Antibody targeting integrin α3β1 or antigen-binding fragment thereof, and use thereof

Abstract

The present invention belongs to the field of antibodies, and specifically relates to an integrin-binding molecule, in particular to an antibody that specifically recognizes integrin and a fragment thereof. In addition, the present invention further relates to a nucleic acid or host cell comprising this type of antibody or fragment thereof, a drug comprising this type of antibody or fragment thereof, and therapeutic and diagnostic methods or uses using these antibodies and fragments.

Description

Antibodies or antigen-binding fragments thereof targeting integrin alpha3beta1 and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410876398.9, filed on July 1, 2024, the disclosure of which is hereby incorporated by reference in its entirety into the present application. TECHNICAL FIELD

[0003] The present application belongs to the field of antibodies and specifically relates to an integrin alpha3beta1 binding molecule, in particular an antibody and fragments thereof specifically recognizing integrin alpha3beta1. Furthermore, the present application relates to nucleic acids or host cells comprising such antibodies or fragments thereof, medicaments comprising such antibodies or fragments thereof, and therapeutic and diagnostic methods or uses applying these antibodies and fragments. BACKGROUND

[0004] Integrins are a family of cell transmembrane adhesion molecules, which act as the connector between extracellular matrix (ECM) and cytoskeleton, and are widely involved in signal transduction between cells and external environment. In addition, integrins play an important role in tumor, autoimmune diseases and other pathological processes. Integrins are heterodimers in structure, containing an alpha subunit and a beta subunit. There are 18 subtypes of alpha subunits and 8 subtypes of beta subunits in mammals, which can generate 24 different heterodimer receptors by combination. According to the types of ligands bound to integrins, integrins can be divided into four families: 1) RGD-binding integrins, which refer to a class of integrins that bind to the Arg-Gly-Asp tripeptide motif in ECM, including alpha v beta 1, alpha v beta 3, alpha v beta 5, alpha v beta 6, alpha v beta 8, alpha 5 beta 1, alpha 8 beta 1 and alpha IIb beta 3, etc.; 2) leukocyte integrins, which are mainly involved in inflammatory response, including alpha 4 beta 1, alpha 9 beta 1, alpha L beta 2, alpha M beta 2, alpha X beta 2, alpha D beta 2, alpha 4 beta 7 and alpha E beta 7, etc.; 3) collagen-binding integrins, which refer to a class of integrins that bind to collagen, including alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1 and alpha 11 beta 1, etc.; 4) laminin-binding integrins, which are a group of integrins that bind to laminin, including alpha 3 beta 1, alpha 6 beta 1, alpha 6 beta 4 and alpha 7 beta 1, etc.

[0005] Integrins are involved in almost all steps of cancer progression, including tumorigenesis, proliferation, invasion, metastasis, immunosuppression, and drug resistance. Integrins are highly upregulated in various tumor tissues, especially the expression of α3β1, α4β1, α5β1, α6β4, αVβ3, αVβ5, αVβ6, and αVβ8 is considered to be associated with metastasis and poor prognosis of patients. For example, a recent study found that integrin α3β1 was upregulated in pancreatic cancer (PDAC), and a special homotrimer collagen I (Col1) produced by tumor cells, by binding to integrin α3β1, changed the tumor microenvironment, inhibited immune response, and accelerated tumor progression, and mediated resistance to gemcitabine treatment. Knocking out the expression of Col1 in PDAC cells significantly inhibited tumor progression and prolonged the survival of mice (Yang Chen, Sujuan Yang 1, Jena Tavormina, et al. Oncogenic collagen I homotrimers from cancer cells bind to α3β1 integrin and impact tumor microbiome and immunity to promote pancreatic cancer. Semin. Cancer Cell. 2022 Aug 8; 40(8): 818-834.e9.). Another study found that in a model of non-small cell lung cancer with EGFR mutations resistant to treatment with erlotinib, integrin α3β1 expression was significantly upregulated. The high-affinity peptide LXY30 targeting integrin α3β1 can specifically enrich in part of the mouse tumor (Wenwu Xiao, Weijie Ma, Sixi Wei, et al. High-affinity peptide ligand LXY30 for targeting α3β1 integrin in non-small cell lung cancer. J Hematol Oncol. 2019 Jun 10; 12(1): 56.).

[0006] Given the widespread expression of integrin α3β1 in tumor cells and the tumor microenvironment, it is of great significance to develop therapeutic drugs targeting this target. Currently, there is no integrin α3β1 specific therapeutic drug entering the clinic. In this study, a series of integrin α3β1 specific antibodies were obtained through mouse immunization and hybridoma fusion screening, which have high affinity and specificity. Based on these antibodies, antibody-drug conjugates (ADCs) were further conjugated to form toxins, which showed excellent preclinical efficacy in multiple animal models. In summary, this study first discovered and verified specific ADCs targeting integrin α3β1, which have great potential for preclinical prospects.

[0007] Therefore, there is a need in the art to develop antibodies or antibody-drug conjugates based thereon that specifically target integrin α3β1. SUMMARY

[0008] The present study is to obtain new integrin α3β1 specific antibodies through mouse hybridoma screening. Meanwhile, the present application is to further conjugate ADC based on the antibodies. The antibodies of the present application and the conjugates based thereon are seen to have significant efficacy in various tumor models, and have very important clinical application prospects.

[0009] Therefore, the present application relates to a binding molecule, such as an antibody or an antigen-binding fragment thereof, or an immunoconjugate, such as an ADC, constructed based thereon, that specifically binds to integrin α3β1.

[0010] In some aspects, the present application discloses an antibody or an antigen-binding fragment thereof that targets integrin α3β1 and the use thereof.

[0011] The present application thus provides a new antibody that binds to integrin α3β1, and an antigen-binding fragment thereof.

[0012] In some embodiments, the anti-integrin α3β1 antibody of the present application has one or more or all of the following properties:

[0013] (1) specifically binds to integrin β1 and integrin α3, i.e., specifically binds to integrin α3β1, such as human integrin α3β1;

[0014] (2) is capable of binding to integrin α3β1, such as human integrin α3β1, expressed on the surface of a cell membrane with high affinity;

[0015] (3) is suitable for constructing an immunoconjugate, such as an antibody-drug conjugate (ADC), for targeted killing of integrin α3β1 positive cells, such as integrin α3β1 positive tumor cells; or for treating tumors such as cancer.

[0016] In some embodiments, the present application provides a nucleic acid encoding the antibody or fragment thereof of the present application, a vector comprising the nucleic acid, and a host cell comprising the vector.

[0017] In some embodiments, the present application provides an antibody-drug conjugate comprising the antibody of the present application.

[0018] The present application provides an antibody-drug conjugate (ADC) having a structure shown in formula (I), Ab-(L-(D) m ) p (I)

[0019] or a pharmaceutically acceptable salt or solvate thereof,

[0020] wherein:

[0021] Ab is an antibody or fragment thereof, in particular an antibody or fragment thereof (e.g. an antigen binding fragment) that specifically binds integrin α3β1 (e.g. human integrin α3β1), e.g. an antibody or antigen binding fragment thereof of the application that targets integrin α3β1;

[0022] L is a linker;

[0023] m is 1-6;

[0024] p is an integer selected from 1 to 16, e.g. an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12;

[0025] wherein D is an anti-tumour compound, e.g. a cytotoxic agent, e.g. selected from the group consisting of: microtubule inhibitors (e.g. MMAE), DNA cross-linking agents, DNA alkylating agents and topoisomerase I inhibitors; for example, the topoisomerase I inhibitor is selected from the group consisting of camptothecins such as camptothecin and its derivatives, e.g. camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, FL118, Dxd, topotecan, belotecan, lurtotecan, irinotecan, exatecan or LD38.

[0026] In some embodiments, the present application provides a method of preparing an antibody or fragment thereof of the application or an antibody drug conjugate thereof.

[0027] In some embodiments, the present application provides a pharmaceutical composition and combination product comprising an antibody or fragment thereof of the application or an antibody drug conjugate thereof.

[0028] The present application also provides a method of preventing or treating an integrin α3β1 -related disease, e.g. a tumour such as a cancer (preferably an integrin α3β1 -positive tumour, e.g. an integrin α3β1 -positive cancer) in a subject using an antibody or fragment thereof of the application or an antibody drug conjugate thereof.

[0029] The present application also relates to a method of detecting integrin α3β1 in a sample. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 shows the binding of five chimeric antibodies chA28, chC8, chC18, chC22 and chC47 to HCT116 and AGS cells.

[0031] Figure 2 shows the mass spectrometry results for the A28 antibody.

[0032] Figure 3 shows reduced binding of antibodies to HCT116 cells after integrin alpha 3 gene silencing.

[0033] Figure 4 shows in vivo activity in the HCT116 CDX model after chA28 conjugated to LD38.

[0034] Figure 5 shows in vivo activity in the PANC-1 CDX model after chA28 conjugated to LD38.

[0035] Figure 6 shows in vivo activity in the HCC827 CDX model after chA28 conjugated to LD38.

[0036] Figure 7 shows in vivo activity in the AGS CDX model after chA28 conjugated to LD38.

[0037] Figure 8 shows in vivo activity in the HCT116 CDX model after chC8 and chC47 conjugated to LD38.

[0038] Figure 9 shows binding of humanized antibody huA28-H2L3 (huA28) to HCT116 and AGS cells.

[0039] Figure 10 shows binding of affinity matured antibodies huA28-comb4, huA28-comb7 and huA28-comb9 to human and monkey integrin alpha 3 beta 1 overexpressing cells.

[0040] DETAILED DESCRIPTION

[0041] For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural; and vice versa. It is to be understood that the present application is not limited to particular methodology, protocols, and reagents described herein, as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0042] The term "about," as used herein when used with a numerical value means a range of numerical values that has a lower limit that is 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit that is 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

[0043] As used herein, the term "and / or" means either or both of the items it connects.

[0044] As used herein, the term "comprising" or "including," or any variation thereof, is intended to mean that the products, compositions and methods described contain the recited elements, integers, steps, or the like, but not to the exclusion of anything not expressly recited. In this document, the terms "comprise" or "comprising" and "include" or "including" when used in this document are used synonymously with the term "comprising" or "including" and are intended to mean the open ended inclusion, that is to say that items, components, or elements which are "comprised" or "included" are not necessarily required, but can be beneficial or useful. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass an antibody variable region consisting of the particular sequence.

[0045] As used herein, the term "anti-", "binds" or "specifically binds" means that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a particular target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biolayer interferometry assay or MSD assay or surface plasmon resonance (SPR) assay.

[0046] The term "anti-integrin α3β1 antibody", "anti-integrin α3β1", "integrin α3β1 antibody" or "antibody that binds integrin α3β1" as used herein refers to an antibody that is capable of binding (e.g., human) integrin α3β1 or a fragment thereof with sufficient affinity to form a complex.

[0047] An "isolated" antibody or molecule is one which has been separated from a component of its natural environment. In some embodiments, an antibody or molecule is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).

[0048] The terms "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having a native immunoglobulin molecule structure. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds. In the case of a heavy chain antibody which has only heavy chains but lacks light chains, a full-length antibody comprises two heavy chains (H) which are interconnected by disulfide bonds. For a conventional four-chain IgG antibody, a full-length antibody heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. A full-length antibody light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody you are offered is a full-length antibody, e.g. a conventional four-chain IgG full-length antibody.

[0049] The term "antibody fragment" includes a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment. The term "antigen-binding fragment" of an antibody refers to a molecule other than a full-length antibody that comprises a portion of an intact antibody that is capable of binding the antigen to which the intact antibody is designed to bind, or competes with the full-length antibody from which the antigen-binding fragment is derived for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g. scFv); single domain antibodies such as VHH, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), nanobodies. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. In addition, pepsin digestion of a full-length antibody under denaturing conditions generates F(ab')2, which is a dimer of two Fab' fragments linked by disulfide bonds, and is a bivalent fragment of an antibody. F(ab')2 can be reduced under non-denaturing conditions to break disulfide linkages in each of the A chains, thereby forming two Fab' fragments. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded for by separate genes, but they can also be made as a single protein chain using recombinant methods, using a synthetic linker that enables them to pair up and form a single chain Fv (scFv), in which the VL and VH regions pair to form an antigen binding site.

[0050] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigenic epitope. The CDRs of a heavy and light chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment schemes, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Accessing the Kabat antibody sequence database by computer; Martin AC; Proteins, 1996 May; 25(1): 130-3; doi: 10.1002 / (SICI)1097-0134(199605)25:1<130::AID-PROT11>3.0.CO;2-L. PMID: 8727325), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (IMGT, the international ImMunoGeneTics information system, http: / / imgt.cines.fr; Lefranc MP; Novartis Found Symp. 2003; 254: 126-36; discussion 136-42, 216-22, 250-2. PMID: 14712935 Review), and North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0051] In some embodiments, the CDRs of the heavy chain variable region of an antibody in the application are determined according to Kabat or IMGT. In some embodiments, the CDRs of the light chain variable region of an antibody in the application are determined according to Kabat or IMGT.

[0052] Unless otherwise indicated herein, amino acid residue positions in an Fc or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242; amino acid residue positions of light chain and heavy chain variable regions are numbered according to the Kabat numbering system (also referred to as the Kabat index) as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242.

[0053] The term "amino acid substitution" is used interchangeably herein to refer to the replacement of at least one amino acid residue in a pre-determined parent amino acid sequence with a different "substituted" amino acid residue. The substituted residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (lie); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Also encompassed within the definition of an amino acid substitution herein is the substitution of one or more non-naturally occurring amino acid residues. A "non-naturally occurring amino acid residue" refers to a residue, in addition to those naturally occurring amino acid residues listed above, that is capable of covalently bonding to an adjacent amino acid residue in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0054] A "conservative alteration" as used herein includes substitutions, deletions or additions to a polypeptide sequence that do not substantially alter the functional activity of the polypeptide sequence. In some embodiments, a conservative alteration is a conservative substitution. Conservative substitutions refer to the substitution of one amino acid for another within the same class, e.g., one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. For example, conservative substitutions often result in the substitution of one amino acid for another that is chemically similar. Conservative substitution tables that provide functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). In some embodiments, the term "conservative alteration" when applied to an antibody molecule amino acid sequence is used to refer to amino acid modifications that do not significantly affect or alter the intended antigen binding characteristics of the antibody molecule of the application containing the amino acid sequence. For example, a conservatively altered variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100-110% or more, binding affinity to the intended antigen relative to the parent antibody.

