ITGβ6-binding protein and use thereof

Abstract

The present invention relates to an ITGβ6-binding protein and the use thereof, and specifically relates to an anti-ITGβ6 antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding same, and a method for preparing same. The anti-ITGβ6 antibody or antigen-binding fragment thereof has high specificity and high affinity for ITGβ6. The present invention further relates to the use of the antibody or antigen-binding fragment thereof in the treatment and diagnosis of diseases.

Description

ITGβ6 binding proteins and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of therapeutic monoclonal antibodies, and more particularly, the present application relates to an antibody against ITGβ6; also relates to the use of the antibody in the treatment and diagnosis of diseases. BACKGROUND

[0002] The integrin superfamily is a class of cell surface transmembrane receptors that mainly recognize extracellular matrix ligands and cell surface ligands (also bind some soluble ligands), mediate cell adhesion and cell movement, establish transmembrane connection with the cytoskeleton, and transmit bidirectional signals between intracellular and extracellular environments. Integrins must form a heterodimer by non-covalent binding of two subunits, alpha and beta, to function. At least 18 alpha subunits and 8 beta subunits have been found in humans, with a total of 24 combinations, each with different ligand specificity, tissue cell distribution, and physiological function. The integrin β6 (ITGβ6) subunit only binds to the αV subunit, and both αV and β6 belong to single transmembrane proteins, consisting of an extracellular domain (head + mobile leg), a transmembrane segment, and a cytoplasmic tail. αVβ6 is only expressed in certain tissue remodeling-related epithelial cells, and αVβ6 performs tissue remodeling by binding to various ligands, plays a role in embryonic development and various physiological and pathological repair events, including: (1) binding to extracellular matrix (ECM) molecules such as fibronectin, tenascin-C, and vitronectin, etc., mediating cell adhesion and migration; (2) locally activating transforming growth factor (TGF-β1), playing an anti-inflammatory monitoring role, maintaining epithelial cell quiescence, and feedback regulating β6 expression. The pathological function of αVβ6 is to utilize the tissue remodeling function to promote tumor cell survival, proliferation, invasion, and metastasis, induce tumor cell resistance to chemotherapy / radiotherapy, and is an adverse prognostic indicator for various tumors, including esophageal cancer, head and neck squamous cell carcinoma, oral squamous cell carcinoma, bladder cancer, breast cancer, gastric cancer, colorectal cancer, cholangiocarcinoma, non-small cell lung cancer, pancreatic cancer, and cervical cancer, etc., and plays a key role in promoting tumor progression and metastasis in the tumor microenvironment.

[0003] Currently, there is no antibody drug targeting ITGβ6 on the market, therefore, it is urgent and necessary to develop an antibody targeting ITGβ6 with higher specificity, lower toxicity and side effects, better clinical efficacy, and more convenient administration, which will provide patients with more medication options. SUMMARY

[0004] In the present application, the inventors have developed high-affinity fully human antibodies with excellent properties, which can specifically recognize / bind ITGβ6, do not bind or substantially do not bind ITGβ1, ITGβ3, ITGβ5, ITGβ8 and / or ITGαV, have good hydrophilicity, and can be efficiently endocytosed into ITGβ6-expressing cells, and can be used in the corresponding therapeutic and diagnostic fields. Thus, the following inventions are completed.

[0005] Antibodies of the present application

[0006] In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to ITGβ6, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0007] (a) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 62, 63, 61 or 3; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 4;

[0008] (b) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 1; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 58, 59, 60 or 2;

[0009] (c) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 5; and / or CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 6;

[0010] or

[0011] (d) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) and / or CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL), wherein the heavy chain variable region (VH) and / or light chain variable region (VL) comprises at least one mutation in one or more CDRs compared to the heavy chain variable region and / or light chain variable region as set forth in any one of (a)-(c), the mutation being a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution;

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0013] (i) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 62; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0014] (ii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 63; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0015] (iii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 61; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0016] (iv) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 3; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0017] (i) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 58;

[0018] (vi) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 59;

[0019] (vii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 60;

[0020] (i) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 2; or

[0021] (ii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 3; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 4; or

[0022] (iii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 5; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 6; or

[0023] (iv) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 7; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 8; or

[0024] (v) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 9; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 10; or

[0025] (vi) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 11; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 12; or

[0026] (vii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 13; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 14; or

[0027] (viii) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) of SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) of SEQ ID NO: 2; or

[0028] (c) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 61; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0029] (d) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 3; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 4;

[0030] (e) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 58;

[0031] (f) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 59;

[0032] (g) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 60;

[0033] (h) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 2; or

[0034] (i) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 5; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) set forth in SEQ ID NO: 6.

[0035] In certain embodiments, the ITGβ6 comprises human ITGβ6 and / or monkey ITGβ6. In certain embodiments, the monkey is Macaca fascicularis.

[0036] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0037] (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0038] (1b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof; CDR-H2 of SEQ ID NO: 14 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or

[0039] (1c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof; CDR-H2 of SEQ ID NO: 40 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof;

[0040] wherein the variant of any one of (1a), (1b), (1c) has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0041] In certain embodiments, an antibody of the application or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0042] (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 48 or a variant thereof; CDR-H2 of SEQ ID NO: 49 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0043] (2b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 46 or a variant thereof; CDR-H2 of SEQ ID NO: 47 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or

[0044] (2c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 50 or a variant thereof; CDR-H2 of SEQ ID NO: 51 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof;

[0045] wherein the variant of any one of (2a), (2b), (2c) has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0047] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 31 or a variant thereof; CDR-H2 of SEQ ID NO: 32 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0048] (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or

[0049] (3c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 44 or a variant thereof; CDR-H2 of SEQ ID NO: 45 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof;

[0050] wherein the variant of any one of (3a), (3b), (3c) has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0051] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0052] (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof; CDR-H2 of SEQ ID NO: 21 or a variant thereof; CDR-H3 of SEQ ID NO: 70, 72, 68, or 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof; CDR-L2 of SEQ ID NO: 24 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof;

[0053] (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 7 or a variant thereof; CDR-H2 of SEQ ID NO: 8 or a variant thereof; CDR-H3 of SEQ ID NO: 9 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 10 or a variant thereof; CDR-L2 of SEQ ID NO: 11 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or

[0054] (4c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof; CDR-H2 of SEQ ID NO: 34 or a variant thereof; CDR-H3 of SEQ ID NO: 35 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 36 or a variant thereof; CDR-L2 of SEQ ID NO: 37 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof;

[0055] wherein the variant of any one of (4a), (4b), (4c) has one or several (e.g. 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0056] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the Chothia numbering system:

[0057] (1a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 26; CDR-H2 of SEQ ID NO: 27; and CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; and CDR-L3 of SEQ ID NO: 25;

[0058] (1b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 13; CDR-H2 of SEQ ID NO: 14; and CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; and CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or

[0059] (1c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 39; CDR-H2 of SEQ ID NO: 40; and CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; and CDR-L3 of SEQ ID NO: 38.

[0060] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system:

[0061] (2a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 48; CDR-H2 of SEQ ID NO: 49; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; CDR-L3 of SEQ ID NO: 25;

[0062] (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 46; CDR-H2 of SEQ ID NO: 47; CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or

[0063] (2c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 50; CDR-H2 of SEQ ID NO: 51; CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; CDR-L3 of SEQ ID NO: 38.

[0064] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the Kabat numbering system:

[0065] (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 31; CDR-H2 of SEQ ID NO: 32; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; CDR-L3 of SEQ ID NO: 25;

[0066] (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 18; CDR-H2 of SEQ ID NO: 19; CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or

[0067] (3c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 44; CDR-H2 of SEQ ID NO: 45; CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; CDR-L3 of SEQ ID NO: 38.

[0068] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0069] (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 20; CDR-H2 of SEQ ID NO: 21; CDR-H3 of SEQ ID NO: 70, 72, 68, or 22; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23; CDR-L2 of SEQ ID NO: 24; CDR-L3 of SEQ ID NO: 25;

[0070] (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; CDR-H3 of SEQ ID NO: 9; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 10; CDR-L2 of SEQ ID NO: 11; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or

[0071] (4c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 33; CDR-H2 of SEQ ID NO: 34; and CDR-H3 of SEQ ID NO: 35; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 36; CDR-L2 of SEQ ID NO: 37; and CDR-L3 of SEQ ID NO: 38.

[0072] In certain embodiments, the antibody or antigen-binding fragment thereof of the application comprises:

[0073] (a) a VH comprising a sequence as set forth in SEQ ID NO: 62, 63, 61 or 3, and / or a VL comprising a sequence as set forth in SEQ ID NO: 4, or a variant thereof;

[0074] (b) a VH comprising a sequence as set forth in SEQ ID NO: 1, or a variant thereof, and / or a VL comprising a sequence as set forth in SEQ ID NO: 58, 59, 60 or 2, or a variant thereof; or

[0075] (c) a VH comprising a sequence as set forth in SEQ ID NO: 5, or a variant thereof, and / or a VL comprising a sequence as set forth in SEQ ID NO: 6, or a variant thereof;

[0076] wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or one or several (e.g. 1, 2, 3, 4 or 5) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; preferably said substitutions are conservative substitutions.

