EDTA). To each well 1 μl of agonist antibody (or isotype control) stock was added at the appropriate concentration. Plates were centrifuged at 1000g for 1 min and incubated for 3 hours at room temperature. Plates were washed three times with 100 μl TBS using an automatic washer. To each well was added 30. Mu.L of staining buffer (TBS-T containing anti-tag antibody HRP conjugate diluted 1:2000) (Invitrogen, waltham, mass., USA) and incubated for 30 minutes at room temperature. The plate was washed three times with 100. Mu.L in an automatic plate washer. To each well was added 30. Mu.L of substrate buffer (TBS containing 100. Mu.M Amplex Red and 4mM hydrogen peroxide) (Biotium, fremont, calif., USA) and developed for 30 minutes at room temperature. Plates were analysed at 563/587nm in a fluorescent microplate reader (Agilent Technologies, SANTA CLARA, CA, USA).

Recombinant integrin function assay (SoLISA), ligand detection

High binding black 384 well microplates (Corning Incorporated, one Riverfront Plaza, NY, USA) were coated overnight at 4 ℃ with 30 μl of TBS containing 4 μg/mL anti-tag antibody. Assay buffer (TBS (Sigma-Aldrich, st.Louis, MO, USA) containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, indicated as TBS-T) was prepared prior to the following steps. The plates were flicked to remove any liquid and blocked by adding 90. Mu.L of TBS-T containing 5% bovine serum albumin (w/v) and then incubated for 1 hour. After incubation, the plates were washed three times with 100 μl TBS using an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). To each well was added 30. Mu.L of capture buffer (TBS-T containing 4. Mu.g/mL labeled recombinant integrin). To each well was added 30. Mu.L of assay buffer (TBS-T containing 8. Mu.g/mL of labeled recombinant ligand and one of 1mM Ca ₂₊/1mM Mg₂₊、1mM Mn₂₊/200μM Ca₂₊, or 10mM EDTA). To each well 1 μl of agonist antibody (or isotype control) stock was added at the appropriate concentration. Plates were centrifuged at 1000g for 1 min and incubated for 3 hours at room temperature. Plates were washed three times with 100 μl TBS using an automatic washer. To each well was added 30 μl of staining buffer (TBS-T containing anti-ligand-tag antibody HRP conjugate diluted 1:2000) (Invitrogen, waltham, MA, USA) and incubated for 30 minutes at room temperature. The plate was washed three times with 100. Mu.L in an automatic plate washer. To each well was added 30. Mu.L of substrate buffer (TBS containing 100. Mu.M Amplex Red and 4mM hydrogen peroxide) (Biotium, fremont, calif., USA) and developed for 30 minutes at room temperature. Plates were analysed at 563/587nm in a fluorescent microplate reader (Agilent Technologies, SANTA CLARA, CA, USA).

Recombinant integrin function assay (SoLISA), antibody detection

High binding black 384 well microplates (Corning Incorporated, one Riverfront Plaza, NY, USA) were coated overnight with 30 μl of TBS containing 8 μg/mL ligand at 4 ℃. Assay buffer (TBS (Sigma-Aldrich, st.Louis, MO, USA) containing 0.05% Triton X-100 (v/v) and 0.025% (v/v) Pluronic F68, indicated as TBS-T) was prepared prior to the following steps. The plates were flicked to remove any liquid and blocked by adding 90. Mu.L of TBS-T (Sigma-Aldrich, st.Louis, MO, USA) containing 5% bovine serum albumin (w/v) and then incubated for 1 hour. After incubation, the plates were washed three times with 100 μl TBS using an automatic plate washer (Agilent Technologies, SANTA CLARA, CA, USA). To each well was added 30. Mu.L of assay buffer (TBS-T containing 4. Mu.g/mL labeled recombinant integrin and one of 1mM Ca ₂₊/1mM Mg₂₊、1mM Mn₂₊/200μM Ca₂₊, or 10mM EDTA). To each well 1 μl of agonist antibody (or isotype control) stock was added at the appropriate concentration. Plates were centrifuged at 1000g for 1 min and incubated for 3 hours at room temperature. Plates were washed three times with 100 μl TBS using an automatic washer. To each well was added 30. Mu.L of staining buffer (TBS-T containing anti-IgG antibody HRP conjugate diluted 1:2000) (Invitrogen, waltham, mass., USA) and incubated for 30 minutes at room temperature. The plate was washed three times with 100. Mu.L in an automatic plate washer. To each well was added 30. Mu.L of substrate buffer (TBS containing 100. Mu.M Amplex Red and 4mM hydrogen peroxide) (Biotium, fremont, calif., USA) and developed for 30 minutes at room temperature. Plates were analysed at 563/587nm in a fluorescent microplate reader (Agilent Technologies, SANTA CLARA, CA, USA).