[0055] As used herein, an "antibody-drug conjugate (ADC)" refers to a compound resulting from the attachment of an antibody to a (small molecule) drug via a linker.

[0056] The term "linker" refers to a structural fragment that links a drug (e.g., a small molecule drug) to an antibody moiety. It will be appreciated that the linker has a functional group that can form a bond with a functional group of the antibody or antigen binding fragment thereof prior to attachment to the antibody or antigen binding fragment thereof. The linkers of the present application can have multiple components (e.g., in some embodiments have a linking group responsible for coupling to the antibody; a degradable peptide unit; and optionally a spacer).

[0057] The term "linker-payload" refers to a compound formed from the attachment of a payload, e.g., a drug (e.g., a small molecule drug), to a linker.

[0058] The term "alkyl" as used herein refers to straight-chain or branched-chain saturated hydrocarbon groups consisting of carbon and hydrogen atoms. Specifically, alkyl groups have 1-10 carbon atoms, for example, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. For example, as used herein, the term "Ci-C6alkyl" refers to straight-chain or branched-chain saturated hydrocarbon groups having from 1 to 6 carbon atoms, examples of which are, for example, methyl, ethyl, propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, or t-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), hexyl (including n-hexyl, 2-methylpentyl, 3-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl), and the like.

[0059] The term "cycloalkyl" as used herein refers to a monocyclic, fused polycyclic, bridged polycyclic, or spirocyclic non-aromatic monovalent hydrocarbon ring structure having the number of ring atoms specified, which can be saturated or unsaturated, for example, containing 1 or more double bonds. Cycloalkyl groups can contain 3 or more carbon atoms in the ring, for example, 3-18, 3-10, or 3-8 carbon atoms, for example, C3-C8cycloalkyl. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl. 3-10 cycloalkyl, C 3-8 cycloalkyl, C 3-6 cycloalkyl, C 5-6 cycloalkyl. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.

[0060] The term "heterocycle" or "heterocyclyl" as used herein refers to a 5-20 membered (e.g., 5-14 membered, 5-8 membered, 5-6 membered) aromatic or non-aromatic monocyclic, bicyclic, or polycyclic ring system having 1-4 ring members independently selected from N, O, or S. One or more N, C, or S atoms in a heterocycle can be oxidized. Preferably, the heterocycle is a 5-10 membered ring system, either monocyclic or fused bicyclic. Representative examples include, but are not limited to, pyrrolidine, azetidine, piperidine, morpholine, tetrahydrofuran, tetrahydropyran, benzofuran, benzothiophene, indole, benzopyrazole, pyrrole, thiophene (thiene), furan, thiazole, imidazole, pyrazole, pyrimidine, pyridine, pyrazine, pyridazine, isothiazole, and isoxazole. It is understood that this term includes heteroaryl as defined herein.

[0061] The term "substituted", as used herein in the definition of various groups, means that the indicated atom can be substituted with for example, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, halo, cyano, nitro, azido, carboxyl, hydroxyl, thiol, amino, mono- or dialkylamino, monocyclo- or dicycloalkylamino, mono- or diarylamino, mono- or diheterocyclylamino, mono- or diheteroarylamino, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- oxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- thio, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- acyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- amido, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- acyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- sulfonyl, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- sulfonyloxy, alkyl- or cycloalkyl- or heterocyclyl- or heteroaryl- or aryl- sulfonylamido, or optionally substituted aminocarbonyl, each of which is further substituted with the remaining optional substituents, and each of which is defined herein. Examples of substituents include, but are not limited to, one or more groups independently selected from halogen, OH, SH, CN, NH2, NHCH3, N(CH3)2, NO2, N3, C(O)CH3, COOH, C(O)-amino, OCOCH3, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamido, methylsulfonylamido, SO, SO2, phenyl, piperidinyl, piperazinyl, and pyrimidinyl.

[0062] The term "substituted" or "substitution", as used herein, means that one or more (e.g., 1, 2, 3, or 4) hydrogens on the designated atom are replaced with the designated group, provided that the designated atom's normal valency is not exceeded and a stable compound results, and that the combination of substituents and variables is in accordance with permitted

[0063] The term "amino acid" refers to naturally occurring and synthetic amino acids. The amino acids can be L or D isomers. The nomenclature of the common amino acids referred to herein follows the conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Also in the present disclosure, the amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, the amino acid can be selected from the group consisting of phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (lie), arginine (Arg), proline (Pro), and glutamine (Gin).

[0064] The terms "optional" or "optionally": mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, when a group or structure is "optionally substituted," the group or structure can be substituted or unsubstituted.

[0065] The terms "pharmaceutically acceptable salt" and "pharmaceutically acceptable salts" are used interchangeably and refer to salts that retain the biological effectiveness and properties of the ADC molecules of the present application and that are not biologically or otherwise undesirable. The ADC molecules of the present application can exist in their pharmaceutically acceptable salt form, including acid addition salts and base addition salts. In the present application, the pharmaceutically acceptable non-toxic acid addition salts are meant to comprise salts of the ADC molecules of the present application formed with inorganic or organic acids which include, but are not limited to, hydrochloric, sulfuric, hydrobromic, hydroiodic, phosphoric, nitric, perchloric, acetic, oxalic, maleic, fumaric, tartaric, benzenesulfonic, methanesulfonic, salicylic, succinic, citric, lactic, propionic, benzoic, p-toluenesulfonic, malic, and the like. The pharmaceutically acceptable non-toxic base addition salts are meant to comprise salts of the ADC molecules of the present application formed with organic or inorganic bases which include, but are not limited to, alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts, such as ammonium salts with organic bases, such as those formed with N-containing groups.

[0066] The term "solvate" refers to an association or complex of one or more solvent molecules and an ADC conjugate of the application. Solvents include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0067] "pharmaceutically acceptable" and "pharmaceutical" are used interchangeably herein.

[0068] The term "drug: antibody ratio" or "DAR" refers to the ratio of drug moieties (D) conjugated to an Ab moiety described herein to the Ab moiety in an ADC. In some embodiments described herein, the DAR can be determined by the product of m and p in formula (I), e.g., the DAR can be 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio of small molecule drug moieties (D) conjugated to an Ab moiety described herein to the Ab moiety in the product as measured by detection methods, e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis, and / or HPLC, which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the application is 1 to 20, e.g., 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and ranges with two of these values as endpoints. It will be understood that when referring to an average DAR value, the ADC of the application refers to a population or mixture of ADC molecules that includes ADC molecules having the same and / or different DAR.

[0069] When referring to "the Ab is derived from an antibody", it is meant that the binding domain constituting the Ab is or is derived from the binding domain of the antibody that specifically binds to the antigen, e.g. the fragment of the Ab that specifically binds to the antigen, e.g. Fab, is or is derived from the corresponding fragment of the antibody, e.g. Fab, or the heavy chain variable region and / or light chain variable region of the binding domain of the Ab is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the binding domain of the Ab are CDRs of the antibody. "The Ab is derived from an antibody" also encompasses that the Ab used to constitute the ADC molecule is the antibody itself, e.g. a full-length antibody.

[0070] The term "therapeutic agent" as described herein encompasses any substance that is effective in the prevention or treatment of a tumor, e.g. a cancer.

[0071] "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer or immune system disorders.

[0072] The term "cytotoxic agent" as used herein refers to an agent that inhibits or prevents the function of cells and / or causes cell death or destruction. Examples of cytotoxic agents include, but are not limited to, auristatins, maytansinoids, camptothecins, ricin, ricin A chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, gelonin, modeccin, modeccin A chain, sarcodictyin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, phytolaccin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoids and other chemotherapeutic agents.

[0073] The term "small molecule drug" refers to an organic compound of low molecular weight that is capable of modulating a biological process. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more able to penetrate cells, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0074] The term "drug" refers to an organic compound capable of modulating a biological process, in particular altering or preventing a pathological process.

[0075] The term "prodrug" refers to an active or inactive compound that is modified by chemical moieties and upon administration to a subject, is converted to an active drug substance by physiological action (e.g., hydrolysis, metabolic biotransformation, etc.) in the body. Techniques for making and using prodrugs are well known to those skilled in the art.

[0076] An "anti-tumor compound" is a pharmaceutically active compound that has an effect on a tumor, which includes but is not limited to a cytotoxic or chemotherapeutic agent, especially a small molecule cytotoxic or chemotherapeutic agent, such as a camptothecin compound, e.g., Exatecan (a topoisomerase I inhibitor), DXd (a novel topoisomerase I inhibitor Exatecan derivative), an auristatin compound, e.g., monomethyl auristatin E (MMAE) or a maytansinoid compound, e.g., a small molecule microtubulin inhibitor DM1. It should be understood that an anti-tumor compound can be substituted with isotopes including but not limited to, e.g., deuterium, tritium, etc. For example, after substitution with deuterium, the carbon-deuterium bond replaces the carbon-hydrogen bond, which is more stable than the latter, and this substitution can directly affect the absorption, distribution, metabolism, and excretion, etc. of certain drugs, thereby improving the efficacy, safety and tolerability of the drugs. Therefore, an "anti-tumor compound" of the present application can encompass a compound substituted with deuterium. When "anti-tumor compound" is mentioned herein, it can be either an element of an immunoconjugate obtained by coupling or otherwise linking with other molecules, e.g., an antibody molecule, or used as a therapeutic agent alone.

[0077] A "camptothecin compound" refers to an anti-tumor compound having the camptothecin parent nucleus structure, which has anti-tumor activity. "Camptothecin compound" is a term routinely used in medicinal chemistry. Those skilled in the art are able to determine whether a compound belongs to a camptothecin compound according to the structure of the compound. In some embodiments, a camptothecin compound encompasses camptothecin or a derivative thereof.

[0078] Similarly, auristatin compounds and maytansinoid compounds are also terms routinely used in medicinal chemistry.

[0079] "Substituted with deuterium" refers to replacement of hydrogen in a molecule with deuterium, e.g., 1 or more hydrogens, e.g., 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) hydrogens are replaced with deuterium.

[0080] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that are typically also contained in the natural environment of the nucleic acid molecule, but the nucleic acid molecule is present outside of the cell or at a chromosomal location different from that of its natural environment.

[0081] An "isolated nucleic acid encoding an anti- integrin α3β1 antibody or fragment thereof" means one or more nucleic acid molecules encoding an antibody heavy or light chain (or fragment thereof, e.g., a heavy chain variable region or a light chain variable region), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0082] The "percent (%) identity" of a given amino acid sequence, when compared to a specific amino acid sequence as shown in the specification, refers to the percentage of amino acid residues in the given sequence that are identical with the amino acid residues in the specific sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative changes as part of the sequence identity. In some embodiments, the present application contemplates variants of the antibody molecules of the present application that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more, to the antibody molecules specifically disclosed herein and their sequences. The variants can include conservative changes.

[0083] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active agent is administered.

[0084] The term "label" as used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a polynucleotide probe or antibody, and that facilitates detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, can catalyze chemical changes to a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., bonding) of a detectable substance to the probe or antibody, as well as indirect labeling by conjugation with a separate, directly labeled alternative reagent.

[0085] The term "combination therapy" or "combined therapy" refers to the administration of two or more therapeutic agents, or treatment modalities, to treat a disease described herein. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses use of each of the therapeutic agents in a multiple- or in separate containers, such as a packet, capsule, powder, and liquid. The powder and / or liquid can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent at different times during the course of the treatment regimen. In any event, the treatment regimen will provide beneficial effects from the combination of the drugs in treating the disorder or condition described herein.

[0086] A "subject / patient / individual sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy or puncture sample; blood or any blood component; a bodily fluid, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time of gestation or development. The tissue sample can contain compounds not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0087] A first aspect of the application provides an antibody or fragment (e.g., antigen binding fragment) thereof that specifically binds integrin a3b1.

[0088] In some embodiments, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises 3 complementarity determining regions from a heavy chain variable region (HCDR) and 3 complementarity determining regions from a light chain variable region (LCDR).

[0089] In some aspects, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises a heavy chain variable region (VH). In some aspects, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises a light chain variable region (VL). In some aspects, the anti-integrin a3b1 antibody or antigen binding fragment thereof of the application comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementarity determining regions from a heavy chain variable region (HCDR), HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementarity determining regions from a light chain variable region (LCDR), LCDR1, LCDR2, and LCDR3.

[0090] In some embodiments, the heavy chain variable region described herein

[0091] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89; or

[0092] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89; or

[0093] (iii) comprises or consists of an amino acid sequence which has 1 or more, preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1, amino acid alterations (e.g. conservative alterations such as amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89, preferably said amino acid alterations do not occur in CDR regions.

[0094] In some embodiments, the light chain variable region described herein

[0095] (i) comprises or consists of an amino acid sequence which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85, or 90; or

[0096] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85, or 90; or

[0097] (iii) comprises or consists of an amino acid sequence which has 1 or more, preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1, amino acid alterations (e.g. conservative alterations such as amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85, or 90, preferably said amino acid alterations do not occur in CDR regions.

[0098] In some embodiments, the three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2 and HCDR3, of the application are selected from the group consisting of

[0099] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as depicted in SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89, or

[0100] (ii) a sequence which comprises, in addition to or instead of the sequence of any one of (i), at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably amino acid substitution, preferably conservative substitution) in said three HCDR regions,

[0101] For example, wherein the HCDRs are determined according to any CDR determination scheme, such as according to the IMGT, AbM, Kabat or Chothia scheme or a combination thereof;

[0102] For example, the HCDR1, 2 and 3 are determined according to IMGT or Kabat, respectively.

[0103] In some embodiments, the three complementarity determining regions from the light chain variable region (LCDRs), LCDR1, LCDR2 and LCDR3 of the present application are selected from

[0104] (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85 or 90, or

[0105] (ii) a sequence comprising in total at least one and not more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i),

[0106] For example, wherein the LCDRs are determined according to any CDR determination scheme, such as according to the IMGT, AbM, Kabat or Chothia scheme or a combination thereof;

[0107] For example, the LCDR1, 2 and 3 are determined according to IMGT or Kabat, respectively.

[0108] In some embodiments, the IMGT-determined HCDR1 described herein comprises or consists of the amino acid sequence as shown in any one of SEQ ID NO: 3, 17 or 77, or the IMGT-determined HCDR1 described herein comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in any one of SEQ ID NO: 3, 17 or 77.