[0077] In certain embodiments, the antibody or antigen-binding fragment thereof of the application comprises:

[0078] (a) a VH comprising a sequence as set forth in SEQ ID NO: 62, and a VL comprising a sequence as set forth in SEQ ID NO: 4;

[0079] (b) a VH comprising a sequence as set forth in SEQ ID NO: 63, and a VL comprising a sequence as set forth in SEQ ID NO: 4;

[0080] (c) a VH comprising a sequence as set forth in SEQ ID NO: 61, and a VL comprising a sequence as set forth in SEQ ID NO: 4;

[0081] (d) a VH comprising a sequence as set forth in SEQ ID NO: 3 and a VL comprising a sequence as set forth in SEQ ID NO: 4;

[0082] (e) a VH comprising a sequence as set forth in SEQ ID NO: 1 and a VL comprising a sequence as set forth in SEQ ID NO: 58;

[0083] (f) a VH comprising a sequence as set forth in SEQ ID NO: 1 and a VL comprising a sequence as set forth in SEQ ID NO: 59;

[0084] (g) a VH comprising a sequence as set forth in SEQ ID NO: 1 and a VL comprising a sequence as set forth in SEQ ID NO: 60;

[0085] (h) a VH comprising a sequence as set forth in SEQ ID NO: 1 and a VL comprising a sequence as set forth in SEQ ID NO: 2; or

[0086] (i) a VH comprising a sequence as set forth in SEQ ID NO: 5 and a VL comprising a sequence as set forth in SEQ ID NO: 6.

[0087] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen, and can include post-translational modifications thereof (e.g., C-terminal lysine clipping in the heavy chain, conversion of N-terminal glutamine or glutamic acid to pyroglutamic acid or pyroglutamate in the heavy chain or light chain), which can occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0088] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain can comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof can be cyclized to pyroglutamic acid. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof can be cyclized to pyroglutamate.

[0089] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0090] In certain embodiments, provided herein are compositions comprising an antibody or antigen binding fragment disclosed herein, wherein the various antibodies or antigen binding fragments can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid, or an N-terminal amino acid cyclized to pyroglutamate.

[0091] In certain embodiments, the N-terminal glutamine of the VH comprising a sequence as set forth in any one of SEQ ID NOs: 62, 63, 61, 3, 1, 5, or a variant thereof, is cyclized to form pyroglutamic acid or pyroglutamate.

[0092] In certain embodiments, the antibody or antigen binding fragment thereof described in any of the above embodiments further has a characteristic selected from the group consisting of:

[0093] (1) specifically binds human or monkey ITGβ6, e.g., as determined by ELISA or flow cytometry;

[0094] (2) specifically binds human or monkey αVβ6, e.g., as determined by ForteBio assay;

[0095] (3) does not bind αVβ1, αVβ3, αVβ5, ITGβ8, and ITGαV, e.g., as determined by flow cytometry;

[0096] (4) has reduced or ablated ADCC activity;

[0097] (5) has reduced CDC activity;

[0098] (6) induces ITGβ6 internalization, e.g., as determined by flow cytometry;

[0099] (7) inhibits cell (e.g., tumor cell) proliferation; and / or

[0100] (8) inhibits tumor growth.

[0101] In certain embodiments, the antibody or antigen binding fragment thereof described in any of the above embodiments can comprise a constant region from or derived from a human immunoglobulin.

[0102] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4). In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or a mutated or chemically modified Fc region that has altered effector function (e.g., reduced ADCC activity and / or CDC activity) compared to the wild-type Fc region. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof of the application comprises a variant of a human IgGl heavy chain constant region that has the following substitutions compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala, and Gly237Ala (positions according to the EU numbering system). In such embodiments, the antibody or antigen-binding fragment thereof of the application has reduced ADCC activity and / or CDC activity. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a human IgGl heavy chain constant region as set forth in SEQ ID NO: 76. In certain embodiments, the heavy chain constant region (CH) as set forth in SEQ ID NO: 76, or variant thereof, lacks the C-terminal lysine. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 76, or variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) compared thereto. In certain embodiments, the heavy chain constant region (CH) as set forth in SEQ ID NO: 76, or variant thereof, lacks the C-terminal lysine.

[0103] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region from or derived from a human immunoglobulin (e.g., kappa or lambda). In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 73, or variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) compared thereto. In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 74, or variant thereof having up to 20 conservative substitutions (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) compared thereto.

[0104] In certain embodiments, the antibody or antigen-binding fragment thereof of the application comprises:

[0105] (1) a heavy chain comprising a VH of SEQ ID NO: 62 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 4 and a light chain constant region (CL) of SEQ ID NO: 74;

[0106] (2) a heavy chain comprising a VH of SEQ ID NO: 63 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 4 and a light chain constant region (CL) of SEQ ID NO: 74;

[0107] (3) a heavy chain comprising a VH of SEQ ID NO: 61 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 4 and a light chain constant region (CL) of SEQ ID NO: 74;

[0108] (4) a heavy chain comprising a VH of SEQ ID NO: 3 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 4 and a light chain constant region (CL) of SEQ ID NO: 74;

[0109] (5) a heavy chain comprising a VH of SEQ ID NO: 1 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 58 and a light chain constant region (CL) of SEQ ID NO: 73;

[0110] (6) a heavy chain comprising a VH of SEQ ID NO: 1 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 59 and a light chain constant region (CL) of SEQ ID NO: 73;

[0111] (7) a heavy chain comprising a VH of SEQ ID NO: 1 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 60 and a light chain constant region (CL) of SEQ ID NO: 73;

[0112] (8) a heavy chain comprising a VH of SEQ ID NO: 1 and a heavy chain constant region (CH) of SEQ ID NO: 77, and, a light chain comprising a VL of SEQ ID NO: 2 and a light chain constant region (CL) of SEQ ID NO: 73; or

[0113] (9) a heavy chain comprising VH as set forth in SEQ ID NO: 5 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising VL as set forth in SEQ ID NO: 6 and a light chain constant region (CL) as set forth in SEQ ID NO: 74.

[0114] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application comprises:

[0115] (1) a heavy chain having a sequence as set forth in SEQ ID NO: 78 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0116] (2) a heavy chain having a sequence as set forth in SEQ ID NO: 80 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0117] (3) a heavy chain having a sequence as set forth in SEQ ID NO: 87 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0118] (4) a heavy chain having a sequence as set forth in SEQ ID NO: 88 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0119] (5) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 89;

[0120] (6) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 90;

[0121] (7) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 91;

[0122] (8) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 92; or

[0123] (9) a heavy chain having a sequence as set forth in SEQ ID NO: 94 and a light chain having a sequence as set forth in SEQ ID NO: 95.

[0124] In certain embodiments, the N-terminal glutamine of the heavy chain having a sequence as set forth in any one of SEQ ID NOs: 78, 80, 87, 88, 93, 94 is subjected to cyclization to form pyroglutamic acid or a pyroglutamate salt.

[0125] In certain embodiments, the C-terminal lysine of the heavy chain having a sequence as set forth in any one of SEQ ID NOs: 78, 80, 87, 88, 93, 94 is lacking.

[0126] In certain embodiments, the N-terminal glutamine of the heavy chain having a sequence as set forth in any one of SEQ ID NOs: 78, 80, 87, 88, 93, 94 is cyclized to form pyroglutamic acid or a salt thereof, and the C-terminal lysine thereof is lacking.

[0127] In certain embodiments, the antibody of the present application comprises:

[0128] (1) a heavy chain having a sequence as set forth in SEQ ID NO: 81 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0129] (2) a heavy chain having a sequence as set forth in SEQ ID NO: 82 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0130] (3) a heavy chain having a sequence as set forth in SEQ ID NO: 83 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0131] (4) a heavy chain having a sequence as set forth in SEQ ID NO: 84 and a light chain having a sequence as set forth in SEQ ID NO: 79;

[0132] (5) a heavy chain having a sequence as set forth in SEQ ID NO: 85 and a light chain having a sequence as set forth in SEQ ID NO: 79; or

[0133] (6) a heavy chain having a sequence as set forth in SEQ ID NO: 86 and a light chain having a sequence as set forth in SEQ ID NO: 79.

[0134] In certain embodiments, the antibody or antigen-binding fragment thereof of any of the above embodiments is a murine, chimeric, humanized antibody.

[0135] In certain embodiments, the variable region of the antibody or antigen-binding fragment thereof of any of the above embodiments is human.

[0136] In certain embodiments, the antibody or antigen-binding fragment thereof of any of the above embodiments is selected from the group consisting of a ScFv, a Fab, a Fab', a F(ab')2, a Fab'-SH, a Fv fragment, a disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0138] The present application also provides the use of the antibody or antigen-binding fragment provided herein or a pharmaceutical composition thereof for tumor treatment.

[0139] Derivatized antibodies

[0140] The antibodies or antigen-binding fragments thereof of the present application can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization of the antibodies or antigen-binding fragments thereof (e.g., labeling) will not adversely affect its binding to ITGβ6, in particular human ITGβ6. Thus, the antibodies or antigen-binding fragments thereof of the present application are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present application can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to produce a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide that can mediate association of the antibody or antigen-binding fragment with another molecule (e.g., an avidin or polyhistidine tag).

[0141] As one of the derivatives of the antibody, the present application provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present application and a conjugating moiety.