Soluble ligand (laminin 511) binding assays by flow cytometry

On the day before the assay, K562 cells expressing integrin in antibiotic-containing medium were counted, washed with 10mL of PBS, centrifuged and resuspended in complete medium without positive selection antibiotic. These cells were cultured overnight at 37 ℃ and 5% CO ₂. laminin-511E 8 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. Mu.g of laminin-511E8 Fc per test group) and incubated in the dark at room temperature for 30 minutes. Cells were centrifuged and resuspended in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, hampton, NH, USA) at 1000 ten thousand cells/mL. Human Fc blockers were added to cells (BD Biosciences, FRANKLIN LAKES, NJ, USA) to a final 25 μg/mL and incubated on ice for 15 min. After incubation, FACS buffer was added to dilute the concentration to 100 tens of thousands of cells/mL. Once the cells were transferred to a V-bottom 96-well plate [ catalog number 290-8116-01V ], 40. Mu.L of assay buffer containing either 1mM Ca ₂₊/1mM Mg₂₊ or 1mM Mn ₂₊/200μM Ca₂₊ or one of 10mM EDTA was added to each respective well followed by agonist antibodies or isoforms and incubated for 5 minutes at room temperature. Next, 5. Mu.L of laminin 511E8 Fc/Ab-AF647 solution was added to each well and incubated at room temperature for 25 minutes. The plates were washed with 200 μl assay buffer. To detect binding of agonist antibodies, cells were resuspended in 100. Mu.L of detection buffer (assay buffer containing 2.5-5.0. Mu.g/mL of anti-IgG) (BD Pharmigen) and incubated for 30 min at room temperature in the dark. Plates were washed with 200. Mu.L assay buffer and pellet resuspended in 100. Mu.L freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [ catalog No. AA47377-9M ] and incubated on ice for 10 min. Finally, the cells were resuspended in PBS and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, pasadena, calif., USA).

Soluble ligand (ligand mimetic) binding assays by flow cytometry

On the day before the assay, K562 cells expressing integrin in antibiotic-containing medium were counted, washed with 10mL of PBS, centrifuged and resuspended in complete medium without positive selection antibiotic. These cells were cultured overnight at 37 ℃ and 5% CO ₂. Cells were centrifuged and resuspended in FACS buffer (PBS containing 2% fetal bovine serum) (Summerlin Scientific, hampton, NH, USA) at 1000 ten thousand cells/mL. Human Fc blockers were added to cells (BD Biosciences, FRANKLIN LAKES, NJ, USA) to a final 25 μg/mL and incubated on ice for 15 min. After incubation, FACS buffer was added to dilute the concentration to 100 tens of thousands of cells/mL. Once the cells were transferred to a V-bottom 96-well plate [ catalog number 290-8116-01V ], 40. Mu.L of assay buffer containing either 1mM Ca ₂₊/1mM Mg₂₊ or 1mM Mn ₂₊/200μM Ca₂₊ or one of 10mM EDTA was added to each respective well followed by agonist antibodies or isoforms and incubated for 5 minutes at room temperature. Next, 5 μl of 10X biotinylated ligand mimetic was added to each well and incubated for 25 minutes at room temperature. The plates were washed with 200 μl assay buffer. To detect binding of agonist antibodies, cells were resuspended in 100. Mu.L of detection buffer (assay buffer containing 2.5-5.0. Mu.g/mL of anti-IgG) (BD Pharmigen). Biotinylated ligand mimics were detected by fluorescence labeled streptavidin and incubated in the dark at room temperature for 30 minutes. Plates were washed with 200. Mu.L assay buffer and pellet resuspended in 100. Mu.L freshly prepared fixation buffer (PBS containing 4% paraformaldehyde) [ catalog No. AA47377-9M ] and incubated on ice for 10 min. Finally, the cells were resuspended in PBS and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, pasadena, calif., USA).