[0109] In some embodiments, the Kabat-determined HCDR1 described herein comprises or consists of the amino acid sequence as shown in any one of SEQ ID NO: 9, 23, 37 or 81, or the Kabat-determined HCDR1 described herein comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence as shown in any one of SEQ ID NO: 9, 23, 37 or 81.

[0110] In some embodiments, an IMGT-determined HCDR2 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 4, 18, 32, 86, or 91, or an IMGT-determined HCDR2 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 4, 18, 32, 86, or 91.

[0111] In some embodiments, a Kabat-determined HCDR2 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 10, 24, 38, 52, 87, or 93, or a Kabat-determined HCDR2 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 10, 24, 38, 52, 87, or 93.

[0112] In some embodiments, an IMGT-determined HCDR3 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 5, 19, 33, 47, 61, 78, or 92, or an IMGT-determined HCDR3 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 5, 19, 33, 47, 61, 78, or 92.

[0113] In some embodiments, a Kabat-determined HCDR3 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 11, 25, 39, 53, 67, 82, or 94, or a Kabat-determined HCDR3 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 11, 25, 39, 53, 67, 82, or 94.

[0114] In some embodiments, an IMGT-determined LCDR1 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 20, or 48, or an IMGT-determined LCDR1 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 6, 20, or 48.

[0115] In some embodiments, a Kabat-determined LCDR1 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 12, 26, 40, 54, or 68, or a Kabat-determined LCDR1 described herein comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 12, 26, 40, 54, or 68.

[0116] In some embodiments, an IMGT-determined LCDR2 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 7, 21, 35, or 79, or an IMGT-determined LCDR2 described herein comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 7, 21, 35, or 79.

[0117] In some embodiments, a Kabat-determined LCDR2 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 13, 27, 41, 55, 83, or 88, or a Kabat-determined LCDR2 described herein comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 13, 27, 41, 55, 83, or 88.

[0118] In some embodiments, an IMGT-determined LCDR3 described herein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 8, 22, 36, 50, or 80, or an IMGT-determined LCDR3 described herein comprises an amino acid sequence having one, two or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 22, 36, 50, or 80.

[0119] In some embodiments, the Kabat-defined LCDR3 described herein comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NO: 8, 22, 36, 50, or 80, or the Kabat-defined LCDR3 described herein comprises an amino acid sequence having one, two, or three alterations, preferably amino acid substitutions, preferably conservative substitutions, compared to the amino acid sequence set forth in any one of SEQ ID NO: 8, 22, 36, 50, or 80.

[0120] In some particular embodiments of the application, the anti- integrin α3β1 antibody of the application or antigen-binding fragment thereof comprises:

[0121] 1) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 1, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 2;

[0122] 2) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 15, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 16;

[0123] 3) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 29, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 30;

[0124] 4) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 43, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 44;

[0125] 5) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 57, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 58;

[0126] 6) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 73, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 74;

[0127] 7) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 76;

[0128] 8) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 84, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 85; or

[0129] 9) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 89, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 90, e.g., wherein the HCDRs and LCDRs can be determined according to any CDR determination scheme, e.g., according to the IMGT, AbM, Kabat or Chothia scheme or a combination thereof, respectively;

[0130] For example, the HCDR1, 2, 3 and LCDR1, 2, 3 are determined according to the IMGT scheme, respectively, or determined according to the Kabat scheme, respectively.

[0131] In some embodiments of the application, the anti- integrin α3β1 antibody or antigen binding fragment thereof of the application comprises: three complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise or consist of the amino acid sequences set forth in the following table SEQ ID NOs, based on the IMGT scheme, respectively:

[0132] or wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise or consist of the amino acid sequences set forth in the following table SEQ ID NOs, based on the Kabat scheme, respectively:

[0133] In some embodiments of the application, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the application comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein the heavy chain variable region, VH, and the light chain variable region, VL, respectively comprise or consist of the amino acid sequence set forth in the SEQ ID NOs listed in the table below:

[0134] In preferred embodiments, the application provides an anti- integrin α3β1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region, VH, and a light chain variable region, VL, wherein the heavy chain variable region, VH, and the light chain variable region, VL, respectively comprise or consist of the amino acid sequence set forth in the SEQ ID NOs listed in the table below:

[0135] In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the application further comprises an antibody heavy chain constant region. In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the application further comprises an antibody light chain constant region. In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the application further comprises a heavy chain constant region and a light chain constant region.

[0136] In some embodiments, the antibody heavy chain constant region of the application is or is from a heavy chain constant region of IgGl, IgG2, IgG3, or IgG4, e.g., a heavy chain constant region of human IgGl, IgG2, IgG3, or IgG4, preferably a heavy chain constant region of human IgGl. In some embodiments, the antibody light chain constant region of the application is or is from a Kappa light chain constant region or a Lambda light chain constant region, e.g., a human Kappa light chain constant region or a human Lambda light chain constant region.

[0137] In some preferred embodiments, the antibody heavy chain constant region described herein

[0138] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71;

[0139] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 71; or

[0140] (iii) comprises or consists of an amino acid sequence having 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (e.g., conservative alterations, such as amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 71.

[0141] In some embodiments, the antibody light chain constant region described herein

[0142] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72;

[0143] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 72; or

[0144] (iii) comprises or consists of an amino acid sequence having 1 or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid alterations (e.g., conservative alterations, such as amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 72.

[0145] In some embodiments, the antibody or fragment thereof of the present application comprises a heavy chain constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71 and a light chain constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 72.

[0146] In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the present application comprises an antibody heavy chain (HC). In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the present application comprises an antibody light chain (LC). In some embodiments, the anti- integrin α3β1 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain and a light chain. In some embodiments, the anti- integrin α3β1 antibody of the present application comprises or consists of two heavy chains and two light chains.

[0147] In some embodiments, the antibody heavy chain described herein comprises or consists of an antibody heavy chain variable region and an antibody heavy chain constant region. In some embodiments, the antibody light chain described herein comprises or consists of an antibody light chain variable region and an antibody light chain constant region.

[0148] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of a heavy chain variable region and a heavy chain constant region, and the light chain comprises or consists of a light chain variable region and a light chain constant region, wherein

[0149] (i). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0150] (ii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0151] (iii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0152] (iv). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0153] (v). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0154] (v). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0155] (vi). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0156] (vii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0157] (viii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; or

[0158] (ix). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72.

[0159] In some embodiments, an anti-Integrin α3β1 antibody or antigen-binding fragment thereof of the present application comprises one of the heavy chains and one of the light chains, or two of the heavy chains and two of the light chains, e.g., two of the same heavy chains and two of the same light chains, or consists of one of the heavy chains and one of the light chains, or consists of two of the heavy chains and two of the light chains, e.g., two of the same heavy chains and two of the same light chains.

[0160] In one embodiment of the application, the amino acid changes described herein are conservative changes. In one embodiment of the application, the amino acid changes described herein include substitution, insertion or deletion of an amino acid or a combination thereof. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid changes described in the application occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described in the application occur in regions outside the heavy chain variable region and / or outside the light chain variable region (e.g., in the heavy chain constant region or the light chain constant region).

[0161] In some embodiments, the substitution is a conservative substitution. A conservative substitution is one in which the amino acid is replaced with one from the same class, e.g., an acidic amino acid is replaced with another acidic amino acid, a basic amino acid is replaced with another basic amino acid, or a neutral amino acid is replaced with another neutral amino acid.

[0162] In certain embodiments, the substitution occurs in a CDR region of the antibody. In general, the resulting variant will have modifications (e.g., improvements) in certain biological properties relative to the parent antibody, for example, increased affinity, and / or will have substantially retained certain biological properties of the parent antibody.

[0163] In certain embodiments, it can be desirable to create an antibody that is engineered with cysteine, e.g., a "ThioMAb", in which one or more residues are substituted with cysteine residues.

[0164] In certain embodiments, the antibodies provided herein can be further modified to contain other nonproteinaceous moieties that are known to one of skill in the art and readily available. Suitable modifications include, but are not limited to, amitization. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol / polypropylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-l,3-dioxan, poly-l,3,6-trioxan, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0165] In addition, to form ADC molecules, the antibodies or antigen-binding fragments thereof of the present application can also be conjugated with linker-payload units using some of the naturally occurring attachment sites thereon. Such naturally occurring attachment sites are the thiol of cysteine and the amino group of lysine. Typically, the interchain disulfide bridges of the antibody are used to achieve a more defined drug-antibody ratio (DAR). Thus, in one embodiment, after reduction of the interchain disulfide bonds of the antibody, linker-payload units are conjugated to the antibody via thiol chemistry to form the ADC conjugate. In addition, it is also contemplated that artificial attachment sites can be introduced into the antibody to achieve more site-directed conjugation.

[0166] In some embodiments, the anti-α3β1 integrin antibodies or antigen-binding fragments thereof of the present application further include antibodies or antigen-binding fragments having one or more of the following properties:

[0167] (1) exhibit the same or similar binding affinity and / or specificity to α3β1 integrin as any of the antibodies prepared in the Examples of the present application;

[0168] (2) inhibit the binding of any of the antibodies prepared in the Examples of the present application to α3β1 integrin;

[0169] (3) bind to the same or overlapping epitope as any of the antibodies prepared in the Examples of the present application; or

[0170] (4) compete with any of the antibodies prepared in the Examples of the present application for binding to α3β1 integrin.

[0171] An antibody that "binds to the same or overlapping epitope" as a reference antibody, as that term is defined herein, refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay, and vice versa.

[0172] An antibody that "competes with a reference antibody for binding to its antigen", as that term is defined herein, refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. And vice versa, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assays, biolayer interferometry (e.g., Fortebio), or surface plasmon resonance (Biacore), etc.

[0173] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen, as defined herein, means an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured in terms of affinity (e.g., equilibrium dissociation constant). Methods of determining affinity are known in the art.

[0174] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody means an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0175] In some embodiments, the anti-α3β1 integrin antibody is a monoclonal antibody.

[0176] In some embodiments, the anti-α3β1 integrin antibody is a chimeric antibody.

[0177] In some embodiments, the anti-α3β1 integrin antibody is a humanized antibody.

[0178] In one embodiment, the anti-α3β1 integrin antibody of the present application also encompasses antibody fragments thereof, such as antigen-binding fragments thereof. In some embodiments, the antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody, or sc(Fv)2.

[0179] In some embodiments, the anti-α3β1 integrin antibody is a full-length antibody.

[0180] In certain embodiments, the anti-α3β1 integrin antibody also encompasses bispecific or multispecific antibody molecules that specifically bind α3β1. In one embodiment, the bispecific antibody can bind to integrin α3β1 and another protein.

[0181] In some embodiments, the anti-α3β1 integrin antibody is the antibody shown under chA28, chC8, chC18, chC22, chC47, huA28-H2L3 (huA28), huA28-comb4, huA28-comb7, or huA28-comb9 numbers prepared in the Examples.

[0182] A second aspect of the present application provides an immunoconjugate, e.g., an antibody drug conjugate, comprising an anti- integrin α3β1 antibody of the present application or antigen binding fragment thereof.

[0183] In some embodiments, the present application provides immunoconjugates, e.g., antibody drug conjugates (ADCs), comprising any of the anti-integrin α3β1 antibodies provided herein and other agents, e.g., therapeutic agents or labels. In some embodiments, the therapeutic agent is a cytotoxic agent or an anti-tumor compound.

[0184] In some embodiments, the antibody drug conjugates of the present application comprise any of the anti-integrin α3β1 antibodies provided herein and a linker-payload. In some embodiments, the payload is a cytotoxic agent or an anti-tumor compound. In some embodiments, the linker-payload in the ADCs of the present application is LD38, see, e.g., PCT / CN2023 / 106385 (WO2025 / 011419, which is incorporated herein by reference in its entirety).

[0185] In some embodiments, the antibodies or antigen binding fragments thereof of the present application are linked to a cytotoxic agent or an anti-tumor compound via a linker to form an antibody drug conjugate.

[0186] In one aspect, one skilled in the art can select a suitable drug molecule as the payload of the ADC, e.g., D in Formula (I), according to the desired mechanism of action of the ADC drug and the cell-killing effect.

[0187] In some embodiments, the present application provides

[0188] An antibody-drug conjugate (ADC) having a structure represented by Formula (I), Ab-(L-(D) m ) p (I)

[0189] or a pharmaceutically acceptable salt or solvate thereof,

[0190] wherein:

[0191] Ab is an antibody or fragment thereof, especially an antibody or fragment (e.g., antigen binding fragment) thereof that specifically binds integrin α3β1 (e.g., human integrin α3β1);

[0192] L is a linker;

[0193] D is a drug, e.g., an anti-tumor compound such as a cytotoxic agent;

[0194] m is 1-6; and

[0195] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12.

[0196] In some embodiments, Ab in formula (I) is an antibody or fragment (e.g., antigen binding fragment) thereof of the present application that specifically binds integrin α3β1.

[0197] To form the ADC molecule, the Ab can be coupled with a linker-payload unit using some of the naturally occurring attachment sites thereon. Such naturally occurring attachment sites are the thiol group of cysteine and the amino group of lysine. Typically, the interchain disulfide bridges of the antibody can be used to achieve a more defined drug-antibody ratio (DAR). Thus, in one embodiment, after reduction of the interchain disulfide bonds of the antibody, a linker-payload unit is coupled to the antibody via thiol chemistry to form the ADC conjugate of formula (I). In addition, it is also contemplated that artificial attachment sites can be introduced into the antibody to achieve a more site-directed coupling.

[0198] In some embodiments, the Ab suitable for the ADC of the present application is an antibody as shown under chA28, chC8, chC18, chC22, or chC47, huA28-H2L3 (huA28), huA28-comb4, huA28-comb7, or huA28-comb9, for example, as characterized and / or prepared in the Examples.

[0199] In one aspect, the skilled person can select a suitable drug molecule as the payload of the ADC, e.g., D in formula (I), according to the desired mechanism of action of the ADC drug and the effect of cell killing.