[0142] In certain embodiments, the conjugating moiety is selected from a detectable label. The detectable label according to the present application can be any substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein (FITC), rhodamine, tetramethylrhodamine (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads (e.g., Dynal beads), biotin, digoxigenin, or a combination thereof. ), a calorimetric label such as gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, such labels can be suitable for use in immunoassays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In certain embodiments, the detectable label as described above can be attached to the antibody of the present application or antigen-binding fragment thereof via linkers of varying lengths to reduce potential steric hindrance.

[0143] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an antitumor agent, such as a cytotoxic agent, a cytokine, a toxin, or a radionuclide.

[0144] In certain embodiments, the conjugated moiety is selected from a substance that can improve the biological properties of the antibody, such as increasing the serum half-life, for example, can be a chemical group, such as polyethylene glycol (PEG), methyl or ethyl, or a sugar group.

[0145] As one of the derivatives of the antibody, the present application provides a multispecific antibody comprising the antibody of the present application or antigen-binding fragment thereof.

[0146] In certain embodiments, the multispecific antibody comprises the antibody of the present application or antigen-binding fragment thereof as a first antigen-binding domain, and further comprises at least one second antigen-binding domain against other targets.

[0147] In certain embodiments, each antigen-binding domain of the multispecific antibody retains the respective original binding specificity.

[0148] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0149] As one of the derivatives of the antibody, the present application provides a chimeric antigen receptor comprising the antibody of the present application or antigen-binding fragment thereof. In certain embodiments, the chimeric antigen receptor comprises the antibody of the present application or antigen-binding fragment thereof (e.g., ScFv) as an extracellular antigen-binding domain that specifically binds to ITGβ6, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present application also provides a host cell (e.g., an immune cell such as a T lymphocyte, an NK cell, a DC cell, a macrophage) containing or expressing the chimeric antigen receptor.

[0150] Preparation of antibodies

[0151] The antibody of the present application can be prepared in various methods known in the art, for example, by obtaining a DNA molecule encoding the heavy and light chain genes of the antibody of the present application through genetic engineering recombination techniques. For example, a DNA molecule encoding the heavy and light chain genes of the antibody of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then a host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed.

[0152] Antigen-binding fragments of the present application can be obtained by proteolytic digestion of intact antibodies (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992); Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can be directly isolated from the culture medium of recombinant host cells. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0153] Accordingly, in another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present application or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In certain embodiments, the nucleotide sequence is codon-degenerate according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0154] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding an antibody heavy chain variable region has a sequence selected from the group consisting of:

[0155] (a) a nucleotide sequence as set forth in SEQ ID NO: 54, 52, or 56, or

[0156] (b) a sequence substantially identical to the nucleotide sequence recited in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity to the nucleotide sequence recited in (a), or a sequence having one or more nucleotide substitutions), or

[0157] (c) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence recited in (a); and / or,

[0158] the nucleic acid molecule encoding the antibody heavy chain variable region has a sequence selected from the group consisting of:

[0159] (d) the nucleotide sequence set forth in SEQ ID NO: 54, or

[0160] (e) a sequence substantially identical to the nucleotide sequence recited in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity to the nucleotide sequence recited in (d), or a sequence having one or more nucleotide substitutions), or

[0161] (f) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence recited in (d).

[0162] In certain embodiments, the nucleic acid molecule encoding the antibody heavy chain variable region and the nucleic acid molecule encoding the antibody light chain variable region have nucleotide sequences as follows:

[0163] (1) the nucleic acid molecule encoding the antibody heavy chain variable region has the nucleotide sequence set forth in SEQ ID NO: 54, and / or the nucleic acid molecule encoding the antibody light chain variable region has the nucleotide sequence set forth in SEQ ID NO: 55;

[0164] (2) the nucleic acid molecule encoding the antibody heavy chain variable region has the nucleotide sequence set forth in SEQ ID NO: 52, and / or the nucleic acid molecule encoding the antibody light chain variable region has the nucleotide sequence set forth in SEQ ID NO: 53; or

[0165] (3) the nucleic acid molecule encoding the antibody heavy chain variable region has the nucleotide sequence set forth in SEQ ID NO: 56, and / or the nucleic acid molecule encoding the antibody light chain variable region has the nucleotide sequence set forth in SEQ ID NO: 57.

[0166] In another aspect of the application, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present application. In certain embodiments, the vector of the present application is, e.g., a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present application in vivo in a subject (e.g., a mammal, e.g., a human).

[0167] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of the present application and a second nucleotide sequence encoding the light chain or light chain variable region thereof, wherein the first and second nucleotide sequences are present on the same or different vectors. When the first and second nucleotide sequences are present on different vectors, the vector of the present application comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0168] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application can be used to construct a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain (e.g., ScFv) that specifically binds to ITGβ6, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the isolated nucleic acid molecule of the present application can comprise a nucleotide sequence encoding a chimeric antigen receptor further comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof (e.g., ScFv) of the present application. In certain embodiments, the isolated nucleic acid molecule of the present application encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of the antibody of the present application.

[0169] In certain embodiments, the antibody or antigen-binding fragment thereof of the present application can be used to construct a chimeric antigen receptor-modified immune cell comprising a chimeric antigen receptor (CAR) and an immune cell (e.g., a T lymphocyte, an NK cell, a DC cell, a macrophage).

[0170] In another aspect of the application, the present application provides a host cell comprising the isolated nucleic acid molecule of the present application or the vector of the present application. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present application is a mammalian cell, e.g., CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, CHO-EBNA).

[0171] In certain embodiments, the host cell of the present application can be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule comprised by the host cell can comprise a nucleotide sequence encoding a chimeric antigen receptor, which further comprises a nucleotide sequence encoding an antibody or antigen binding fragment thereof (e.g., ScFv) of the present application. In certain embodiments, the isolated nucleic acid molecule comprised by the host cell encodes a chimeric antigen receptor comprising an antigen binding fragment (e.g., ScFv) of an antibody of the present application.

[0172] In another aspect of the present application, a method of making an antibody or antigen binding fragment thereof of the present application is provided, which comprises culturing a host cell of the present application under conditions permitting expression of the antibody or antigen binding fragment thereof, and recovering the antibody or antigen binding fragment thereof from the cultured host cell culture.

[0173] In certain embodiments, the host cell used in the method of making is a Chinese hamster ovary cell.

[0174] In certain embodiments, the present application provides an antibody or antigen binding fragment thereof obtainable by the aforementioned method of making.

[0175] Therapeutic applications

[0176] In another aspect of the present application, a pharmaceutical composition comprising an antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor of the present application, and a pharmaceutically acceptable carrier and / or excipient is provided.

[0177] In certain embodiments, the pharmaceutical composition of the present application comprises an antibody or antigen binding fragment thereof of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0178] In certain embodiments, the pharmaceutical compositions of the present application comprise a vector or host cell of the present application, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule comprised by the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, which further comprises a nucleotide sequence encoding an antibody or antigen binding fragment thereof (e.g., ScFv) of the present application; and the host cell comprises an isolated nucleic acid molecule or vector as previously described. In certain embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen binding fragment (e.g., ScFv) of an antibody of the present application. In certain embodiments, the host cell is an immune cell, such as a T cell. In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0179] In certain embodiments, the pharmaceutical compositions can further comprise an additional pharmaceutically active agent. In certain embodiments, the additional pharmaceutically active agent is an agent having anti-tumor activity. In certain embodiments, the additional pharmaceutically active agent is selected from an ITGβ6 inhibitor, a TROP2 inhibitor, a B7H3 inhibitor, a PTK7 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof. In certain embodiments, the antibody or antigen binding fragment thereof of the present application is provided as an independent component or as a mixed component with the additional pharmaceutically active agent. Thus, the antibody or antigen binding fragment thereof of the present application and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0180] In certain embodiments, the antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor of the present application in the pharmaceutical compositions of the present application is sufficient to (e.g., in a subject):

[0181] (a) inhibit cell (e.g., tumor cell) proliferation;

[0182] (b) inhibit tumor growth;

[0183] (c) inhibit ITGβ6 and / or αVβ6-mediated signaling;

[0184] (d) induce ITGβ6 internalization;

[0185] (e) treat an ITGβ6 and / or αVβ6-mediated disease / disorder; or

[0186] (f) any combination of (a)-(e).

[0187] In certain embodiments, the ITGβ6 and / or αVβ6 mediated disease / condition is a tumor, e.g., a tumor expressing ITGβ6 and / or αVβ6. In certain embodiments, the tumor is selected from colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or cholangiocarcinoma. In certain embodiments, the tumor is selected from non-small cell lung cancer lung cancer, head and neck squamous carcinoma, bladder cancer, esophageal cancer, pancreatic cancer, cervical cancer.

[0188] In another aspect of the present application, there is provided use of an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition of the present application in the manufacture of a medicament for treating and / or adjuvant treating a tumor.

[0189] In another aspect of the present application, there is provided a method of inhibiting cell proliferation, comprising contacting the cell with an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition of the present application. In certain embodiments, the cell is an ITGβ6 expressing cell, e.g., a tumor cell. In certain embodiments, the method is performed in vitro.

[0190] In another aspect of the present application, there is provided a method for treating and / or adjuvant treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition of the present application.

[0191] In certain embodiments, the method further comprises administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nano therapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy can be applied simultaneously, separately or sequentially with the above method.