Wound healing/scratch assay

The previous day, flat-bottomed 96-well plates treated with tissue culture were coated with 100. Mu.L of 2.0. Mu.g/mL laminin-511 (iMatrix) prepared in 1XPBS under sterile conditions and left overnight at 4 ℃. 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 isolate cells for inoculation for scratch assays, SKOV3 cell layers were treated with 0.25% trypsin-EDTA and 30,000 cells were inoculated into 100 μl of warm serum-free medium per well. Plates were centrifuged at 500g for 5min to pellet cells and incubated overnight in a cell incubator. The next day, a vertical wound/scratch was created in the middle of the well using a sterile p200 tip. Agonist or isotype antibody treatments were prepared in warmed complete medium with 100 μl of treatment added per well. Finally, ca ₂₊Mg₂₊ or Mn ₂₊ was added per well to a final concentration of 0.5mM in 10. Mu.L. The wound healing process was observed at 24 hours to determine wound closure, when the closure was approached by treatment with complete medium alone, the medium was aspirated and the cell layer was washed with 200 μl PBS and fixed with 4% PFA at 4 ℃ for 10 minutes. 200. Mu.L of 0.5% crystal violet was added to each well and stained at room temperature for 30 minutes.

Transient transfection of 293HEK with integrin alpha subunit chimeric DNA construct

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 2mL of PBS, and then 800. Mu.L of Opti-MEM was gently added to the cells. Transfection reagents were prepared with 2.5. Mu.g of DNA and 3. Mu.L of lipofectamine 2000 in 250. Mu.L of Opti-MEM, and then incubated for 5 minutes at room temperature. The solution was dispensed drop-wise into wells, incubated overnight, and then the complete medium was changed. Ab74 binding to cells analyzed by flow cytometry was bound and detected by anti-human IgG1 antibody conjugated to a fluorophore.

Additional sequences

Human recombinant integrin α3β1:

ITGA3 sp|P26006|33-991, protein sequence: 1034aa

MGWSCIILFLVATATGVHSFNLDTRFLVVKEAGNPGSLFGYSVALHRQTERQQRYLLLAGAPRELAV

PDGYTNRTGAVYLCPLTAHKDDCERMNITVKNDPGHHIIEDMWLGVTVASQGPAGRVLVCAHRYT

QVLWSGSEDQRRMVGKCYVRGNDLELDSSDDWQTYHNEMCNSNTDYLETGMCQLGTSGGFTQ

NTVYFGAPGAYNWKGNSYMIQRKEWDLSEYSYKDPEDQGNLYIGYTMQVGSFILHPKNITIVTGA

PRHRHMGAVFLLSQEAGGDLRRRQVLEGSQVGAYFGSAIALADLNNDGWQDLLVGAPYYFERKE

EVGGAIYVFMNQAGTSFPAHPSLLLHGPSGSAFGLSVASIGDINQDGFQDIAVGAPFEGLGKVYIYH

SSSKGLLRQPQQVIHGEKLGLPGLATFGYSLSGQMDVDENFYPDLLVGSLSDHIVLLRARPVINIVH

KTLVPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESA

VFHGFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALE

NHTEVQFQKECGPDNKCESNLQMRAAFVSEQQQKLSRLQYSRDVRKLLLSINVTNTRTSERSGED

AHEALLTLVVPPALLLSSVRPPGACQANETIFCELGNPFKRNQRMELLIAFEVIGVTLHTRDLQVQL

QLSTSSHQDNLWPMILTLLVDYTLQTSLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQV

GPMGEGLVGLGTLVLGLEWPYEVSNGKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRP

SSPQRRRRQLDPGGGQGPPPVTLAAAKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKAR

VWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENAQLEKELQALEKENAQLEWELQALEKELAQGGDYKDDDDK(SEQ ID NO:65).

ITGB1 sp|P05556|21-728 protein sequence 781aa

MGWSCIILFLVATATGVHSQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDD

LEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTL

KFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTT

PAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIG

WRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNI

QTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYC

KNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEG

IPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQ

CVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCE

ASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQE

CSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDDTSGLLEVLFQGPGKNAQLKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHHHH(SEQ ID NO:66).

Human recombinant integrin α3β1 domain Calf-Calf 2:

ESNLQMRAAFVSEQQQKLSRLQYSRDVRKLLLSINVTNTRTSERSGEDAHEALLTLVVPPALLLSSV

RPPGACQANETIFCELGNPFKRNQRMELLIAFEVIGVTLHTRDLQVQLQLSTSSHQDNLWPMILTLL

VDYTLQTSLSMVNHRLQSFFGGTVMGESGMKTVEDVGSPLKYEFQVGPMGEGLVGLGTLVLGLE

WPYEVSNGKWLLYPTEITVHGNGSWPCRPPGDLINPLNLTLSDPGDRPSSPQRRRRQLDPGGGQGP

PPVTLAAAKKAKSETVLTCATGRAHCVWLECPIPDAPVVTNVTVKARVWNSTFIEDYRDFDRVRVNGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENHHHHHH(SEQ ID NO:67).