[0200] In a particular embodiment of the present application, D in formula (I) of the present application can be any anti-tumor compound, as long as it is a compound having an anti-tumor effect and having a substituent or moiety structure that can be attached to the linker structure, without particular limitation. For example, the anti-tumor compound can be a pharmaceutically active compound that has an effect on tumors. For the anti-tumor compound, part or all of the linker is preferably cleaved within the tumor cell to free the anti-tumor compound moiety, thereby exhibiting an anti-tumor effect. Upon cleavage of the linker from the linking moiety of the drug, the anti-tumor compound is freed in its unmodified structure and can exert its original anti-tumor effect.

[0201] In some embodiments, the drug D of the antibody-drug conjugate is a drug or prodrug thereof that is toxic or inhibitory to cells, e.g., a cytotoxic agent.

[0202] In the art, a variety of different mechanisms of cytotoxic agents suitable for use as payloads have been reported, including, but not limited to,

[0203] (1) Tubulin inhibitors / stabilizing disruptors: e.g., auristatins, e.g., monomethyl auristatin (MMAE), maytansine derivatives (e.g., DM2, DM4), Tubulysins, Cryptomycins, etc.;

[0204] (2) DNA synthesis inhibitors, e.g., methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, etc.;

[0205] (3) Topoisomerase inhibitors: e.g., topoisomerase I inhibitors (e.g., camptothecins) and topoisomerase II inhibitors (e.g., dactinomycin, doxorubicin, mitoxantrone).

[0206] In some embodiments, the drug D of the antibody-drug conjugate is a monomethyl auristatin (MMAE) of the formula:

[0207] In some embodiments, the drug D of the antibody-drug conjugate is a topoisomerase I inhibitor, e.g., but not limited to, a camptothecin compound, camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, FL118, Exatecan, topotecan, irinotecan, or derivatives thereof, etc.

[0208] In some embodiments of the present application, the drug D of the antibody-drug conjugate is selected from a camptothecin compound, e.g., camptothecin, and derivatives thereof, e.g.,

[0209] wherein R A is selected from, e.g., hydrogen, C 1-6 alkyl, hydroxyC 1-6 alkyl, aminoC 1-6 alkyl, etc.

[0210] In some embodiments, the drug D of the antibody-drug conjugate is 10,11- methylenedioxy camptothecin (also referred to simply as FL118) or derivatives thereof, e.g., those disclosed in WO2019 / 195665 (herein incorporated by reference in its entirety), e.g.,

[0211] wherein R B is selected from: hydrogen, C 1-8 alkyl, C 1-8 haloalkyl, C 3-8 cycloalkyl, C 3-8 cycloalkyl1-4 alkyl, phenyl and phenyl C 1-4 alkyl.

[0212] In some embodiments, the drug D of the antibody-drug conjugate is 10-hydroxycamptothecin (also referred to as HCPT) or a derivative thereof, for example,

[0213] wherein R C selected from H and optionally substituted C 1-6 alkyl; R C selected from H, C 1-6 alkyl-C(=0)-, or optionally substituted heterocyclyl-C(=0)-, R D selected from H and optionally substituted C 1-6 alkyl.

[0214] In one embodiment, the drug D of the antibody-drug conjugate is irinotecan or topotecan:

[0215] In one embodiment, the drug D of the antibody-drug conjugate is exatecan or a derivative thereof, for example,

[0216] wherein R E selected from, for example, H, optionally substituted C 1-6 alkyl-C(=0)-, said substituents being selected from -OH or substituted or unsubstituted amino.

[0217] In one embodiment, the drug D of the antibody-drug conjugate is:

[0218] In some specific embodiments, the linker L in formula (I) of the present application can be any linker capable of effecting conjugation of the antibody in formula (I) to the drug. The addition of the linker is suitably such that the ADC of the present application is sufficiently stable in the circulation system of the subject and can provide rapid and efficient release of the payload in formula (I) in the target site (e.g. tumor cell or tumor environment).

[0219] In some embodiments, the linker in formula (I) of the present application is a non-degradable linker. Examples of non-degradable linkers include, but are not limited to, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Typically, ADCs comprising such linkers must be internalized by the cell, the antibody portion of the ADC is degraded by lysosomal proteases within the cell, releasing the drug active molecule.

[0220] In some embodiments, the linker in Formula (I) of the present application is a degradable linker. An ADC comprising such a linker triggers drug release by the nature of the cleavage site in the linker. Thus, the cleavage site of such a linker can be designed according to the characteristics of the target therapeutic site (e.g., tumor lysosome and / or tumor environment). In most cases, the degradable linker can consist of a coupling moiety, a degradable moiety, and optionally a spacer moiety. The coupling moiety is responsible for the linkage of the antibody to the linker-drug unit, which can be selected according to the antibody conjugation chemistry desired to be employed. The degradable moiety will comprise a peptide or a peptide analog that can be recognized by an enzyme, e.g., an oligopeptide or oligopeptide analog that can be degraded by a proteolytic enzyme, such as Val-Ala, Val-Cit, Phe-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Gly-Gly-Phe-Gly, cyclobutyl-Ala, cyclobutyl-Cit, etc., under an enzyme-based release mechanism. In some cases, the properties of the ADC, e.g., the stability of the ADC in blood circulation and / or the potency of the ADC at the target site, can be improved by introducing a modification at the position of a peptide residue adjacent to the enzyme cleavage site in the linker. In some cases, a spacer can also be introduced between the degradable moiety of the linker and the drug D, as desired, to facilitate the release of the drug active molecule from the rest of the conjugate (especially, traceless release), e.g., a p-aminobenzyl carbamate (PABA) or an aminomethyl (-NHCH2-) spacer that can be eliminated spontaneously in an acidic medium. In addition, when the drug is highly hydrophobic, in some cases, it can be considered (but not necessary) to add, e.g., a PEG unit to improve the properties of the ADC, e.g., to reduce precipitation and aggregation. Linkers that can be suitable for use in the present application include, but are not limited to, the linkers disclosed in WO2022 / 170971 (which is hereby incorporated by reference in its entirety).

[0221] In some embodiments, the linker L- in Formula (I) of the present application has the following structure: 1- E-L2-L 3-

[0222] wherein Z is a linker to Ab, L3 is absent, or is a spacer for linking to drug D.

[0223] Preferably,

[0224] Z is selected from

[0225] L1 is -(CH2) n -C(=O)-, -(CH2) n -C(=O)-NH-(CH2) n -C(=O)-, -(CH2) n-(O(CH2)2) t (CH2) n -C(=O)- or -(CH2) n -C(=O)-NH-(CH2) n -(O(CH2)2) t (CH2) n -C(=O)-;

[0226] E is absent or a peptide residue comprising 2-10 amino acids, wherein the amino acids are natural or unnatural amino acids and are optionally substituted, and the C-terminus of the peptide residue is covalently linked to L2;

[0227] L2 is absent or

[0228] L3 is absent or -C(=O)-*, wherein * indicates that the terminus is covalently linked to D;

[0229] n is an integer from 0-10,

[0230] t is an integer from 1-10.

[0231] In some embodiments, E in said -L- is selected from Lys, Gly, Asp, Asn, Glu, Gin, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Glu-Gly, Gly-Asp, Gly-Asn, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Phe-Gly, (Gly)2-Glu-Gly, or any combination of fragments of the above; preferably, E is selected from Val-Cit, Ala-Ala, Gly-Glu, Glu-Gly, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Glu-Gly, (Gly)2-Phe-Gly.

[0232] In some embodiments, E in said -L- is selected from:

[0233] wherein s = 0-8.

[0234] In some preferred embodiments, L in formula (I) of the present application is a linker selected from the following structures:

[0235] In some implementations, the connector-payload is LD38.

[0236] In some embodiments, the present invention provides ADC conjugates having the following structures or pharmaceutically acceptable salts or solvates thereof:

[0237] The antibody Ab unit is the anti-integrin α3β1 antibody or its antigen-binding fragment of the present invention.

[0238] p represents the average DAR value from 1 to 20, for example, an average DAR value of approximately 2, 3, 4, 6, 7, or 8.

[0239] In some embodiments, the S atom in the ADC linked to the Ab comes from the antibody Ab. In some embodiments, the Ab breaks its disulfide bond under the action of a reducing agent such as TCEP to generate a thiol group -SH, which is then linked to the terminal functional group of the linker, such as the maleimide moiety.

[0240] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 7-8.

[0241] In some embodiments, the ADC according to the present invention has at least one or more of the following advantages:

[0242] - Compared to the distribution of ADCs in the bloodstream, it significantly promotes the distribution and accumulation of ADCs in the tumor environment;

[0243] - Effectively limits premature release of the payload into the plasma, exhibiting high circulatory stability.

[0244] - It can provide effective release of the active form of toxic drugs in the tumor environment;

[0245] - It imparts a highly effective tumor-killing effect in animals; and

[0246] - Good animal tolerance.

[0247] In some embodiments, the ADC according to the present invention may also have at least one or more of the following advantages:

[0248] -The addition of the linker-payload does not induce aggregation and can achieve high drug loading, with DAR up to 8; and

[0249] - Acceptable pharmacokinetic (PK) characteristics.

[0250] In a third aspect of the invention, a nucleic acid, such as an isolated nucleic acid molecule, is provided that encodes the anti-integrin α3β1 antibody of the invention or a fragment thereof or any strand thereof.

[0251] For example, the nucleic acid of the application comprises a nucleic acid encoding an amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NO: 1, 2, 15, 16, 29, 30, 43, 44, 57, 58, 73, 74, 75, 76, 84, 85, 89, or 90, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NO: 1, 2, 15, 16, 29, 30, 43, 44, 57, 58, 73, 74, 75, 76, 84, 85, 89, or 90. As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the application can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0252] When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting human integrin α3β1 antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in-frame to a nucleic acid encoding the constant region of the heavy chain and / or light chain, such that when expressed from a suitable expression vector, the nucleic acid encodes the antibody heavy chain and / or.

[0253] To facilitate production and purification, a secretory signal peptide can be fused at the N-terminus of the heavy chain and / or light chain of the antibody, and / or a tag peptide facilitating purification, such as a his-tag.

[0254] In a fourth aspect of the application, one or more vectors comprising the nucleic acid of the application are provided.

[0255] The term "vector", as used herein, when used in reference to a nucleic acid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector that comprises a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting DNA control regions that direct the transcription and translation of the relevant nucleotide sequence in a host cell. Additional elements can be included in expression vectors to allow for

[0256] In one embodiment, the vector is an expression vector, for example a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pCDNA vector, for example pCDNA3.4.

[0257] In a fifth aspect of the application, there is provided a host cell comprising a nucleic acid encoding an antibody or antigen binding fragment thereof of the application, or a vector comprising said nucleic acid.

[0258] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny of the original transformant that have a non-identical genotype, irrespective of the number of passages. Progeny can not contain the mutation present in the parent cell, but can contain a mutation that was not present in the original cell. Mutant progeny that have the same function or biological activity as the originally transformed cell are included herein. Host cells are any type of cell system that can be used to produce an antibody molecule of the application, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, E. coli cells. Host cells include cells in culture as well as cells in a transgenic animal, transgenic plant or in a cultured plant tissue or animal tissue.

[0259] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g. a CHO cell (e.g. CHO-S or CHO-K or CHO-K1) or a 293 cell (e.g. 293T, 293F or HEK293 cell)) or other cell suitable for making an antibody or fragment thereof. In one embodiment, the host cell is prokaryotic, for example is a bacterium, for example E. coli.

[0260] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell or other cell suitable for making an antibody or fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antibodies. Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK 293, 293F or 293T cells); etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO-S cells or CHO-K or CHO-K1, etc.; and myeloma cell lines such as NS0 and Sp2 / 0. Mammalian host cell lines suitable for production of antibodies are known in the art.

[0261] In a sixth aspect of the application, the present application provides a method of making an anti- integrin α3β1 antibody or fragment thereof (preferably an antigen binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expression of the nucleic acid encoding the antibody or fragment thereof (preferably an antigen binding fragment) or either or both chains thereof, and optionally isolating the antibody or fragment thereof (preferably an antigen binding fragment). In a certain embodiment, the method further comprises recovering the anti-integrin α3β1 antibody or fragment thereof (preferably an antigen binding fragment) from the host cell.

[0262] The polynucleotides encoding the polypeptide chains of the antibodies or antigen binding fragments thereof of the present application can be inserted into one or more vectors for further cloning and / or expression in a host cell. The expression vectors can be constructed using methods well known to those skilled in the art. Once the expression vector comprising the nucleic acid molecule(s) of the present application has been prepared for expression, the expression vector can be transfected or introduced into an appropriate host cell. A variety of techniques can be used for this purpose, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, transduction by retroviruses, viral transfection, biolistics, liposome-based transfection, or other conventional techniques.

[0263] Antibodies prepared as described herein can be purified by known art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, e.g., Protein A, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art.

[0264] The purity of the antibody molecules of the present application can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0265] In a seventh aspect of the application, there are provided methods or assays for identifying, screening or characterizing the physical / chemical properties and / or biological activities of the anti-integrin α3β1 antibodies provided herein.

[0266] In one aspect, the antibodies of the present application are tested for their antigen binding activity, for example, by known methods such as Bio-Layer Interferometry, ELISA, and the like. Methods known in the art can be used to determine binding to integrin α3β1, for example, using a radioimmunoassay (RIA) or a Bio-Layer Interferometry assay (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) measurements.

[0267] The present application also provides assays for identifying anti- integrin α3β1 antibodies having biological activity. Biological activity is selected from properties of the antibodies of the present application, and can include, for example, binding to integrin α3β1 (e.g., binding to human integrin α3β1), etc.

[0268] For example, binding activity of an antibody molecule of the present application to integrin α3β1 or cells expressing integrin α3β1 can be determined by methods known in the art, e.g., Fortebio, flow cytometry, Octet, or plasmon resonance (Biacore), etc., or exemplary methods disclosed in Example 1 herein.

[0269] For example, killing activity or anti-tumor activity of an antibody molecule of the present application or an antibody drug conjugate comprising the same to cells (e.g., integrin α3β1 -positive cells, e.g., tumor cells) can be determined by methods known in the art, e.g., cell killing assays, such as in vitro cell killing assays, or in vivo animal model assays, such as exemplary methods disclosed in Examples 7 or 8 or 9 herein.

[0270] Cells for use in any of the above in vitro assays include cell lines that naturally express integrin α3β1 or are engineered to express integrin α3β1. Such cells also include cell lines that express integrin α3β1 and are transfected with DNA encoding integrin α3β1 that are not normally expressing integrin α3β1, e.g., HCT116, PANC-1, HCC827, or AGS cells.