[0192] In any of the above embodiments, the tumor to which the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present application is directed to can be any tumor type. In certain embodiments, the tumor to which the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present application is directed to is an ITGβ6-positive tumor. In certain embodiments, the tumor to which the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present application is directed to is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or cholangiocarcinoma. In certain embodiments, the tumor is selected from the group consisting of non-small cell lung cancer lung cancer, head and neck squamous carcinoma, bladder cancer, esophageal cancer, pancreatic cancer, cervical cancer.

[0193] The antibody or antigen-binding fragment thereof of the present application, the pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the antibody of the present application in the required amount in an appropriate solvent with one or more of the other ingredients enumerated above, as desired. The sterile injection solutions can be prepared as a solution, suspension, or emulsion. The pH and exact constitution of the solution are selected to ensure the solubility of the active ingredient and to preserve the sterility of the solution. The sterile injection solutions can be filtered, for example, through a bacterial-retaining filter, prior to, or following, the addition of the various ingredients to the solution. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for reconstitution with a suitable carrier, for example, sterile pyrogen-free water, prior to use. Such sterile lyophilized powders can be dispensed in unit or multi-dose sealed containers, for example, ampules or vials.

[0194] In addition, the antibody or antigen-binding fragment thereof of the present application can be present in unit dosage form in a pharmaceutical composition, to facilitate administration.

[0195] The antibody or antigen-binding fragment thereof, pharmaceutical composition of the present application can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebroventricular, inguinal, intravesical, local (e.g., powder, salve or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended goal. In certain preferred embodiments, the antibody or antigen-binding fragment thereof, pharmaceutical composition of the present application is administered by intravenous infusion or injection.

[0196] The pharmaceutical composition of the present application can include a "therapeutically effective amount" of the antibody or antigen-binding fragment thereof of the present application. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present application can vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight, and sex, the mode of administration of the drug, and other therapies simultaneously administered, and the like.

[0197] In the present application, the dosing regimen can be adjusted to achieve the best response for the intended purpose (e.g., therapeutic response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0198] In the present application, the subject can be a mammal, e.g., a human.

[0199] Detection applications

[0200] The antibody or antigen-binding fragment thereof of the present application is capable of specifically binding to ITGβ6, and thus can be used to detect the presence of or level of ITGβ6 in a sample.

[0201] Accordingly, in another aspect, the present application provides a kit comprising the antibody or antigen-binding fragment thereof of the present application. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application is labeled with a detectable label. In certain preferred embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present application. Preferably, the second antibody also comprises a detectable label.

[0202] In the present application, the detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels are amenable to immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), acridinium esters, magnetic beads (e.g., Dynal® thermometric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin (e.g., streptavidin) modified to bind the above labels. In certain embodiments, the detectable label as described above can be linked to the antibodies of the present application via linkers of varying lengths to reduce potential steric hindrance.

[0203] In another aspect, the present application provides a method of detecting the presence or level of ITGβ6 in a sample comprising the step of using an antibody or antigen-binding fragment thereof of the present application. In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present application is further labeled with a detectable label. In other preferred embodiments, the method further comprises using a reagent labeled with a detectable label to detect the antibody or antigen-binding fragment thereof of the present application. The method can be used for diagnostic purposes, or non-diagnostic purposes (e.g., the sample is a cell sample, rather than a sample from a patient).

[0204] In certain embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of the present application under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and ITGβ6, and detecting the formation of the complex.

[0205] In view of the low or no expression of ITGβ6 in normal tissues, and expression or high expression in some cancers, a tumor can be diagnosed by detecting the presence or level of ITGβ6 in a sample. Accordingly, in certain embodiments, the method is used to diagnose a tumor, e.g., an ITGβ6-positive tumor, e.g., a colorectal cancer, a gastric cancer, a lung cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, an ovarian cancer, a thyroid cancer, a bladder cancer, an endometrial cancer, a breast cancer, a liver cancer, a prostate cancer, a skin cancer, or a bile duct cancer, or any combination thereof.

[0206] In certain embodiments, the method comprises detecting the expression level of ITGβ6 in a test sample from a subject, and comparing the expression level to a reference value (e.g. healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

[0207] In another aspect, there is provided use of an antibody or antigen-binding fragment thereof of the application in the manufacture of a kit for detecting the presence of or level of ITGβ6 in a sample and / or diagnosing a tumor.

[0208] In another aspect, the present application provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, or a multispecific antibody of the present application. Optionally, the diagnostic or therapeutic kit can further comprise instructions for use. The kit can also comprise a delivery device for local administration in the therapeutic kit. The delivery device can include a drug-loaded syringe or a needle-free device.

[0209] All the technical features disclosed in the specification, or the steps in all the methods or processes disclosed, can be combined in any manner, except for mutually exclusive technical features and / or steps.

[0210] The antibody of the present application has high binding affinity and specificity to ITGβ6, can induce efficient endocytosis after binding to cells expressing ITGβ6, can carry toxin proteins to efficiently kill tumor cells, and has good physicochemical properties and stability, and can have better quality and in vivo metabolic properties, and is suitable for conjugation and other advantages. Therefore, the antibody of the present application has the potential to be used for treating and / or adjuvant therapy of tumors. In addition, the antibody of the present application is a humanized antibody, which has a lower risk of causing an immunogenic reaction when administered to a human subject, and has higher safety. Therefore, the antibody of the present application has great clinical value.

[0211] Abbreviations

[0212] CDR Complementarity-determining region in immunoglobulin variable region

[0213] FR Framework region of antibody: amino acid residues in antibody variable region other than CDR residues

[0214] VH Heavy chain variable region of antibody

[0215] VL Light chain variable region of antibody

[0216] IgG Immunoglobulin G

[0217] IMGT The international ImMunoGeneTics information system (IMGT)) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0218] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0219] Chothia The immunoglobulin numbering system proposed by Chothia et al., which is based on the location of structural loop regions to identify the boundaries of CDR regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0220] AbM The AbM CDR definition method is derived from related work by Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0221] mAb Monoclonal antibody

[0222] EC 50 Concentration producing 50% efficacy or binding

[0223] IC 50 Concentration producing 50% inhibition

[0224] ELISA Enzyme-linked immunosorbent assay

[0225] PCR Polymerase chain reaction

[0226] HRP horseradish peroxidase

[0227] K D equilibrium dissociation constant

[0228] Ka association rate constant

[0229] Kd dissociation rate constant

[0230] ADCC antibody-dependent cellular cytotoxicity

[0231] FACS flow cytometry

[0232] CDR-H1 complementarity determining region 1 in an immunoglobulin heavy chain variable region

[0233] CDR-H2 complementarity determining region 2 in an immunoglobulin heavy chain variable region

[0234] CDR-H3 complementarity determining region 3 in an immunoglobulin heavy chain variable region

[0235] CDR-L1 complementarity determining region 1 in an immunoglobulin light chain variable region

[0236] CDR-L2 complementarity determining region 2 in an immunoglobulin light chain variable region

[0237] CDR-L3 complementarity determining region 3 in an immunoglobulin light chain variable region

[0238] definitions

[0239] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the cell culture, biochemical, nucleic acid chemistry, immunological laboratory procedures, and the like, used herein are in accordance with conventional techniques well known in the respective fields. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0240] As used herein, the term "antibody" is used in the broadest sense, and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can be comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a kind of kappa (kappa) and lambda (lambda) light chain. The heavy chains can be assigned to mu, delta, gamma, alpha, or epsilon, and define a class of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site. Assignment of amino acids to each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0241] Herein, unless the context clearly indicates otherwise, the term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies.

[0242] The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the variable region of the antibody has cyclized to a pyroglutamate. Thus, in compositions comprising antibodies disclosed herein, the various antibodies therein can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or an N-terminal amino acid cyclized to a pyroglutamic acid or pyroglutamate.

[0243] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, e.g., according to the definition in the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system. And, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0244] In the present application, the CDRs contained by the antibodies of the present application or antigen-binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies of the present application or antigen-binding fragments thereof are preferably determined by the Chothia, AbM, Kabat, Contact, or IMGT numbering system.

[0245] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0246] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0247] As used herein, the term "antigen binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. For example, the antigen binding fragment can be a polypeptide fragment of a full-length antibody that retains the ability to specifically bind to the same antigen as the full-length antibody to which it is derived, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0248] As used herein, the term "full-length antibody" means an antibody that consists of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". Wherein, a "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that consists of, in N-terminal to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, optionally, a heavy chain constant region CH4 domain when the full-length antibody is of IgE isotype. Preferably, a "full-length heavy chain" is a polypeptide chain that consists of, in N-terminal to C-terminal direction, VH, CH1, HR, CH2, and CH3. A "full-length light chain" or "light chain" is a polypeptide chain that consists of, in N-terminal to C-terminal direction, a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains. The full-length antibody of the present application can be from a single species, e.g., human; can also be a chimeric antibody or a humanized antibody. The full-length antibody of the present application comprises two antigen binding sites formed by pairs of VH and VL, respectively, which specifically recognize / bind to the same antigen.

[0249] As used herein, the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CHI domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fab' fragment" means the fragment obtained after reduction of the disulfide bond connecting the two heavy chain fragments in a F(ab')2 fragment, consisting of one complete light chain and a Fd fragment of a heavy chain (consisting of a VH and CHI domain); the term "Fab'-SH" refers to an Fab fragment containing a free sulfhydryl group.