Human recombinant integrin α3β1 domain Thigh:

MGWSCIILFLVATATGVHSMDYKDDDDKGGGGSGGGGSGGGGSLEVLFQGPLRARPVINIVHKTL

VPRPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFAGSESAVFH

GFFSMPEMRCQKLELLLMDNLRDKLRPIIISMNYSLPLRMPDRPRLGLRSLDAYPILNQAQALENHTEVQFQLEVLFQGPGGGGSGGGGSGGGGSHHHHHH(SEQ ID NO:68).

Mouse recombinant integrin α3β1:

Mouse alpha 3ECD sequence, protein sequence 1036aa

MGWSCIILFLVATATGVHSFNLDTRFLVVKEAVNPGSLFGYSVALHRQTERQQRYLLLAGAPRDLAV

GDDYTNRTGAVYLCPLTAHKDDCERMDISEKSDPDHHIIEDMWLGVTVASQGPAGRVLVCAHRYT

KVLWSGLEDQRRMVGKCYVRGNDLQLDPGDDWQTYHNEMCNSNTDYLQTGMCQLGTSGGFTQ

NTVYFGAPGAYNWKGNSYMIQRKDWDLSEYSYRGSEEQGNLYIGYTVQVGNAILHPTDIITVVTG

APRHQHMGAVFLLKQESGGDLQRKQVLKGTQVGAYFGSAIALADLNNDGWQDLLVGAPYYFER

KEEVGGAVYVFMNQAGASFPDQPSLLLHGPSRSAFGISIASIGDINQDGFQDIAVGAPFEGLGKVYI

YHSSSGGLLRQPQQIIHGEKLGLPGLATFGYSLSGKMDVDENLYPDLLVGSLSDHIVLLRARPVINIL

HRTLVARPAVLDPALCTATSCVQVELCFAYNQSAGNPNYRRNITLAYTLEADRDRRPPRLRFARSQS

SVFHGFFSMPETHCQTLELLLMDNVRDKLRPIVIAMNYSLPLRMPDRLKLGLRSLDAYPVLNQAQ

AMENHTEVHFQKECGPDNKCDSNLQMRAAFLSEQLQPLSRLQYSRDTKKLFLSINVTNSPSSQRA

GEDAHEALLTLEVPSALLLSSVRPSGTCQANNETILCELGNPFKRNQRMELLIAFEVIGVTLHTRDL

PVLLQLSTSSHQDNLQPVLLTLQVDYTLQASLSLMNHRLQSFFGGTVMGEAAMKTAEDVGSPLKY

EFQVSPVGDGLAALGTLVLGLEWPYEVTNGKWLLYPTEITIHSNGSWPCQPSGNLVNPLNLTLSDP

GVTPLSPQRRRRQLDPGGDQSSPPVTLAAAKKAKSETVLTCSNGRARCVWLECPLPDTSNITNVTV

KARVWNSTFIEDYKDFDRVRVDGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGTGGLLEVLFQGPGENAQLEKELQALEKENAQLEWELQALEKELAQGGDYKDDDDK(SEQ ID NO:69).

Mouse beta 1ECD sequence, protein sequence 781aa

MGWSCIILFLVATATGVHSQTDKNRCLKANAKSCGECIQAGPNCGWCTNTTFLQEGMPTSARCDD

LEALKKKGCQPSDIENPRGSQTIKKNKNVTNRSKGMAEKLRPEDITQIQPQQLLLKLRSGEPQKFTL

KFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTT

PAKLRNPCTSEQNCTSPFSYKNVLSLTDRGEFFNELVGQQRISGNLDSPEGGFDAIMQVAVCGSLIG

WRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNVYTMSHYYDYPSIAHLVQKLSENNI

QTIFAVTEEFQPVYKELKNLIPKSAVGTLSGNSSNVIQLIIDAYNSLSSEVILENSKLPDGVTINYKSY

CKNGVNGTGENGRKCSNISIGDEVQFEISITANKCPNKESETIKIKPLGFTEEVEVVLQFICKCNCQS

HGIPASPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVC

GQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCRCRVCECYPNYTGSACDCSLDTGP

CLASNGQICNGRGICECGACKCTDPKFQGPTCETCQTCLGVCAEHKECVQCRAFNKGEKKDTCAQ

ECSHFNLTKVESREKLPQPVQVDPVTHCKEKDIDDCWFYFTYSVNGNNEAIVHVVETPDCPTGPDDTSGLLEVLFQGPGKNAQLKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHHHH(SEQ ID NO:70).