[0271] It is understood that any of the above assays can be performed using an antibody drug conjugate of the present application in place of or in addition to an anti-integrin α3β1 antibody.

[0272] It is understood that any of the above assays can be performed using an anti-integrin α3β1 antibody or antigen binding fragment thereof in combination with another active agent.

[0273] In an eighth aspect of the present application, a method of making an ADC is provided, the method comprising conjugating an antibody (Ab) of the present application to one or more payloads, e.g., active substances D, via one or more linkers (L) as defined in the present application.

[0274] In some embodiments, the method comprises preparing an Ab (anti-integrin α3β1 antibody of the present application or antigen binding fragment thereof) for use in an ADC as described herein, comprising culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or a plurality of polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for expression of the Ab or chains thereof, as provided above, and optionally recovering and / or purifying the Ab from the host cell (or host cell culture medium).

[0275] In some embodiments, the method comprises the steps of:

[0276] (a) adding an antibody to a buffered solution, adding a reducing agent, followed by incubation;

[0277] (b) adding a linker-payload to the reaction solution in step (a) for conjugation to obtain a crude product; and

[0278] (c) optionally purifying the crude product to obtain an antibody-drug conjugate of the application;

[0279] wherein Ab and linker-payload are as defined above.

[0280] In some embodiments, the buffered solution of step a) is a PBS buffer, preferably having a pH of 5.0-9.0, such as 6.0-8.0.

[0281] In some embodiments, the reducing agent of step a) is TCEP or a salt thereof, such as the hydrochloride salt of TCEP.

[0282] In some embodiments, the steps are carried out under the specific reaction conditions disclosed in the examples.

[0283] It should be noted that embodiments within the scope of 100%, 80%, 60%, 40%, 20% or 10% of the specific reaction conditions disclosed in the examples are also contemplated by the present application.

[0284] In a ninth aspect of the application, there are provided methods or assays for identifying, screening or characterizing the physical and / or chemical properties and / or biological activities of the ADCs provided herein.

[0285] For example, the killing or anti-tumor activity of an ADC molecule of the application on cells (e.g. integrin α3β1 -positive cells, such as integrin α3β1 -positive tumor cells) or an anti-tumor model comprising said cells can be determined by methods known in the art, such as cell killing assays, e.g. in vitro cell killing assays, or in vivo animal model assays, such as the exemplary methods disclosed in Examples 5-9 herein.

[0286] Cells for use in any of the above in vitro assays include cell lines that naturally express integrin α3β1 or have been engineered to express integrin α3β1. Such cells also include cell lines that express integrin α3β1 and have been transfected with DNA encoding integrin α3β1 that is not normally expressed by the cell, such as HCT116, PANC-1, HCC827 or AGS cells.

[0287] It will be appreciated that any of the above assays can be carried out using a combination of an ADC molecule of the application and another active agent.

[0288] In a tenth aspect of the application, a composition, such as a pharmaceutical composition or a pharmaceutical preparation, comprising an antibody of the application or an antigen-binding fragment thereof or an immunoconjugate thereof, such as an antibody drug conjugate, is provided.

[0289] The term "pharmaceutical composition" as used herein refers to a composition that is in a form suitable for its intended use, and that contains an active ingredient in an effective amount for achieving its intended biological activity, and that does not contain additional ingredients that are unacceptable to the subject to whom the composition is administered.

[0290] In some embodiments, the pharmaceutical composition or pharmaceutical preparation further comprises pharmaceutical auxiliaries, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art. As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents, and the like, which are physiologically compatible.

[0291] For the use of pharmaceutical auxiliaries and their uses, see also "Handbook of Pharmaceutical Excipients", 8thEdition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0292] The compositions or preparations of the application can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), powders or suspensions, liposomal formulations, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0293] Pharmaceutical preparations comprising the antibodies described herein can be prepared by mixing an antibody of the application or an antigen-binding fragment thereof or an immunoconjugate thereof, such as an antibody drug conjugate, having the desired degree of purity with one or more pharmaceutically acceptable excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0294] The pharmaceutical compositions or preparations of the application can further comprise more than one active ingredient, which are required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to provide other therapeutic agents. The active ingredients are suitably combined in amounts that are effective for the purpose intended.

[0295] In an eleventh aspect of the application, a pharmaceutical combination or combination product is provided, comprising an anti-integrin α3β1 antibody or a fragment thereof, preferably an antigen-binding fragment, of the application, or an immunoconjugate thereof, such as an antibody drug conjugate, and one or more other therapeutic agents.

[0296] The terms "pharmaceutical combination" or "combination product" as used herein refer to either a non-fixed combination or a fixed combination, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the application or antigen-binding fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, and (ii) the other therapeutic agent) are presented in a separate entity and are administered simultaneously, within a short period of time, or sequentially within the same day, or at different time intervals, wherein such administration provides therapeutically effective levels of the two or more active agents in the body of the patient. The term "fixed combination" means that the two or more active agents are administered in a single entity. Preferably, the dosage amounts and / or time intervals of the two or more active agents are selected so that a combined use of the individual parts results in an effect that is greater than the effect achieved by the use of any of the parts alone in the treatment of the disease or condition. It will be appreciated that the individual components of the pharmaceutical combination can be in separate formulations or kits. The individual components can each be in a separate formulation, which can be the same or different.

[0297] In a twelfth aspect of the application, a kit-of-parts is provided comprising a pharmaceutical combination of the application, preferably said kit is in the form of a pharmaceutical dosage unit. Thereby dosage units can be provided according to a dosing regimen or pharmaceutical administration interval.

[0298] In one embodiment, the kit-of-parts of the application comprises in the same package:

[0299] - a first container containing a pharmaceutical composition comprising an anti- integrin α3β1 antibody or fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate;

[0300] - a second container containing a pharmaceutical composition comprising the other therapeutic agent.

[0301] In a thirteenth aspect of the application, a method of preventing or treating an integrin α3β1 -related disease in a subject is provided, comprising administering to the subject an effective amount (e.g., a prophylactically effective amount or a therapeutically effective amount) of an anti-integrin α3β1 antibody or antigen-binding fragment thereof, antibody drug conjugate, pharmaceutical composition or combination product of the application.

[0302] As used herein, "treatment" refers to slowing, interrupting, arresting, reversing, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indicia of a disease, or relieving an illness, condition, or disorder, or preventing or inhibiting further development of the disease, condition, or disorder.

[0303] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder.

[0304] The term "effective amount" refers to an amount or dose of an antibody of the application or antigen-binding fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product, which elicits the intended effect, upon administration to a patient in a single or multiple doses, in the patient requiring treatment or prevention. Depending on the intended effect, this can include both "therapeutically effective amounts" and "prophylactically effective amounts".

[0305] A "therapeutically effective amount" as used herein refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody of the application or antigen-binding fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 30%, even more preferably by at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject, with respect to a measurable parameter (e.g., tumor volume).

[0306] A "prophylactically effective amount" as used herein refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Generally, the prophylactically effective amount will be less than the therapeutically effective amount since the prophylactic dose is used before the disease is full-blown, or at an earlier stage of the disease.

[0307] An "individual" or "subject" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice). In some embodiments, the individual or subject is a human.

[0308] In some embodiments, the integrin α3β1 -related disease described herein includes a tumor, e.g., a cancer. The cancer can be in an early, intermediate, or advanced stage or be a metastatic cancer. In some embodiments, the cancer can be a solid tumor or a hematological tumor (encompassing lymphomas). In some embodiments, the cancer is selected from colon cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), or gastric cancer (e.g., gastric adenocarcinoma).

[0309] In some embodiments, the tumor is an integrin-positive tumor, e.g., an integrin alpha 3 beta 1 -positive tumor. In one embodiment, the tumor refers to having integrin (e.g., integrin alpha 3 beta 1) expression or having integrin (e.g., integrin alpha 3 beta 1) nucleic acid in the tumor tissue or tumor cells of the individual, e.g., compared to adjacent normal tissue or normal cells (e.g., normal cells in the tissue) of the individual or the same tissue or cells therein of a healthy individual, or, compared to, the protein level (e.g., expression) of integrin alpha 3 beta 1 is elevated, or the nucleic acid level of integrin alpha 3 beta 1 is elevated. Thus, in some embodiments, an integrin-positive tumor refers to having upregulated expression of integrin, or overexpression of integrin in the tumor tissue or cells.

[0310] In some embodiments, the antibody or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product of the application delays the onset of the disorder and / or symptoms associated with the disorder.

[0311] In some embodiments, the antibody or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product of the application is capable of inhibiting tumor tissue or tumor cell growth. In some embodiments, the application relates to a method of inhibiting tumor tissue or tumor cell (e.g., tissue or cells of a tumor as defined herein) growth, i.e., administering the antibody or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product of the application.

[0312] In some embodiments, the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual the antibody or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product of the application, in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents.

[0313] In some embodiments, the treatment modalities include surgery, radiation therapy (e.g., external beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiated area is designed), local irradiation (e.g., irradiation directed to a preselected target or organ) or focused irradiation, etc.

[0314] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., as separate antibodies, or linked (e.g., as a bispecific or multispecific antibody molecule) when administered.

[0315] The combination therapies of the application encompass combined administration (e.g., two or more therapeutic agents are included in the same formulation or separate formulations), and separate administration, in which case, administration of the antibody of the application or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product, etc., can occur prior to, simultaneously with, and / or following, administration of the other therapeutic agent and / or agents.

[0316] The anti- integrin α3β1 antibodies of the application (and immunoconjugates comprising the same, e.g., antibody drug conjugates, compositions, pharmaceutical compositions, formulations, combination products, etc.) can be administered by any suitable method, including parenterally, and, if local treatment is desired, intralesionally. Parenteral injections or infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous injections or infusions. Depending on whether the medication is short- or long-term, it can be administered by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single dosing or multiple dosing at multiple time points, bolus dosing, and pulse infusion.

[0317] In other aspects, the application provides the use of an antibody of the application or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product, in the manufacture or preparation of a medicament for use in a use described herein, e.g., for the prevention or treatment of a relevant disease or condition mentioned herein.

[0318] In other aspects, the application provides an antibody of the application or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product, for use in therapy, e.g., for the treatment of a relevant disease or condition mentioned herein or for a use mentioned herein.

[0319] In other aspects, the application also provides the use of an antibody of the application or antigen-binding region fragment thereof or immunoconjugate thereof, e.g., antibody drug conjugate, or composition or pharmaceutical combination / combination product, for a method described herein, e.g., for the prevention or treatment of a relevant disease or condition mentioned herein.

[0320] In a fourteenth aspect of the application, the application also relates to methods of and compositions comprising the antibodies of the application or antigen-binding fragments thereof for use in diagnosis and detection.

[0321] In certain embodiments, any of the anti- integrin α3β1 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of integrin α3β1 in a biological sample.

[0322] The term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is from tumor tissue or cancer tissue.

[0323] In one embodiment, an anti- integrin α3β1 antibody or antigen-binding fragment thereof is provided for use in a diagnostic or detection method.

[0324] In another aspect, a method of detecting the presence of integrin α3β1 in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of integrin α3β1 protein in a biological sample. In certain embodiments, the integrin α3β1 is human integrin α3β1 or mouse integrin α3β1 or cynomolgus monkey integrin α3β1. In certain embodiments, the method comprises contacting a biological sample with an anti-integrin α3β1 antibody as described herein under conditions that allow the anti-integrin α3β1 antibody to bind to integrin α3β1 and detecting whether a complex is formed between the anti-integrin α3β1 antibody and integrin α3β1. Formation of a complex is indicative of the presence of integrin α3β1. The method can be an in vitro or in vivo method. In one embodiment, the anti-integrin α3β1 antibody is used to select a subject suitable for treatment with an anti-integrin α3β1 antibody, e.g., where integrin α3β1 is a biomarker for selecting the subject.

[0325] In certain embodiments, labeled antibodies or fragments thereof are provided. Labels include, but are not limited to, labels or moieties that are directly detected, such as fluorescent labels, chromophoric labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by enzymatic reaction or molecular interaction.

[0326] In some embodiments provided herein, the sample is obtained prior to treatment with an antibody or fragment thereof of the application. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0327] In some embodiments, integrin α3β1 is detected prior to treatment, e.g., prior to initiation of treatment or prior to a treatment interval.

[0328] In some embodiments, a method of treating an integrin α3β1 -related disease of the application is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample, e.g., tumor tissue or cells) for the presence of integrin α3β1, thereby determining an integrin α3β1 value, comparing the integrin α3β1 value to a control value (e.g., a value in the same tissue or cells of a healthy individual or a value in a proximal normal tissue or cells of the patient to be tested), and if the integrin α3β1 value is greater than the control value, administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the application or an immunoconjugate thereof, e.g., an antibody drug conjugate, or a composition or pharmaceutical combination / combination product thereof, optionally in combination with one or more other therapies, thereby treating the disease.

[0329] In a fifteenth aspect of the application, the application relates to the following specific embodiments:

[0330] 1. An antibody or antigen-binding fragment thereof that specifically binds to integrin α3β1, the antibody comprising

[0331] 1) three complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised in a VH as shown in SEQ ID NO: 1, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised in a VL as shown in SEQ ID NO: 2;

[0332] 2) three complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised in a VH as shown in SEQ ID NO: 15, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised in a VL as shown in SEQ ID NO: 16;

[0333] 3) three complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised in a VH as shown in SEQ ID NO: 29, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised in a VL as shown in SEQ ID NO: 30;

[0334] 4) three complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised in a VH as shown in SEQ ID NO: 43, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised in a VL as shown in SEQ ID NO: 44;

[0335] 5) three complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised in a VH as shown in SEQ ID NO: 57, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised in a VL as shown in SEQ ID NO: 58;

[0336] 6) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 73, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 74;

[0337] 7) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 76;

[0338] 8) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 84, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 85; or

[0339] 9) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 89, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 90.

[0340] For example, wherein the HCDRs and the LCDRs are determined according to the IMGT or the Kabat scheme, respectively.