[0250] As used herein, the term "Fv fragment" means an antibody fragment consisting of a VL and VH domain of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that is capable of forming a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although its affinity can be lower than that of a complete binding site.

[0251] As used herein, the term "Fc fragment" means an antibody fragment formed by disulfide bond binding of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0252] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and The Pharmacology of Monoclonal Antibodies, Vol. 113, Martin Roseburg and Gordon P. Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS (SEQ ID NO: 96) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 97) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al., (2001), Cancer Immunol, 50(1):51-59, among others. In some cases, a disulfide bond can also exist between the VH and VL of the scFv.

[0253] As used herein, the term "diabody" means that the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complement domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0254] Each of the above-mentioned antibody fragments is capable of specifically binding to the same antigen bound by the full-length antibody from which it is derived, and / or competes with the full-length antibody for specific binding to the antigen. In this context, one skilled in the art can obtain an antigen-binding fragment of an antibody (e.g., an antibody fragment as described above) from a given antibody (e.g., an antibody provided herein) using known and routine techniques, and screen the antigen-binding fragment of the antibody for specificity in the same manner as used for the intact antibody.

[0255] As used herein, the term "multispecific antibody" refers to an antibody having multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody having two different antigen-binding specificities formed by conjugation of a first antibody (or fragment thereof) and a second antibody (or fragment thereof) or antibody analog via a conjugation arm, which includes but is not limited to chemical reaction, genetic fusion, and enzymatic. A "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, a trispecific antibody being an antibody having three different antigen-binding specificities, and a tetraspecific antibody being an antibody having four different antigen-binding specificities.

[0256] As used herein, the terms "monoclonal antibody," "monoclonal," "mAb" have the same meaning and are used interchangeably, and refer to one antibody or one fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of antibody molecules that are identical except for naturally-occurring mutations that can arise during production. Monoclonal antibodies are highly specific, being directed against a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies, and generally comprise at least 2 or more different antibodies, which generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" is not to be construed as a requirement for using any particular method for producing the antibody.

[0257] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and the heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and the heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which retains the binding activity of the antigen of interest. For example, the term "chimeric antibody" can include an antibody whose heavy and light chain variable regions are from a first antibody (e.g., murine), while the heavy and light chain constant regions are from a second antibody (e.g., human).

[0258] As used herein, the term "humanized antibody" refers to an antibody that can be prepared by replacing a portion of a non-human antibody prepared by immunization of a mammal other than a human with a portion of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human source antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (recipient antibody). For example, a humanized antibody can be prepared by grafting CDR sequences derived from a germline of another mammalian species onto a human framework sequence.

[0259] As used herein, the term "variant", in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that comprises an amino acid sequence that has been altered by the introduction of an amino acid residue substitution, deletion, or addition. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0260] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) or half maximal effective concentration (EC50) of the interaction. 50 ) represents.

[0261] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "on rate constant" (kaor kon) and the "off rate constant" (kdisor koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 : 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon, and kdisvalues can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition to this, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0262] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell assumes the genetic material carried by the vector and expresses the polynucleotide carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0263] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence is the origin of replication, or autonomously replicating sequence, which permits the vector to replicate independent of the host chromosomal DNA. The term "expression vector" as used herein refers to a vector that comprises a recombinant polynucleotide operably linked to expression control sequences necessary for the expression of the nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; additional elements can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained within liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0264] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, or CHO-EBNA cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0265] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently accomplished by use of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program ALIGN available from DNAstar, Inc., using, for example, a program parameter of an affine gap cost of 1 for the gap open penalty and 0.5 for the gap extension penalty. Percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated in the GCG software package (available at www.gcg.com).

[0266] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue has an atom or a group of atoms that is similar in size, shape, charge, chemical properties, etc. to the atom or group of atoms of the original amino acid residue. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0267] The nomenclature used herein to refer to the twenty conventional amino acids follows 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. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the single and three letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0268] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, physiologically and / or pharmacologically, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0269] As used herein, the term "preventing" refers to an approach taken to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor) in a subject. As used herein, the term "treating" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of the present application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0270] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor, or is at risk of having a disease described above.

[0271] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an amount effective to prevent a disease (e.g., a tumor) means an amount that is sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor); an amount effective to treat a disease means an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic uses would depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., the age, weight, and sex of the patient, the mode of administration of the drug, and other therapies being administered to the patient, etc.

[0272] As used herein, the term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with the isotype of an antibody. Examples of antibody effector functions include but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), down regulation of cell surface receptors (e.g., B cell receptor), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods of altering the effector function of an antibody are known in the art, e.g., by introducing mutations in the Fc region.

[0273] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, or macrophages) and causes these cytotoxic effector cells to bind specifically to an antigen-attached target cell, which is then killed by the cytotoxic cells.

[0274] As used herein, the term "complement-dependent cytotoxicity (CDC)" refers to a form of cytotoxicity involving complement, i.e., the classical complement pathway is activated by the binding of specific antibodies to the corresponding antigens on the cell membrane surface, and the resulting membrane attack complex exerts a lytic effect on the target cell.

[0275] As used herein, the term "antibody-dependent cellular phagocytosis (ADCP)" refers to a form of cytotoxicity in which the Fab fragment of an antibody drug can target a marker on the surface of a tumor cell, and the Fc fragment can bind to Fc receptors on phagocytic cells (such as macrophages, monocytes, and neutrophils), thereby facilitating the phagocytosis of the cancer cell by the phagocytic cells in vivo.

[0276] In the present context, "combination" includes therapies that can be administered separately, e.g., separately dispensed (e.g., can be provided in separate kits), as well as therapies that can be administered together in a single dispensation, i.e., "co-dispensation". In certain embodiments, the anti-ITGβ6 antibodies or antigen-binding fragments thereof of the present application can be administered sequentially. In other embodiments, the anti-ITGβ6 antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-ITGβ6 antibodies or antigen-binding fragments thereof of the present application can be used in combination with at least one other (active) agent in any manner.

[0277] In such combination therapies, the various active agents often have different mechanisms of action, and the combination therapy can result in a synergistic effect. Combination therapies include therapeutic agents that affect the immune response, e.g., enhance or activate the response, and therapeutic agents that affect, e.g., inhibit or kill, tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies can allow for a reduction in the dose of one or more of the agents, to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies can have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.

[0278] In the present context, ITGβ6 positivity is obtained by immunohistochemistry and evaluation of staining intensity by a specialized clinical pathologist.

[0279] The terms "cancer" "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues, and can metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0280] Embodiments of the application will be described in detail with reference to the drawings and the following detailed description below, but it is to be understood that only preferred embodiments of the application are described and illustrated for the purposes of clarity and understanding, and it is to be understood that the application is not limited to the embodiments described and illustrated herein. Various objects and advantageous aspects of the application will become apparent to those skilled in the art from the following detailed description, taken in combination with the accompanying drawings and the preferred embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0281] Figure 1A: Binding activity assay of anti-human ITGβ6 fully human antibodies to human breast ductal carcinoma cells HCC70.

[0282] Figure 1A: Binding activity assay of anti-human ITGβ6 fully human antibodies to human breast ductal carcinoma cells HCC70.

[0283] Figure 1C: Binding activity assay of anti-human ITGβ6 fully human antibodies to human esophageal carcinoma cells TE-4.

[0284] Figure 1D: Binding activity assay of anti-human ITGβ6 fully human antibody to human esophageal cancer cell TE-4.

[0285] Figure 2A: Endocytosis activity assay of anti-human ITGβ6 fully human antibody to human breast ductal carcinoma cell HCC70.

[0286] Figure 2B: Endocytosis activity assay of anti-human ITGβ6 fully human antibody to human breast ductal carcinoma cell HCC70.

[0287] Figure 2C: Endocytosis activity assay of anti-human ITGβ6 fully human antibody to human esophageal cancer cell TE-4.

[0288] Figure 2D: Endocytosis activity assay of anti-human ITGβ6 fully human antibody to human esophageal cancer cell TE-4.

[0289] Figure 2E: Endocytosis activity assay of anti-human ITGβ6 fully human antibody to human pancreatic cancer cell Capan-2.

[0290] Figure 3A: Binding activity assay of anti-human ITGβ6 fully human mAb2 affinity matured antibody to human breast ductal carcinoma cell HCC1954.

[0291] Figure 3B: Binding activity assay of anti-human ITGβ6 fully human mAb1 affinity matured antibody to human breast ductal carcinoma cell HCC1954.

[0292] Figure 4A: Endocytosis activity assay of anti-human ITGβ6 fully human mAb2 affinity matured antibody to human breast ductal carcinoma cell HCC1954.

[0293] Figure 4B: Endocytosis activity assay of anti-human ITGβ6 fully human mAb1 affinity matured antibody to human breast ductal carcinoma cell HCC1954.

[0294] Figure 4C: Endocytosis activity assay of anti-human ITGβ6 fully human mAb1 affinity matured antibody to human breast ductal carcinoma cell HCC1954.

[0295] Sequence information

[0296] Information of the sequences involved in the present application is described in Table 1 below:

[0297] Table 1: Sequence description DETAILED DESCRIPTION

[0298] The present application will now be described with reference to the following examples, which are intended to be illustrative only and not limiting of the present application.