Mouse recombinant integrin α3β1 domain Calf-Calf 2:

MGWSCIILFLVATATGVHSDSNLQMRAAFLSEQLQPLSRLQYSRDTKKLFLSINVTNSPSSQRAGED

AHEALLTLEVPSALLLSSVRPSGTCQANNETILCELGNPFKRNQRMELLIAFEVIGVTLHTRDLPVL

LQLSTSSHQDNLQPVLLTLQVDYTLQASLSLMNHRLQSFFGGTVMGEAAMKTAEDVGSPLKYEFQ

VSPVGDGLAALGTLVLGLEWPYEVTNGKWLLYPTEITIHSNGSWPCQPSGNLVNPLNLTLSDPGVT

PLSPQRRRRQLDPGGDQSSPPVTLAAAKKAKSETVLTCSNGRARCVWLECPLPDTSNITNVTVKAR

VWNSTFIEDYKDFDRVRVDGWATLFLRTSIPTINMENKTTWFSVDIDSELVEELPAEIEGENHHHHHH(SEQ ID NO:71)。

The above examples are provided to illustrate the disclosure and not to limit its scope. Other variations of the present disclosure will be apparent to those of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, internet resources, patents, patent applications, and accession 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:

2. The isolated antibody of claim 1, wherein:

(1) The CDR H1 comprises the sequence of either 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 a 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 either 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 a 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 comprising a 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 includes a 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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 of 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. The isolated antibody of 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. The isolated antibody of any one of claims 1-13, 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. The isolated antibody of any one of claims 1 to 14, wherein the antibody comprises HCDR1 having the sequence of SEQ ID No. 1, HCDR2 having the sequence of SEQ ID No. 6, HCDR3 having the sequence of SEQ ID No. 11, LCDR1 having the sequence of SEQ ID No. 21, LCDR2 having the sequence of SEQ ID No. 25, and LCDR3 having the sequence of SEQ ID No. 28.

16. The isolated antibody of 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. The isolated antibody of 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. The isolated antibody of any one of claims 1 to 14, wherein the antibody comprises HCDR1 having the sequence of SEQ ID No. 2, HCDR2 having the sequence of SEQ ID No. 7, HCDR3 having the sequence of SEQ ID No. 12, LCDR1 having the sequence of SEQ ID No. 22, LCDR2 having the sequence of SEQ ID No. 25, and LCDR3 having the sequence of SEQ ID No. 29.

19. The isolated antibody of 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. The isolated antibody of 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. The isolated antibody of any one of claims 1 to 14, wherein the antibody comprises HCDR1 having the sequence of SEQ ID No. 3, HCDR2 having the sequence of SEQ ID No. 8, HCDR3 having the sequence of SEQ ID No. 13, LCDR1 having the sequence of SEQ ID No. 21, LCDR2 having the sequence of SEQ ID No. 25, and LCDR3 having the sequence of SEQ ID No. 30.

22. The isolated antibody of 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 of 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 of any one of claims 1 to 14, wherein the antibody comprises HCDR1 having the sequence of SEQ ID No. 4, HCDR2 having the sequence of SEQ ID No. 9, HCDR3 having the sequence of SEQ ID No. 14, LCDR1 having the sequence of SEQ ID No. 23, LCDR2 having the sequence of SEQ ID No. 26, and LCDR3 having the sequence of SEQ ID No. 31.

25. The isolated antibody of 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 of 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 of any one of claims 1 to 14, wherein the antibody comprises HCDR1 having the sequence of SEQ ID No. 5, HCDR2 having the sequence of SEQ ID No. 10, HCDR3 having the sequence of SEQ ID No. 15, LCDR1 having the sequence of SEQ ID No. 24, LCDR2 having the sequence of SEQ ID No. 27, and LCDR3 having the sequence of SEQ ID No. 32.

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

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

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

34. The isolated antibody of claim 33, wherein the antibody binds to a sequence within the thigh-genu region in the alpha 3 portion.

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

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

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

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

40. An isolated nucleic acid encoding the isolated antibody of any one of claims 1 to 39.

41. An expression vector comprising the nucleic acid of claim 40.

42. An isolated host cell comprising the vector of claim 41.

43. A pharmaceutical composition comprising the isolated antibody of 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 the isolated antibody of any one of claims 1-38.

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

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

47. The method of claim 44 or 45, wherein the kidney disease is glomerular disease.

48. The method of claim 47, wherein the glomerular disease is a nephritis, a kidney 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 ligand mimetic peptides and/or antibodies;

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

3) Counter-selecting antibodies binding to said integrin alpha 3 beta 1 against immobilized beta 1 agonist antibodies or ligand mimetic peptides alone, and

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

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