[0341] 2. An antibody or antigen binding fragment thereof that specifically binds to integrin alpha3beta1, said antibody comprising three complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise or consist of the amino acid sequences set forth in the following table SEQ ID NO based on the IMGT scheme, respectively:

[0342] or wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise or consist of the amino acid sequences set forth in the following table SEQ ID NO based on the Kabat scheme, respectively:

[0343] 3. The antibody or antigen-binding fragment thereof that binds integrin α3β1 of embodiment 1 or 2, said antibody comprising a heavy chain variable region, wherein said heavy chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89.

[0344] 4. The antibody or antigen-binding fragment thereof that binds integrin α3β1 of any one of embodiments 1-3, said antibody comprising a light chain variable region, wherein said light chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85, or 90.

[0345] 5. The antibody or antigen-binding fragment thereof that binds integrin α3β1 of any one of embodiments 1-4, said antibody comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region and light chain variable region comprise or consist of the amino acid sequences set forth in the following table SEQ ID NO:

[0346] 6. The antibody or antigen-binding fragment thereof that binds integrin α3β1 of any one of embodiments 1-5, said antibody comprising a heavy chain constant region, e.g., a heavy chain constant region of (human) IgGl, IgG2, IgG3, or IgG4, e.g., a heavy chain constant region of IgGl, e.g., said heavy chain constant region

[0347] (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71; or

[0348] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 71.

[0349] 7. The antibody or antigen-binding fragment thereof that binds integrin α3β1 according to any one of embodiments 1-6, said antibody comprising a light chain constant region, e.g. a (human) lambda or kappa light chain constant region, e.g. the light chain constant region

[0350] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72; or

[0351] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0352] 8. The antibody or antigen-binding fragment thereof that binds integrin α3β1 according to any one of embodiments 1-7, said antibody comprising a heavy chain constant region and a light chain constant region, wherein

[0353] the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 71; and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0354] 9. The antibody or antigen-binding fragment thereof that binds integrin α3β1 according to any one of embodiments 1-8, said antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of a heavy chain variable region and a heavy chain constant region, and the light chain comprises or consists of a light chain variable region and a light chain constant region, wherein

[0355] (i). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0356] (ii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0357] (iii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0358] (iv). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0359] (v). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0360] (vi). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0361] (vii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72;

[0362] (viii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; or

[0363] (ix). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72.

[0364] 10. The antibody or antigen-binding fragment thereof that binds integrin α3βl of any one of embodiments 1-8, which has one or more of the following properties:

[0365] (1) specifically binds to integrin βl and integrin α3, i.e., specifically binds to integrin α3βl, e.g., human integrin α3βl;

[0366] (2) is capable of binding integrin α3βl, e.g., human integrin α3βl, expressed on the surface of a cell membrane with high affinity;

[0367] (3) is suitable for use in constructing an immunoconjugate (e.g., an antibody drug conjugate (ADC)) for targeted killing of integrin α3βl -positive cells, e.g., integrin α3βl -positive tumor cells; or for treating a tumor, e.g., a cancer;

[0368] (4) inhibits the binding of any of the antibodies listed in embodiment 9 to integrin α3βl;

[0369] (5) binds to the same or an overlapping epitope as any of the antibodies set forth in embodiment 9; or

[0370] (6) competes with any of the antibodies set forth in embodiment 9 for binding to integrin α3βl.

[0371] 11. The antibody or antigen-binding fragment thereof of any one of embodiments 1-10, wherein the antibody is a monoclonal antibody.

[0372] 12. The antibody or antigen-binding fragment thereof of any one of embodiments 1-11, wherein the antibody is a chimeric antibody or a humanized antibody.

[0373] 13. The antibody or antigen-binding fragment thereof of any one of embodiments 1-12, wherein the antigen-binding fragment is an antibody fragment selected from a Fab, a Fab', a Fab'-SH, an Fv, a single chain antibody scFv, a (Fab')2 fragment, a single domain antibody, a diabody, or a linear antibody.

[0374] 14. The antibody or antigen-binding fragment thereof of any one of embodiments 1-13, wherein the antibody is a bispecific antibody or a multispecific antibody.

[0375] 15. An isolated nucleic acid encoding the anti- integrin α3β1 antibody or antigen binding fragment thereof of any one of embodiments 1 to 14.

[0376] 16. An expression vector comprising the nucleic acid of embodiment 15, for example the expression vector is a pCDNA vector, for example a pCDNA3.4 vector.

[0377] 17. A host cell comprising the nucleic acid of embodiment 25 or the expression vector of embodiment 26, for example the host cell is prokaryotic or eukaryotic, for example the host cell is selected from an E. coli cell, a yeast cell, a mammalian cell or other cell suitable for making an antibody or antigen binding fragment thereof, for example the host cell is a 293 cell or a CHO cell.

[0378] 18. A method of making an anti- integrin α3β1 antibody or antigen binding fragment thereof, the method comprising culturing the host cell of embodiment 17 under conditions suitable for expression of the nucleic acid encoding the anti- integrin α3β1 antibody or antigen binding fragment thereof of any one of embodiments 1 to 14, optionally isolating the antibody or antigen binding fragment thereof, optionally the method further comprises recovering the anti- integrin α3β1 antibody or antigen binding fragment thereof from the host cell.

[0379] 19. An immunoconjugate comprising the antibody or antigen binding fragment thereof of any one of embodiments 1 to 14 and another therapeutic agent, preferably the immunoconjugate is an antibody drug conjugate.

[0380] 20. The immunoconjugate of claim 19, wherein the other therapeutic agent is a cytotoxic agent or an anti-tumor compound.

[0381] 21. An antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt or solvate thereof, Ab-(L-(D) m ) p (I)

[0382] wherein:

[0383] Ab is the antibody or antigen binding fragment thereof of any one of embodiments 1 to 14,

[0384] L is a linker;

[0385] D is a drug, preferably an anti-tumor compound or a cytotoxic agent;

[0386] m is 1-6; and

[0387] p is 1 to 16, for example an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0388] 22. The antibody-drug conjugate of embodiment 21, or a pharmaceutically acceptable salt or solvate thereof, wherein D is selected from the group consisting of a microtubule inhibitor (e.g., MMAE), a DNA cross-linking agent, a DNA alkylating agent, and a topoisomerase I inhibitor; for example, the topoisomerase I inhibitor is selected from the group consisting of camptothecin class compounds such as camptothecin and its derivatives, e.g., camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, FL118, Dxd, topotecan, belotecan, lurtotecan, irinotecan, exatecan; or L-D is LD38.

[0389] 23. The antibody-drug conjugate of embodiment 21 or 22, or a pharmaceutically acceptable salt or solvate thereof, having an average DAR of 2-10, e.g., 7-9 or 7-8.

[0390] 24. The antibody-drug conjugate of any one of embodiments 21-23, or a pharmaceutically acceptable salt or solvate thereof, wherein D is camptothecin and its derivatives, having the structure:

[0391] wherein R A is selected from the group consisting of hydrogen, C 1-6 alkyl, hydroxy C 1-6 alkyl, amino C 1-6 alkyl;

[0392] or

[0393] wherein R B is selected from the group consisting of hydrogen, C 1-8 alkyl, C 1-8 haloalkyl, C 3-8 cycloalkyl, C 3-8 cycloalkyl C 1-4 alkyl, phenyl and phenyl C 1-4 alkyl;

[0394] or

[0395] wherein R C is selected from the group consisting of H and optionally substituted C 1-6 alkyl; R C is selected from the group consisting of H, C 1-6 alkyl-C(=O)- or optionally substituted heterocyclyl-C(=O)-; R D is selected from the group consisting of H and optionally substituted C 1-6 alkyl;

[0396] or

[0397] wherein R E selected from, for example, H, optionally substituted C 1-6 alkyl-C(=0)-, said substituent being selected from -OH or substituted or unsubstituted amino.

[0398] 25. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to embodiment 24, wherein D has the following structure:

[0399] 26. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 21-23, wherein D is monomethyl auristatin (MMAE) of the following formula:

[0400] 27. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to embodiments 21-26, wherein -L- has the following structure: -Z-L 1- E-L2-L3-

[0401] wherein Z is attached to Ab and L3 is attached to D;

[0402] Z is selected from

[0403] L1is -(CH2) n -C(=0)-, -(CH2) n -C(=0)-NH-(CH2) n -C(=0)-, -(CH2) n -(0(CH2)2) t (CH2) n -C(=0)- or -(CH2) n -C(=0)-NH-(CH2) n -(0(CH2)2) t (CH2) n -C(=0)-;

[0404] E is absent or a peptide residue comprising 2-10 amino acid residues, wherein the amino acid residues are natural or unnatural amino acid residues and are optionally substituted, and the C-terminal end of the peptide residue is covalently linked to L2; preferably, E is selected from Lys, Gly, Asp, Asn, Glu, Gin, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Glu-Gly, Gly-Asp, Gly-Asn, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Asp-Gly, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Phe-Gly, (Gly)2-Glu-Gly, or any combination of fragments of the above; preferably, E is selected from Val-Cit, Ala-Ala, Gly-Glu, Glu-Gly, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Glu-Gly, (Gly)2-Phe-Gly;

[0405] L2 is absent or

[0406] L3 is absent or -C(=0)-*, wherein * indicates that the terminal end is covalently linked to D;

[0407] n is an integer from 0 to 10,

[0408] t is an integer from 1 to 10.

[0409] 28. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein E in -L- is selected from:

[0410] wherein s = an integer from 0 to 8.

[0411] 29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein -L- is selected from the following structures

[0412] 30. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 21-29, having an average DAR value of 2-10, such as 4-8, 6-8, or preferably 7-8.

[0413] 31. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 21-23, wherein the antibody-drug conjugate is selected from

[0414] wherein Ab is an antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-14, p is as defined in any one of embodiments 21-23;

[0415] Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8 or 6-8 or 7-8.

[0416] 32. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or an immunoconjugate of embodiment 19 or 20 or an antibody-drug conjugate of any one of embodiments 21-31 or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutical excipient.

[0417] 33. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or an immunoconjugate of embodiment 19 or 20 or an antibody-drug conjugate of any one of embodiments 21-31 or a pharmaceutically acceptable salt or solvate thereof, and another therapeutic agent.

[0418] 34. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or an immunoconjugate of embodiment 19 or 20 or an antibody-drug conjugate of any one of embodiments 21-31 or a pharmaceutically acceptable salt or solvate thereof, and another therapeutic agent.

[0419] 35. Use of an effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments 1 to 14 or an immunoconjugate of embodiment 19 or 20 or an antibody-drug conjugate of any one of embodiments 21-31 or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition of claim 32 in the manufacture of a medicament for preventing or treating a tumor in a subject or individual.

[0420] 36. The use of embodiment 35, wherein the tumor is a solid tumor or a hematological tumor, for example the tumor is a cancer, for example the cancer is selected from colon cancer, pancreatic cancer, lung cancer (e.g. non-small cell lung cancer), or gastric cancer (e.g. gastric adenocarcinoma).

[0421] 37. The use of embodiment 35 or 36, further comprising administering to the subject one or more other therapies, for example the therapies comprise a therapeutic modality and / or another therapeutic agent.

[0422] 38. A method of detecting integrin α3β1 in a sample, the method comprising

[0423] (a) contacting a sample with any of the anti- integrin α3β1 antibodies or antigen-binding fragments thereof of any of embodiments 1 to 14; and

[0424] (b) detecting the formation of a complex between the anti-integrin α3β1 antibody or antigen-binding fragment thereof and integrin α3β1; optionally, the anti-integrin α3β1 antibody is detectably labeled.

[0425] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Any or all of the features discussed in the above description can be combined in various embodiments of the application. In addition, materials, methods, and examples herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims. Examples:

[0426] Example 1 Mouse hybridoma screening, human-mouse chimeric antibody construction and FACS detection

[0427] After immunization of mice with SKOV3 and A375 cells, the spleen cells were fused with myeloma cells, and the hybridoma cells after fusion were obtained by two to three rounds of screening and cloning. After ELISA and FACS detection, five positive clones were finally obtained, and the sequence results were obtained after sequencing identification.

[0428] IgG antibody preparation

[0429] The coding nucleic acids of the light and heavy chain variable region sequences of the antibodies were respectively cloned into the expression vector pCDNA 3.4 containing the coding nucleic acids of the heavy chain constant region (SEQ ID NO: 71) or the light chain constant region (SEQ ID NO: 72) (see the table below for combination), and were co-transfected into CHO suspension cells for expression. The cell supernatant was collected and purified by Protein A column. The purified antibodies were detected by SDS-PAGE and HPLC-SEC for size and purity.

[0430] Detection of IgG antibody and cell binding activity

[0431] The tumor cells were trypsinized, and 2*10 5The cells were resuspended in 100 μΐ of U-bottom 96-well plates. Then, each antibody (chA28, chC8, chC18, chC22, chC47) was diluted with PBS, with 80 nM as the initial concentration, 3-fold dilution, and a total of 8 concentrations. 100 μΐ of the diluted antibody was added to the resuspended cells in the cell plate, which was incubated at 4°C for 1 h, and washed with PBS for 3 times. The secondary antibody Anti-Human IgG H&L (FITC, Abeam, ab6854) was diluted with PBS at 1:2000, 100 μΐ of the diluted secondary antibody was added to the resuspended cells in the cell plate, which was incubated at 4°C for 1 h, and washed with PBS for 3 times. 120 μΐ of PBS was added to resuspend the cells, and the binding signals were detected by FACS, and the data were analyzed by Graphpad.

[0432] The results are shown in Figure 1, and the five antibodies chA28, chC8, chC18, chC22, and chC47 can bind to HCT116 and AGS cells, and the EC50 values for binding to HCT116 cells are 1.45 nM, 4.60 nM, 2.74 nM, 2.27 nM, and 1.79 nM, respectively, and the EC50 values for binding to AGS cells are 1.28 nM, 4.04 nM, 0.84 nM, 1.23 nM, and 1.08 nM, respectively.

[0433] Table 1: Integrin α3β1 specific antibodies

[0434] Example 2: Finding the antibody target point by mass spectrometry

[0435] The cell lysate of PANC-1 (Coastbio, Cat: CBP60545) cells was subjected to CO-IP using the anti-α3β1 antibodies obtained in the above example, and 30 kD, 45 kD, 60 kD, and 150 kD bands were identified as significant bands compared with the PANC-1 cell blank group (Figure 2A). According to the results of the silver staining experiment, the protein bands of the PANC-1+ch-A28 group were significantly labeled compared with the blank group (blue arrow). The bands at 30 kD, 45 kD, 60 kD, and 150 kD correspond to different proteins in the mass spectrometry data, and the information in the MW [kDa] column can be viewed. The specific identification results are shown in the mass spectrometry data.