[0299] Unless otherwise indicated, the molecular biology and immunological techniques utilized in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present application by way of example only and are not intended to limit the scope of the present application claimed.

[0300] Those skilled in the art know that the examples describe the present application by way of example only and are not intended to limit the scope of the present application claimed. The experimental methods in the examples are conventional methods unless otherwise specified. The specific conditions in the examples are performed according to the conventional conditions or the conditions recommended by the manufacturer unless otherwise specified. The reagents or instruments used are conventional products that can be obtained commercially unless otherwise specified.

[0301] Example 1: Preparation of ITGβ6 antigen, control antibody protein and construction of cell lines

[0302] Human ITGαV & ITGβ6 (αVβ6), monkey αVβ6, rat αVβ6 and mouse αVβ6 proteins were purchased from acrobiosystems. According to the humanized 2A2 HCLG antibody sequence in the patent CN 114828887 A, the gene synthesis, plasmid extraction and expression purification were entrusted to Shanghai Baiying Biological Technology Co., Ltd. to obtain the control antibody mAb4. The negative control antibody hIgG1 is an anti-chicken lysozyme antibody, the heavy chain variable region of which is fused with the human hIgG1 heavy chain constant region, and the light chain variable region of which is fused with the human Kappa light chain constant region. Similarly, the gene synthesis, plasmid extraction and expression purification were entrusted to Shanghai Baiying Biological Technology Co., Ltd. to obtain the control antibody hIgG1.

[0303] Full length sequences of human ITGβ6 (Uniprot: P18564), monkey ITGβ6 (Uniprot: A0A2K5TZ36), mouse ITGβ6 (Uniprot: Q9Z0T9), human ITGαV (Uniprot: P06756), monkey ITGαV (Uniprot: A0A2K5WCD3), mouse ITGαV (Uniprot: P43406), human ITGβ1 (Uniprot: P05556), human ITGβ3 (Uniprot: P05106), human ITGβ5 (Uniprot: P18084) and human ITGβ8 (Uniprot: P26012) were gene synthesized by Genscript and constructed into pLVX vector, after plasmid extraction, virus was packaged for constructing overexpression cell lines CHOS-human ITGβ6, CHOS-monkey ITGβ6, CHOS-mouse ITGβ6, CHOS-human αVβ6, CHOS-monkey αVβ6, CHOS-mouse αVβ6, CHOS-human ITGαV, CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5 and CHOS-human ITGβ8.

[0304] Example 2: Phage library panning of anti-human ITGβ6 antibodies

[0305] Phage library was constructed based on human PBMC, in order to obtain phage targeting and recognizing ITGβ6 antigen, full human antibodies can be obtained from the library by panning, the simple screening process and method are as follows.

[0306] 2.1 First round of cell panning

[0307] 2.1.1 Preparation of phage library: the 21 full human natural phage sub-libraries constructed were mixed in equal amounts and the titer was determined.

[0308] 2.1.2 Phage negative panning: 4E7 CHOS cells in logarithmic growth phase were taken and washed with PBS for 3 times; after washing, they were divided into two equal parts, and the phage mixed library was subjected to negative panning at 10 rpm / 4°C / 1 h, and the negative panning was performed twice.

[0309] 2.1.3 Cell blocking: 1E7 CHOS-human αVβ6 cells in logarithmic growth phase were taken and washed with PBS for 3 times, and after washing, they were blocked with 2% MPBS (2% skimmed milk powder dissolved in PBS) at 10 rpm / 4°C / 1 h; after blocking, they were centrifuged at 500 g for 3 min, the supernatant was discarded, and the cells were resuspended with 5 ml of fresh 2% MPBS.

[0310] 2.1.4 Phage positive selection: After the completion of phage library negative selection, 500g, 3min centrifugation to obtain phage supernatant. The completed blocked CHOS-human αVβ6 cells were added to the supernatant, mixed evenly, and positive selection was performed at 10 rpm / 4°C / 1h;

[0311] 2.1.5 Washing: After the completion of positive selection, the cells were washed 5 times with 5ml PBS, 5 times with 5ml 0.1% PBST, and finally 1 time with 5ml PBS, with each washing being 500g / 4°C / 3min, and the supernatant was discarded;

[0312] 2.1.6 Elution: The cell pellet was added with 1ml 1M TEA, quickly blown and mixed evenly and vortexed for 1min, then rotated at 10rpm at room temperature for 10min, neutralized with 500μl 1M Tris-HCl (pH 7.4), and 1.5ml eluate was obtained, labeled as R1E;

[0313] 2.1.7 Elution titer determination: TG1 was inoculated in advance, and when OD600 reached 0.6-0.8, it could be used for titer determination. As follows: 10μl R1E of 2.1.6 was added to 990μl 2YT (dilution 10E2), and 10-fold dilution was obtained based on the 10E2 dilution, 100μl of each dilution of 10E2 / 10E3 was added to 200μl TG1, i.e. dilution of 10E3 / 10E4, 37°C for 30min, and then plated on ampicillin plates, 37°C overnight, and the next day the number of colonies in the plates was counted, which was the elution titer of the first round (Round 1).

[0314] 2.1.8 Infection: After the titer was determined, the eluate was used to infect TG1 (OD600 was about 0.6-0.8), and after 37°C for 40min, 3500g centrifugation for 10min, the supernatant was discarded, 3ml 2YT was added and blown evenly, and then plated on 150mm ampicillin plates, and incubated at 30°C overnight;

[0315] 2.1.9 Phage packaging: The next day, the plates were scraped with 2YT (100μg / ml ampicillin and 1% glucose) 5ml / plate, and 80ml 2YT (100μg / ml ampicillin) was added according to the volume ratio of 1:100, and incubated at 37°C until OD600 was 0.6-0.8, then M13K07 helper phage was added for packaging, and incubated at 37°C for 30min, then kanamycin (50μg / ml) was added, and incubated at 30°C at 230rpm overnight;

[0316] 2.1.10 Bacteriophage precipitation: centrifuge the overnight culture, 15000g for 15min, filter the supernatant with 0.45um filter membrane, add 1 / 4 volume of PEG / NaCl, i.e. 10ml, and let it stand on ice for 4h; after standing, discard the supernatant after centrifugation at 15000g for 15min, and resuspend the precipitate with 2ml PBS to obtain the packaging library, which is labeled as R1A;

[0317] 2.1.11 Titer of the packaging library: the titer determination method is as described above, and the final dilution gradient is 10E8 and 10E9, and the plates are coated with ampicillin, and the titer is calculated the next day.

[0318] 2.2 Second round of protein liquid phase panning

[0319] 2.2.1 Blocking of the bacteriophage library and magnetic beads: blocking of the bacteriophage library: take 500ul of R1A from the first round and add it to 2% BSA (BSA dissolved in PBS) blocking solution, mix well, and let it stand at 37℃ for 1h; blocking of the magnetic beads: take 50ul of resuspended magnetic beads with a pipette into two new centrifuge tubes (negative selection tube A and positive selection tube B), add 1ml of 0.05% PBST, resuspend for 20s, and magnetically separate for 2min, and remove the supernatant (wash twice). Add 1ml of 2% BSA blocking solution, and repeat the resuspension and magnetic separation; add 1ml of blocking solution again, and let it stand in a rotary shaker at room temperature for 1h at 20rpm.

[0320] 2.2.2 Antigen-magnetic bead binding and negative panning of the bacteriophage library:

[0321] (1) Antigen-magnetic bead binding: after the B tube is instantaneously separated, magnetically separate for 2min, remove the supernatant, wash twice with 0.05% PBST, add 5ug of biotinylated antigen (diluted with 0.05% PBST to a concentration of 1mg / ml), and let it stand in a rotary shaker at room temperature for 1h at 20rpm;

[0322] (2) Negative panning of the bacteriophage library: after the A tube is instantaneously separated, magnetically separate for 2min, remove the supernatant, wash twice with 0.05% PBST, add the blocked bacteriophage library to the A tube, and let it stand in a rotary shaker at room temperature for 1h at 20rpm. Magnetically separate for 2min, and transfer the supernatant to a new EP tube, which is the blocked and negatively panned bacteriophage library.

[0323] 2.2.3 Positive panning of the bacteriophage

[0324] (1) Antigen-antibody binding: "B tube-positive selection", instantaneously separate, magnetically separate, wash three times with 0.05% PBST, add the blocked and negatively panned bacteriophage library, and let it stand in a rotary shaker at 37℃ for 0.5-1h at 20rpm.

[0325] (2) Wash: "B tube - positive selection" instant separation, and magnetic separation, 1 ml 0.1% PBST resuspended 20 s, magnetic separation, discard the supernatant, repeat washing 15 times;

[0326] (3) Elution and neutralization: B tube, after magnetic separation, PBS washing 1 time, add 1 ml Gly-HCl (pH 2.2), placed in a rotating mixer, room temperature 20 rpm for 10 min, magnetic separation, suction of the supernatant, and add the new 1M Tris-HCl (pH 7.4) 2 ml EP tube, labeled as R2E.

[0327] (4) Elution titer determination, as described above, the final label is R2A.

[0328] 2.3 Third round of cell selection

[0329] The selection steps are described in 2.1, but the following steps are changed: the phage library is used as the second round of selection amplification library R2A200μl; overexpressed cells CHOS-human αVβ6 cells are blocked with 1% casein; the washing condition is that the cells are washed with 4 ml PBS for 5 times, then washed with 5 ml 0.1% PBST for 10 times, and finally washed with 4 ml PBS once, each time centrifuged at 500g / 3min / 4℃, and finally the supernatant is discarded. Finally, the library is eluted with TEA and labeled as R3E, and the titer of R3E is determined.

[0330] Example 3: Identification of anti-ITGβ6 phage clones

[0331] 3.1 Cell ELISA identification of phage positive clones

[0332] 3.1.1 Single clone packaging: single clones are picked from the R3E titer determination plate to a 96 deep well plate, and 300μl / well of 2YTA (100μg / ml ampicillin) is added to the deep well plate, which is cultured at 37℃ 300rpm until OD600 is about 0.6-0.8. 200μl of the bacterial solution is added to the diluted helper phage M13K07 for packaging, 50μl / well; 37℃ for 30min, 37℃ for 30min, then kanamycin (50μg / ml) is added, 30℃, 300rpm overnight culture;

[0333] 3.1.2 Cell preparation: 3E7 of logarithmic growth phase cells CHOS-human ITGαV and CHOS-human αVβ6 cells are taken respectively, washed with PBS for 3 times; 2% MPBS blocking solution 10 ml resuspend the cell pellet, according to 3E5 / well plated into the sharp bottom plate, 37℃ for 1h; 500g centrifugation for 3min, discard the supernatant;

[0334] 3.1.3 First antibody incubation: Centrifuge the packaged monoclonal deep well plate at 4000g for 10 min, resuspend the cell pellet of 3.1.2 with 50 μl supernatant, incubate at 4°C for 1 h; centrifuge at 500g for 3 min, discard the supernatant;

[0335] 3.1.4 Second antibody incubation: Gently mix the cell pellet with 150 μl 0.1% PBST per well and wash 3 times, discard the supernatant; resuspend the cell pellet with 100 μl 1% casein diluted HRP-M13 secondary antibody (1:20000) per well, incubate at 4°C for 30 min; centrifuge at 500g for 3 min, discard the supernatant;

[0336] 3.1.5 Color development termination: Gently mix the cell pellet with 150 μl 0.1% PBST per well and wash 3 times, discard the supernatant; resuspend the cell pellet with 100 μl TMB per well, color develop for a few minutes, then centrifuge at 500g for 3 min, take 50 μl per well to a new plate containing 50 μl stop solution (2M H2SO4), immediately read the OD450 value. Finally select the positive clones which bind to CHOS-human αVβ6 cells but not to CHOS-human ITGαV cells, the results are shown in Table 2 below.

[0337] Table 2: Binding detection of single positive clones by cell ELISA

[0338] 3.2 Flow cytometry detection of positive clones

[0339] Prepare cells CHOS-human ITGαV and CHOS-human αVβ6 at 2E5 cells per well, wash the cells with PBS twice, resuspend the cells with 50 μl 1% BSA per well and plate into the eppendorf plate; take 50 μl 3.1 positive phage packaging supernatant to 50 μl cells, gently mix, incubate at 4°C for 1 h; wash with PBS twice, centrifuge at 500g for 3 min, discard the supernatant; resuspend the cell pellet with 50 μl 1% BSA diluted PE-M13 secondary antibody (1:200) per well, incubate at 4°C for 30 min; wash with PBS twice, centrifuge at 500g for 3 min, discard the supernatant, resuspend the cell pellet with 120 μl PBS, then detect on the machine. According to the analysis data of the detected fluorescence signal, the relative fluorescence intensity is obtained: MFI ratio = MFI (phage staining) ÷ MFI (blank cells), MFI is the mean fluorescence intensity. Finally obtain the positive clones which specifically bind to CHOS-human αVβ6 / CHOS-mouse αVβ6, and do not bind to CHOS-human ITGαV and CHOS-human αVβ3 / CHOS-human αVβ5 of the same family, as shown in Table 3 below:

[0340] Table 3: Detection of positive clones by flow cytometry

[0341] 3.3 Anti-human ITGβ6 antibody sequence identification

[0342] Phage clones mAb2, mAb1 and mAb3 were sequenced, and the sequences were analyzed by CDR Numbering Definition. The antibody variable region sequences and CDR sequences are shown in Table 4:

[0343] Table 4: Antibody variable region sequences and CDR sequences

[0344] Example 4: Evaluation of anti-human ITGβ6 fully human antibodies

[0345] 4.1 Expression of anti-human ITGβ6 fully human antibodies

[0346] The sequences of phage clones mAb2, mAb1 and mAb3 were provided to Shanghai Baiying Biotechnology Co., Ltd. for gene synthesis, plasmid extraction and expression purification, and antibodies mAb2, mAb1 and mAb3 were obtained. Among them, the heavy chain constant region of antibody mAb2 is shown as SEQ ID NO: 77, and the light chain constant region is shown as SEQ ID NO: 73; the heavy chain constant region of antibody mAb1 is shown as SEQ ID NO: 77, and the light chain constant region is shown as SEQ ID NO: 74; the heavy chain constant region of antibody mAb3 is shown as SEQ ID NO: 77, and the light chain constant region is shown as SEQ ID NO: 74. The heavy chain constant region shown as SEQ ID NO: 77 has the following substitutions relative to the sequence of the wild type IgG1 heavy chain constant region: Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering system), so as to reduce the ADCC activity and / or CDC activity of the antibody.

[0347] 4.2 Hydrophilic HIC and isoelectric point (PI) detection of anti-human ITGβ6 fully human antibodies

[0348] The isoelectric point (PI) of the candidate antibodies was determined by isoelectric focusing. The method is briefly described as follows: the Maurice isoelectric focusing system of ProteinSimple Company was used for analysis in combination with its capillary cartridge. The antibody was diluted with water, and the pH gradient was formed by using 4% final concentration of amphoteric electrolyte 3-10 (GE Company) in the detection system.

[0349] Liquid phase HIC detection method: the equipment is Agilent 1260, the analysis column is TSKgel Butyl-NPR, 4.6*100mm, the column temperature is 30℃, the detection wavelength is 280nm, the flow rate is 0.5ml / min, the mobile phase A is 1.5mol / L (NH4)2SO4; the mobile phase B is 25mmol / L Na2HPO4, pH = 7.0, 25% isopropyl alcohol. Take the test product in an appropriate amount, dilute it with diluent (0.75mol / L (NH4)2SO4) to prepare a 1.0mg / ml solution as the test sample solution; take control 1 and control 2 respectively, dilute them with diluent to prepare a 1mg / ml solution as the system suitability solution. Inject about 40μg, analyze the gradient elution: 0-3min, maintain 95% of the mobile phase A and 5% of the mobile phase B; 3-40min, increase the mobile phase B from 5% to 100%; 40-45min, maintain 95% of the mobile phase A and 5% of the mobile phase B. After detection, calculate the hydrophobic value of the test product according to the control sample, HIC value = (test product RT-hydrophilic control RT) / (hydrophobic control RT-hydrophilic control RT), RT is the retention time of peak. The isoelectric point and HIC determination results of the anti-human ITGβ6 fully human antibody are shown in Table 5, mAb2, mAb1 and mAb3 have good hydrophilicity, and the isoelectric point is within the normal range.

[0350] Table 5: Isoelectric point and HIC determination results of anti-human ITGβ6 fully human antibody

[0351] 4.3 Dynamic affinity detection of anti-human ITGβ6 fully human antibody

[0352] The dynamic affinity of the anti-human ITGβ6 fully human antibody to human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 protein was detected by ForteBio (Pall life sciences). The specific method is as follows: the antibody to be tested is diluted to 5μg / ml with PBST (0.02% Tween-20), and human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 protein is gradient diluted to 200nM, 100nM, 50nM, 25nM, 12.50nM, 6.25nM, 3.125nM, 0nM, then the antibody to be tested is captured by Protein A Sensor (Pall life sciences) for 60s in PBST (0.02% Tween-20) solution, after equilibration for 60s in PBST equilibration solution, then combined with the four proteins for 90s respectively, then the results were analyzed by global fitting to obtain the affinity constant. The results are shown in Table 6, which show that the phage screening antibodies mAb2, mAb1 and mAb3 can bind to human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 protein.

[0353] Table 6: Anti-human ITGβ6 fully human antibody dynamic affinity detection results

[0354] 4.4 Anti-human ITGβ6 fully human antibody cell affinity, species cross and specificity detection

[0355] The affinity of the anti-human ITGβ6 fully human antibody to human breast ductal carcinoma cells HCC70 (Nanjing Kebai) and human esophageal carcinoma cells TE-4 (Nanjing Kebai) was detected by a flow cytometer (Beckman, model Cytoflex); the affinity of the anti-human ITGβ6 fully human antibody to CHOS-human ITGβ6 and CHOS-human αVβ6 was detected; the species cross of the anti-human ITGβ6 fully human antibody was detected, including the affinity to CHOS-monkey ITGβ6, CHOS-monkey αVβ6, CHOS-mouse ITGβ6 and CHOS-mouse αVβ6; whether the anti-human ITGβ6 fully human antibody specifically binds to ITGβ6 was detected, including the affinity to CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8 and CHOS-human ITGαV.