[0436] The entire lane was cut off for in-gel digestion, and mass spectrometry identification experiments were performed. After searching the secondary spectrum of the mass spectrometry results against the protein theoretical data library, 1248 specific peptides were matched. After searching and matching the peptides, 365 quantifiable proteins were identified. In Thermo Proteome Discoverer 2.5, four groups of data, Abundance Antibody, Log2; Abundance NegCtrl, Log2, were calculated for Abundance ratio, Log2: Antibody / NegCtrl, and significance analysis was performed, and 75 significantly different up-regulated proteins were analyzed, and venn analysis was performed, and two high PSM peptide segments, Integrin beta-1 and Integrin alpha-3, were identified (Figure 2B).

[0437] Example 3 Confirmation of target by gene silencing of HTC116 cells

[0438] HCT116 cells were trypsinized, and the cells were diluted to 1.25 million cells per milliliter with complete medium, and the cell suspension was ready for use.

[0439] Lipofectamine 3000 reagent (Therrno, L3000008) was used for siRNA transfection of cells. 100 nM siRNA (Gibco, sense (5'-3'): CCAGGAUGGAUUUCAGGAUTT, antisense (5'-3'): AUCCUGAAAUCCAUCCUGGTT) was incubated with Opti-MEM medium (Gibco, 31985070) containing Lipofectamine 3000 at room temperature for 15 minutes. Then, the siRNA complex was added to the cell plate, and 2 mL of cell suspension was co-cultured for 48 h.

[0440] After 48 h of siRNA transfection, the tumor cells were trypsinized, and 2 x 10 5Each antibody (chA28, chC8, chC22, chC47) was diluted with PBS to a concentration of 15 pg / mL. 100 pL of the diluted antibody was added to the resuspended cells in the cell plate, which was incubated at 4°C for 1 h, and then washed three times with PBS. The secondary antibody Anti-Human IgG H&L (FITC, Abeam, ab6854) and Anti-Mouse IgG H&L (FITC, Abeam, ab6785) were diluted with PBS at a ratio of 1:2000. 100 pL of the diluted secondary antibody was added to the resuspended cells in the cell plate, which was incubated at 4°C for 1 h, and then washed three times with PBS. 120 pL of PBS was added to resuspend the cells, and the binding signal was detected by FACS. FIG. 3A shows that the integrin a3 protein was down-regulated after siRNA treatment of HCT116. FIGS. 3B-3F show that the binding activity of the integrin a3 protein after down-regulation with the five test antibodies was all down-regulated, thereby determining that integrin a3 is the target protein.

[0441] Example 4 Preparation of integrin a3b1 antibody conjugated drugs

[0442] The antibodies chA28, chC8, and chC47 were reduced by adding an appropriate amount of reducing agent (TCEP-HCl, Thermo, CAS No.: 51805-45-9) to expose the sulfhydryl groups between the antibody chains. The Linker + payload (LD38, Shijian Bio, LD-38-20230612, synthesis see PCT / CN2023 / 106385 (WO2025 / 011419)) was dissolved and configured into a 10 mM solution with DMSO.

[0443] The dissolved LD38 was added according to a molar ratio of 10:1 between the antibody and the linker-payload, and a Michael addition reaction occurred between the sulfhydryl groups and the maleimide groups on the linker to conjugate the antibody and the LD38 together.

[0444] The mixture after conjugation was subjected to buffer exchange to remove residual LD38. The purity and DAR (drug / antibody ratio) values of the antibody conjugate after the reaction were determined by HPLC-SEC (volume exclusion chromatography) and HPLC-HIC (hydrophobic chromatography), respectively. The conjugation results of different candidate molecules are shown in Table 4, and the antibody conjugate drug detection method is shown in Table 5.

[0445] Table 4: Preparation of integrin a3b1 antibody conjugated drugs

[0446] Table 5: Detection method of ADC purity and DAR value

[0447] HPLC-SEC method:

[0448] HPLC-HIC method:

[0449] The obtained ADCs coupled with LD38, chA28-LD38, chC8-LD38 or chC47-LD38 are as follows:

[0450] wherein Ab is chA28, chC8 or chC47, respectively.

[0451] Example 5 In vivo efficacy of HCT116 CDX model

[0452] HCT116 human colon cancer cells (Nanjing Kebai, Cat: CBP60028) were used to construct a subcutaneous xenograft tumor model in BALB / c-nude mice (purchased from Shanghai Slek Experimental Animal Co., Ltd.) (Shanghai City Experimental Animal Quality Certificate: 20220004029470), and the in vivo efficacy of chA28-LD38 antibody drug conjugate was evaluated.

[0453] Specifically, logarithmic growth phase HCT116 cells were collected, the culture solution was removed and washed twice with PBS, then inoculated on the right side of the back of BALB / c-nude mice, with an inoculation amount of 10*10^6 / 200 μL / one (without adding matrix glue). After 11 days of inoculation, the tumor grew to an appropriate size, and the mice with tumor volume reaching the standard (average tumor volume ≈225 mm 3 ) were randomly divided into 2 groups, 6 animals in each group. The blank control group (DPBS only) and the chA28-LD38 group, respectively, and the vehicle was DPBS.

[0454] Drug administration started on the day of grouping (day 0), with an i.p. administration mode, a 5 mg / kg administration dose, and a once administration frequency on day 0 and day 7, respectively.

[0455] After starting the grouping and administration, the mouse body weight and tumor volume were measured 2-3 times per week. Each time, the mouse tumor size was measured using a vernier caliper, and the volume calculation formula was V=(LxW^2) / 2, where L was the longest diameter of the tumor, and W was the short diameter perpendicular to the longest diameter. On day 17 after grouping and administration, the experiment was terminated, and all mice were euthanized.

[0456] The results are shown in Figure 4, and the chA28-LD38 group had a significant tumor growth inhibition effect, and there was a statistical difference compared with the blank control group (p<0.0001).

[0457] The final data showed that the tumor volume of the blank control group was 1417.1 ± 118.0 mm 3 ; the tumor volume of the chA28-LD38 group was 180.2 ± 20.2 mm 3 . The above results show that the ADC drug chA28-LD38 has strong tumor inhibition activity.

[0458] Example 6 In vivo efficacy of PANC-1 CDX model

[0459] PANC-1 human pancreatic cancer cells (Nanjing Kebai, Cat: CBP60545) were used to construct a subcutaneous xenotransplant tumor model in BALB / c-nude mice (purchased from Shanghai Sylab Experimental Animal Co., Ltd.) (Shanghai City Experimental Animal Quality Certificate: 20220004027956), and the in vivo efficacy of the chA28-LD38 antibody drug conjugate was evaluated.

[0460] Specifically, PANC-1 cells in the logarithmic growth phase were collected, the culture solution was removed and washed twice with PBS, and then inoculated into the right dorsal part of BALB / c-nude mice at a dose of 10*10^6 / 200 μL / one (matrix glue and DPBS mixed at a ratio of 1:1). After 29 days of inoculation, the tumor grew to an appropriate size, and the mice with a tumor volume reaching the standard (average tumor volume ≈207 mm 3 ) were randomly divided into 2 groups, 4 animals in each group. The blank control group (only DPBS) and the chA28-LD38 group, respectively, and the solvent was DPBS.

[0461] Drug administration started on the day of grouping (day 0), with an i.p. administration mode, a dose of 5 mg / kg, and a frequency of administration on days 0, 7, and 14, respectively.

[0462] After starting the grouping and administration, the mouse body weight and tumor volume were measured 2-3 times per week. Each time, the mouse tumor size was measured using a vernier caliper, and the volume calculation formula was V = (L x W^2) / 2, where L was the longest diameter of the tumor, and W was the short diameter perpendicular to the longest diameter. On day 21 after grouping and administration, the experiment was terminated, and all mice were euthanized.

[0463] The results are shown in Figure 5, and the chA28-LD38 group had a significant tumor growth inhibition effect, and there was a statistically significant difference compared with the blank control group (p<0.0001).

[0464] The final data showed that the tumor volume of the blank control group was 550 ± 67.1 mm3; the tumor volume of the chA28-LD38 group was 17.6 ± 12.8 mm3. The above results show that the ADC drug chA28-LD38 has strong tumor inhibition activity.

[0465] Example 7 In vivo efficacy of HCC827 CDX model

[0466] HCC827 human non-small cell lung cancer cells (Nanjing Kebai, Cat: CBP60101) were used to construct a subcutaneous xenograft tumor model in BALB / c-nude mice (purchased from Jiangsu Jicui Yekang Biotechnology Co., Ltd.) (Jiangsu Province Experimental Animal Quality Certificate: A202309070025), and the in vivo efficacy of the chA28-LD38 antibody drug conjugate was evaluated.

[0467] Specifically, logarithmic growth phase HCC827 cells were collected, the culture solution was removed and washed twice with PBS, and then inoculated into the right dorsal of BALB / c-nude mice at a dose of 7*10^6 / 200 μL / one (without adding matrix glue). After 11 days of inoculation, the tumor grew to an appropriate size, and the mice with tumor volume reaching the standard (average tumor volume ≈ 158 mm 3 ) were randomly divided into 2 groups, 4 animals in each group. The blank control group (DPBS only) and the chA28-LD38 group, respectively, and the vehicle was DPBS.

[0468] Drug administration started on the day of grouping (day 0), with an i.p. administration mode, a dose of 5 mg / kg, and a frequency of administration of once on day 0 and day 6.

[0469] After starting the grouping and administration, the body weight and tumor volume of the mice were measured 2-3 times a week. Each time, the size of the mouse tumor was measured using a vernier caliper, and the volume was calculated using the formula V = (L x W^2) / 2, where L is the longest diameter of the tumor and W is the short diameter perpendicular to the longest diameter. On day 35 after grouping and administration, the experiment was terminated and all mice were euthanized.

[0470] The results are shown in Figure 6, and the chA28-LD38 group had a significant tumor growth inhibition effect, and there was a statistically significant difference compared with the blank control group (p<0.0001).

[0471] The final data showed that the tumor volume of the blank control group was 1636.4 ± 391.9 mm 3 , and the tumor volume of the chA28-LD38 group was 8.3 ± 4.8 mm 3 . The above results show that the ADC drug chA28-LD38 has strong tumor inhibition activity.

[0472] Example 8 In vivo efficacy of AGS CDX model

[0473] AGS human gastric adenocarcinoma cells (experimented by Nanjing Yunqiao Purui Biological Technology Co., Ltd.) were used to construct a subcutaneous xenograft tumor model in NCG mice (purchased from Jiangsu Jicai Yaoke Biological Technology Co., Ltd.) (Jiangsu Province Experimental Animal Quality Certificate: 20230003003295), and the in vivo efficacy of chA28-LD38 antibody drug conjugate was evaluated.

[0474] Specifically, logarithmic growth phase AGS cells were collected, the culture solution was removed and washed twice with PBS, then inoculated into the right dorsal of BALB / c-nude mice at a dose of 10*10^6 / 200 μL / mouse (without adding matrix glue). After 46 days of inoculation, the tumor grew to the appropriate size, and the mice with tumor volume reaching the standard (average tumor volume ≈230 mm 3 ) were randomly divided into 2 groups, 5 animals in each group. The blank control group (DPBS only) and the chA28-LD38 group, respectively, and the vehicle was DPBS.

[0475] Drug administration started on the day of grouping (day 0), with an i.p. administration mode, a 5 mg / kg dose, and a QW frequency, administered once on days 0, 7, 14, and 21. After the start of grouping and drug administration, the body weight and tumor volume of the mice were measured 2-3 times per week. The size of the mouse tumor was measured each time using a vernier caliper, and the volume was calculated using the formula V = (L x W^2) / 2, where L is the longest diameter of the tumor and W is the short diameter perpendicular to the longest diameter. On day 28 after grouping and drug administration, the experiment was terminated and all mice were euthanized.

[0476] The results are shown in Figure 7. The chA28-LD38 group had a significant tumor growth inhibition effect, and there was a statistically significant difference compared with the blank control group (p<0.0001).

[0477] The final data showed that the tumor volume of the blank control group was 1919.1 ± 399.0 mm3; the tumor volume of the chA28-LD38 group was 242.4 ± 34.4 mm 3 The above results show that the ADC drug chA28-LD38 has strong tumor inhibition activity.

[0478] Example 9 HCT116 CDX model in vivo efficacy

[0479] HCT116 human colon cancer cells (Nanjing Kebai, Cat: CBP60028) were used to construct a subcutaneous xenograft tumor model in BALB / c-nude mice (purchased from Shanghai Lingchang Biological Technology Co., Ltd.) (Shanghai City Experimental Animal Quality Certificate: 20230003005991), and the in vivo efficacy of chC8-LD38 and chC47-LD38 antibody drug conjugates was evaluated.

[0480] Specifically, the HCT116 cells in logarithmic growth phase were collected, the culture solution was removed and washed twice with PBS, and then inoculated into the right dorsal of BALB / c-nude mice at a dose of 10*10^6 / 200 μL / mouse (without adding Matrigel). After 9 days of inoculation, the tumors grew to an appropriate size, and the mice with tumor volume reaching the standard (average tumor volume ≈ 120 mm 3 ) were randomly divided into 3 groups, 5 animals in each group. The blank control group (DPBS only), chC8-LD38 and chC47-LD38 groups, respectively, and the solvent was DPBS.

[0481] The administration started on the day of grouping (day 0), and the administration mode was i.v., the administration dose was 3 mg / kg, and the administration frequency was once on day 0, day 7 and day 15, respectively.

[0482] The body weight and tumor volume of the mice were measured 2-3 times a week after the start of administration. The size of the mouse tumor was measured each time using a vernier caliper, and the volume calculation formula was V = (L x W^2) / 2, where L was the longest diameter of the tumor, and W was the short diameter perpendicular to the longest diameter. On day 21 after grouping and administration, the experiment was terminated, and all mice were euthanized.

[0483] The results are shown in Figure 8, and the administration groups chC8-LD38 and chC47-LD38 had significant tumor growth inhibition, and there were statistical differences compared with the blank control group (p < 0.0001).

[0484] The final data showed that the tumor volume of the blank control group was 1217.2 ± 193.9 mm 3 , and the tumor volumes of the chC8-LD38 and chC47-LD38 groups were 47.7 ± 12.3 mm 3 and 165.9 ± 45.8 mm 3 , respectively. The above results showed that the ADC drugs chC8-LD38 and chC47-LD38 both had strong tumor inhibition activity.

[0485] Example 10 Humanization of Antibody

[0486] The antibody humanization design was performed by classic CDR grafting and back mutation of key amino acids, which was briefly described as follows. First, the antibody germline with high sequence homology was selected to provide the antibody framework region (FR). The antigen binding fragment complementarity determining region (CDRs) in the murine variable region according to the Kabat naming method was transplanted to the former to form the humanized antibody variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To better ensure the physicochemical activity and drugability of the antibody, the antibody germline was preferably selected from the antibody sequence with high frequency in human body and verified by clinical tests. Second, to ensure better antibody affinity, the key amino acids of the human framework region were selected for back mutation. The mutation points were generally selected from the hydrophobic core residues located at the interface of VH-VL or the conservative amino acids that were close to CDR in space and possibly involved in antigen binding. The humanized antibody huA28-H2L3 of chA28 was obtained. For the convenience of naming, the humanized antibody huA28-H2L3 was abbreviated as huA28 in the subsequent experiments. The sequence is shown in the antibody sequence table.

[0487] The humanized antibody was prepared as described in Example 1 and its binding activity with cells was detected. The humanized antibody was constructed into IgG1, expressed in CHO-k1 cells, and subjected to humanized antibody binding activity determination with HCT116 and AGS cells.

[0488] The results are shown in Figure 9. The humanized antibody huA28-H2L3 of chA28 maintained the binding activity to human tumor cells HCT116 and AGS comparable to that of the chimeric antibody.

[0489] Example 11 Antibody affinity maturation improves binding to monkey overexpressed cells

[0490] Since the antigen binding activity of huA28 to monkey antigen was weaker than that to human antigen, the classic CDR amino acid single-point saturation mutation and combination mutation method was planned to improve its binding to monkey antigen overexpressed cells. The method is briefly described as follows. In the first round of single-point saturation mutation, first, 72 CDR amino acids of heavy and light chains were determined based on the Kabat rule. Except for cysteine, 18 different mutants were constructed for each single-point amino acid, totaling 1296. After transfection of CHO cells, the expression supernatant of different single-point mutants was screened with monkey overexpressed cells, and the single-point mutant with higher binding activity relative to huA28 was screened. In the second round, combination mutation was designed according to the potential single-point mutation. The corresponding antibodies were prepared as described in Example 1, and their binding activity to human and monkey integrin α3β1 overexpressed cells was detected. IgG1 antibodies were expressed in CHO cells, and the binding activity of the combination mutant antibodies to human and monkey integrin α3β1 overexpressed cells was evaluated by flow cytometry, respectively.

[0491] As a result, three affinity matured antibodies were identified that had comparable binding activity to human and monkey integrin α3β1 overexpressing cells (Table 6), designated huA28-comb4, huA28-comb7 and huA28-comb9, respectively. The sequences are detailed in the antibody sequence listing.

[0492] Table 6: huA28 affinity matured antibodies binding to human and monkey overexpressing cells

[0493] 293-human Integrin α3β1 cells were constructed by transfecting human integrin α3 (P26006) and β1 (P05556) into 293 cells;

[0494] 293-cyno Integrin α3β1 cells were constructed by transfecting cynomolgus monkey integrin α3 (A0A2K5VFM7) and β1 (A0A7N9D0D7) into 293 cells.

[0495] SEQUENCE LISTING

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds integrin α3β1, the antibody comprising 1) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 1, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 2; 2) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 15, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 16; 3) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 29, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 30; 4) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 43, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 44; 5) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 57, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 58; 6) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 73, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 74; 7) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 75, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 76; 8) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 84, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO: 85; or 9) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in a VH as set forth in SEQ ID NO: 89, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in a VL as set forth in SEQ ID NO:

90. For example, wherein the HCDRs and the LCDRs are determined according to the IMGT or Kabat scheme, respectively.

2. An antibody or antigen-binding fragment thereof that specifically binds integrin α3β1, the antibody comprising three complementarity determining regions of a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity determining regions of a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise or consist of the amino acid sequences set forth in the following table SEQ ID NO based on the IMGT scheme, respectively: or wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are based on the Kabat scheme and comprise or consist of the amino acid sequences set forth in the following table SEQ ID NO:

3. The antibody or antigen-binding fragment thereof that binds integrin a3b1 of claim 1 or 2, the antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 15, 29, 43, 57, 73, 75, 84, or 89.

4. The antibody or antigen-binding fragment thereof that binds integrin a3b1 of any one of claims 1-3, the antibody comprising a light chain variable region, wherein the light chain variable region comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 16, 30, 44, 58, 74, 76, 85, or 90.

5. The antibody or antigen-binding fragment thereof that binds integrin α3βl of any one of claims 1-4, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise or consist of the amino acid sequence set forth in Table SEQ ID NO:

6. The antibody or antigen-binding fragment thereof that binds integrin a3b1 of any one of claims 1-5, the antibody comprising a heavy chain constant region, e.g., a heavy chain constant region of (human) IgGl, IgG2, IgG3, or IgG4, e.g., a heavy chain constant region of human IgGl, e.g., the heavy chain constant region (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

71.

7. The antibody or antigen-binding fragment thereof that binds integrin a3b1 of any one of claims 1-6, the antibody comprising a light chain constant region, e.g., a (human) lambda or kappa light chain constant region, e.g., the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

72.

8. The antibody or antigen-binding fragment thereof that binds integrin a3b1 of any one of claims 1-7, the antibody comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 71; and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

72.

9. The antibody or antigen-binding fragment thereof that binds integrin alpha3beta1 of any one of claims 1-8, comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of a heavy chain variable region and a heavy chain constant region, and the light chain comprises or consists of a light chain variable region and a light chain constant region, wherein (i). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (ii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (iii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (iv). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (v). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (vi). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (vii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 76, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; (viii). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72; or (ix). the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71, and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO:

72.

10. The antibody or antigen-binding fragment thereof that binds integrin α3β1 of any one of claims 1-8, which has one or more of the following properties: (1) specifically binds integrin β1 and integrin α3, i.e., specifically binds integrin α3β1, e.g., human integrin α3β1; (2) is capable of binding integrin α3β1, e.g., human integrin α3β1, expressed on the surface of a cell membrane with high affinity; (3) is suitable for constructing an immunoconjugate (e.g., an antibody drug conjugate (ADC)) for targeted killing of integrin α3β1 -positive cells, e.g., integrin α3β1 -positive tumor cells; or for treating a tumor, e.g., a cancer; (4) inhibits the binding of any of the antibodies listed in claim 9 to integrin α3β1; (5) binds to the same or an overlapping epitope as any of the antibodies shown in claim 9; or (6) competes with any of the antibodies shown in claim 9 for binding to integrin α3β1.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody is a monoclonal antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody is a chimeric antibody or a humanized antibody.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antigen-binding fragment is an antibody fragment selected from a Fab, Fab', Fab'-SH, Fv, single-chain antibody scFv, (Fab')2 fragment, a single domain antibody, a diabody, or a linear antibody.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody is a bispecific antibody or a multispecific antibody.

15. An isolated nucleic acid encoding the anti- integrin α3β1 antibody or antigen-binding fragment thereof of any one of claims 1 to 14.

16. An expression vector comprising the nucleic acid of claim 15, for example the expression vector is a pCDNA vector, for example a pCDNA3.4 vector.

17. A host cell comprising the nucleic acid of claim 15 or the expression vector of claim 16, for example the host cell is prokaryotic or eukaryotic, for example the host cell is selected from an E. coli cell, a yeast cell, a mammalian cell or other cell suitable for making an antibody or antigen-binding fragment thereof, for example the host cell is a 293 cell or a CHO cell.

18. A method of making an anti-integrin α3β1 antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of claim 17 under conditions suitable for expression of the nucleic acid encoding the anti-integrin α3β1 antibody or antigen-binding fragment thereof of any one of claims 1 to 14, optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprises recovering the anti-integrin α3β1 antibody or antigen-binding fragment thereof from the host cell.

19. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 14 and another therapeutic agent, preferably the immunoconjugate is an antibody drug conjugate.

20. The immunoconjugate of claim 19, wherein the other therapeutic agent is a cytotoxic agent or an anti-tumor compound.

21. An antibody-drug conjugate of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, Ab-(L-(D) m ) p (I) wherein: Ab is the antibody or antigen-binding fragment thereof of any one of claims 1 to 14, L is a linker; D is a drug, preferably an anti-tumor compound or a cytotoxic agent; m is 1-6; and p is 1 to 16, for example an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

22. The antibody-drug conjugate of claim 21, or a pharmaceutically acceptable salt or solvate thereof, wherein D is selected from the group consisting of a microtubulin inhibitor (e.g. MMAE), a DNA cross-linking agent, a DNA alkylating agent, and a topoisomerase I inhibitor; for example, the topoisomerase I inhibitor is selected from the group consisting of camptothecin class compounds such as camptothecin and its derivatives, for example, camptothecin (CPT), hydroxycamptothecin, 9-aminocamptothecin, FL118, Dxd, topotecan, belotecan, lurtotecan, irinotecan, exatecan; or L-D is LD38.

23. The antibody-drug conjugate of claim 21 or 22, or a pharmaceutically acceptable salt or solvate thereof, having an average DAR of 2-10, for example 7-9 or 7-8.

24. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 21-23, wherein D is camptothecin or its derivative, having the following structure: wherein, R A selected from: hydrogen, C 1-6 alkyl, hydroxyC 1-6 alkyl, aminoC 1-6 alkyl; or wherein R B is selected from the group consisting of hydrogen, C 1-8 alkyl, C 1-8 haloalkyl, C 3-8 cycloalkyl, C 3-8 cycloalkyl C 1-4 alkyl, phenyl and phenyl C 1-4 alkyl; or wherein R is selected from H and optionally substituted C C alkyl; R is selected from H, C 1-6 alkyl; R is selected from H, C C alkyl; R is selected from H, C 1-6 alkyl-C(=O)- or optionally substituted heterocyclyl-C(=O)-; R is selected from H, C D alkyl; R is selected from H and optionally substituted C 1-6 alkyl; or wherein R E is selected from, for example, H, optionally substituted C 1-6 alkyl-C(=O)-, said substituent being selected from -OH or substituted or unsubstituted amino.

25. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 24, wherein D has the following structure:

26. The antibody-drug conjugate, or pharmaceutically acceptable salt or solvate thereof, of any one of claims 21-23, wherein D is monomethyl auristatin E (MMAE) of the formula:

27. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claims 21-26, wherein -L- has the structure: -Z-L 1- E-L2-L3- wherein Z is attached to Ab, and L3 is attached to D; Z is selected from L1is -(CH2) n -C(=O)-, -(CH2) n -C(=O)-NH-(CH2) n -C(=O)-, -(CH2) n -(O(CH2)2) t (CH2) n -C(=O)- or -(CH2) n -C(=O)-NH-(CH2) n -(O(CH2)2) t (CH2) n -C(=O)-; E is absent or a peptide residue comprising 2-10 amino acid residues, wherein the amino acid residues are natural or unnatural amino acid residues and are optionally substituted, and the C-terminus of the peptide residue is covalently linked to L2; preferably, E is selected from Lys, Gly, Asp, Asn, Glu, Gin, Val-Cit, Val-Ala, Ala-Ala, Gly-Glu, Glu-Gly, Gly-Asp, Gly-Asn, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Asp-Gly, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Phe-Gly, (Gly)2-Glu-Gly, or any combination of fragments of the above; preferably, E is selected from Val-Cit, Ala-Ala, Gly-Glu, Glu-Gly, Asn-Asn, Asn-Asn-Gly, Asp-Glu, Gly-Glu-Gly, Glu-Gly-Gly, Lys-Ala-Ala, (Gly)2-Glu-Gly, (Gly)2-Phe-Gly; L2 is absent or is L3 is absent or -C(=0)-*, wherein * indicates that the terminal end is covalently linked to D; n is an integer from 0 to 10, t is an integer from 1 to 10.

28. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 27, wherein, E in said -L- is selected from: wherein s is an integer from 0 to 8.

29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof of claim 27, wherein, said -L- is selected from the group consisting of 30. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 21-29, having an average DAR value of 2-10, such as 4-8, 6-8, or preferably 7-8.

31. The antibody-drug conjugate, or pharmaceutically acceptable salt or solvate thereof, of any one of claims 21-23, wherein the antibody-drug conjugate is selected from wherein Ab is an antibody or antigen-binding fragment thereof as defined in any one of claims 1-14, and p is as defined in any one of claims 21-23; Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8 or 6-8 or 7-8.

32. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an immunoconjugate according to claim 19 or 20 or an antibody-drug conjugate according to any one of claims 21-31 or a pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutical excipient.

33. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an immunoconjugate according to claim 19 or 20 or an antibody-drug conjugate according to any one of claims 21-31 or a pharmaceutically acceptable salt or solvate thereof, and another therapeutic agent.

34. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or an immunoconjugate according to claim 19 or 20 or an antibody-drug conjugate according to any one of claims 21-31 or a pharmaceutically acceptable salt or solvate thereof, and another therapeutic agent.

35. Use of an effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1 to 14 or immunoconjugate of claim 19 or 20 or antibody-drug conjugate of any one of claims 21-31, or a pharmaceutically acceptable salt or solvate thereof or pharmaceutical composition of claim 32 in the manufacture of a medicament for preventing or treating a tumor in a subject or individual in a subject.

36. The use of claim 35, wherein the tumor is a solid tumor or a hematological tumor, for example the tumor is a cancer, for example the cancer is selected from colon cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), or gastric cancer (e.g., gastric adenocarcinoma).

37. The use of claim 35 or 36, further comprising administering one or more other therapies to the subject, for example the therapies comprise a therapeutic modality and / or other therapeutic agent.

38. A method of detecting integrin α3β1 in a sample, the method comprising (a) contacting the sample with any anti-integrin α3β1 antibody or antigen-binding fragment thereof of any one of claims 1 to 14; and (b) detecting the formation of a complex between the anti-integrin α3β1 antibody or antigen-binding fragment thereof and integrin α3β1; optionally, the anti-integrin α3β1 antibody is detectably labeled.

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