[0356] The cells growing adherently were digested with a Trypsin-EDTA (0.25%) (Thermo) solution, counted and adjusted to a cell density of 4.0 x 10 6 / ml, washed twice with 1% BSA and resuspended in a 1% BSA solution, 50 μl of the cell suspension was added to each well of a 96-well sharp bottom plate (2 x 10 5 The candidate antibody and the negative control antibody hIgG1 were diluted with 1% BSA, with a starting final concentration of 200 nM and a 3-fold gradient dilution, for a total of 11 concentration points, 50 μl of the diluted antibody was added to the sharp bottom plate containing the cells, and incubated at 4°C for 60 min; the cells were washed twice with 1% BSA, and then 50 μl of the diluted secondary antibody was added to each well, mixed well, and incubated at 4°C for 30 min; the cells were washed twice with 1% BSA, and then resuspended in 200 μl of 1% BSA, and detected by flow cytometry. Data processing: the Median PE or Median FITC values were exported, and then imported into data analysis software to calculate the EC 50 .

[0357] The HCC70 and TE-4 tumor cell affinity results of the anti-human ITGβ6 fully human antibody are shown in FIGS. 1A, 1B, 1C, 1D and Table 7, respectively, and the affinity of mAb2, mAb1 and mAb3 to tumor cells is stronger than that of mAb4.

[0358] Table 7: Results of affinity assay of anti-human ITGβ6 fully human antibodies to HCC70 and TE-4 cells

[0359] The results of affinity of anti-human ITGβ6 fully human antibodies to CHOS-human ITGβ6, CHOS-human αVβ6 cells are shown in Table 8, the affinities of mAb2, mAb1 and mAb3 on these two overexpressed cells are stronger than that of mAb4, and the affinities of all antibodies to CHOS-human αVβ6 cells are stronger than that to CHOS-human ITGβ6 cells.

[0360] Table 8: Results of affinity assay of anti-human ITGβ6 fully human antibodies to CHOS-human ITGβ6, CHOS-human αVβ6 cells

[0361] The results of affinity of anti-human ITGβ6 fully human antibodies to CHOS-monkey ITGβ6, CHOS-monkey αVβ6, CHOS-mouse ITGβ6 and CHOS-mouse αVβ6 cells are shown in Table 9, the results show that mAb2, mAb1, mAb3 and mAb4 can all bind to CHOS-monkey ITGβ6 and CHOS-monkey αVβ6 cells; mAb2, mAb1 and mAb3 can bind to CHOS-mouse ITGβ6 and CHOS-mouse αVβ6 cells, and mAb4 does not bind to CHOS-mouse ITGβ6 cells and weakly binds to CHOS-mouse αVβ6 cells.

[0362] Table 9: Results of affinity assay of anti-human ITGβ6 fully human antibodies to CHOS-monkey ITGβ6, CHOS-monkey αVβ6, CHOS-mouse ITGβ6, CHOS-mouse αVβ6 cells

[0363] The results of affinity of anti-human ITGβ6 fully human antibodies to CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8 and CHOS-human ITGαV cells are shown in Table 10, the results show that mAb2, mAb1, mAb3 and mAb4 do not bind to CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8 and CHOS-human ITGαV cells, indicating that the binding of all antibodies to ITGβ6 is specific.

[0364] Table 10: Results of affinity assay of anti-human ITGβ6 fully human antibodies to CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8, CHOS-human ITGαV cells

[0365] 4.5 Detection of endocytosis activity of anti-human ITGβ6 fully human antibodies

[0366] The endocytosis activity of anti-human ITGβ6 fully human antibodies on human breast ductal carcinoma cells HCC70 (Nanjing Keygen Biotech), human esophageal carcinoma cells TE-4 (Nanjing Keygen Biotech) and human pancreatic carcinoma cells Capan-2 (Nanjing Keygen Biotech) was detected by flow cytometry (Thermo, model Attune NxT).

[0367] The adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted, and the cell density was adjusted to 1 x 10 5 cells / ml with complete culture medium, 100 μl of cell suspension (1 x 10 4 cells / well) was added to a 96-well plate, and the 96-well plate was incubated in a 37°C, CO2 incubator for 24 h. The 96-well plate was taken out, and the culture medium was aspirated. 50 μl of fresh complete culture medium was added to each well. The test antibodies and the negative control antibody hIgG1 were diluted with complete culture medium, with a starting final concentration of 8 nM and a 3-fold gradient dilution, for a total of 8 concentration points. PHrodo red reagent was diluted with complete culture medium to 12 μg / ml (the final concentration of PHrodo red was 3 μg / ml). The gradient-diluted test antibodies were mixed with the diluted PHrodo reagent at a ratio of 1:1 (30 μl:30 μl) and incubated at room temperature for 30 min in the dark. 50 μl of the test antibody and PHrodo reagent mixture was added to the 96-well plate, which was incubated at 37°C, 5% CO2 for 24 h. The 96-well plate was taken out, washed once with sterile PBS, and the cells were digested with 100 μl of Trypsin-EDTA (0.25%) per well, followed by the addition of 100 μl of complete culture medium for neutralization. After the cells in the wells were dispersed by blowing, they were detected by FACS. Data processing: the Median YL1-H value was exported, and then imported into data analysis software to calculate the EC 50 , as shown in FIGS. 2A, 2B, 2C, 2D, 2E and Table 11. mAb2, mAb1 and mAb3 had good endocytosis activity.

[0368] Table 11: Endocytosis activity assay of anti-human ITGβ6 fully human antibodies

[0369] Example 5: Affinity maturation of mAb2 and mAb1 antibodies

[0370] Affinity maturation is a process by which antibodies gain increased affinity and anti-pathogen activity, hoping to increase the affinity of the clones while obtaining more high endocytosis activity clones. On the basis of the obtained mAb2 and mAb1 clones, the light chain variable region 3 (CDR-L3) of mAb2 and the heavy chain variable region 3 (CDR-H3) of mAb1 were randomly mutated, and two mutated phage libraries were established respectively, and the simple process and method are as follows.

[0371] 5.1 mAb2 and mAb1 phage mutant library construction

[0372] In the heavy chain variable region 3 of mAb1 antibody and the light chain variable region 3 of mAb2 antibody, partial overlapping NNK randomization primers were designed for each 6 amino acid sites, and each 6 amino acids of the two CDRs were saturated mutated. Then, the random mutation fragments of the heavy chain variable region 3 of mAb1 antibody and the light chain variable region 3 of mAb2 antibody were amplified by PCR respectively, and the PCR products were gel purified. Then, the phagemid vector and the two PCR fragments were cut by restriction endonuclease and gel purified, and then the products were connected respectively and the TG1 competent cells were independently electrotransformed, and the ampicillin plates were separately coated.

[0373] 5.1.1 Phage packaging: the next day, 5 mL / plate of 2YT (with 100 μg / mL ampicillin and 1% glucose) was used to scrape the plates of the two groups electrotransformed the day before, and then 40 mL of 2YT (100 μg / mL ampicillin) was added at a volume ratio of 1:100, and the culture was incubated at 37°C until the OD600 was 0.6-0.8. M13K07 helper phage was added for packaging, and the culture was incubated at 37°C for 30 min, then 37°C, 180 rpm, 30 min, then kanamycin (50 μg / mL) was added, and the culture was incubated at 30°C, 230 rpm overnight.

[0374] 5.1.2 Phage precipitation: centrifuge the overnight culture at 15000g for 15 min, filter the supernatant with a 0.45 μm filter membrane, add 1 / 4 of the volume of PEG / NaCl, i.e. 10 mL, and let it stand on ice for 4 h; after standing, centrifuge at 15000g for 15 min, discard the supernatant, and resuspend the precipitate with 2 mL of PBS to obtain the packaging library, which is labeled as mAb2 affinity maturation library-1 and-2; the same procedure was used to construct the mAb1 affinity maturation library, and the mAb1 affinity maturation library-1 and-2 were obtained.

[0375] 5.1.3 Titer of packaging library: the titer was determined as described above, with a final dilution gradient of 10E8, 10E9, ampicillin plates were coated, and the titer was calculated the next day.

[0376] 5.2 Affinity maturation phage library screening of anti-ITGβ6 antibody

[0377] ITGβ6 antigen panning was performed using mAb2 affinity maturation library-1 / -2 and mAb1 affinity maturation library mutant library-1 / -2, the simple screening process and method are as follows:

[0378] 5.2.1 First round of protein liquid phase panning

[0379] Take mAb2 affinity maturation library or mAb1 affinity maturation library, and perform the first round of protein liquid phase panning, the steps are the same as those in step 2.2, and the final eluate is neutralized with 1M Tris-HCl (pH 7.4), and is labeled as mAb2_R1E or mAb1_R1E. Elution titer determination, as described in 2.1.7, and finally labeled as mAb2_R1A or mAb1_R1A.

[0380] 5.2.2 Second round of cell panning

[0381] Take mAb2_R1A or mAb1_R1A for the second round of cell panning, and the cell panning steps are the same as those in step 2.3, and the washed cells are eluted with TEA and neutralized with 1M Tris-HCl (pH 7.4), and the library is labeled as mAb2_R2E or mAb1_R2E, and the titers of the two R2E are determined.

[0382] 5.3 Identification of affinity matured phage clones of anti-ITGβ6 antibodies

[0383] Cell ELISA identification of phage positive clones

[0384] Single clones were picked from the mAb2_R2E or mAb1_R2E panning titer determination plate into 96-deep well plates, and phage packaging and cell affinity tests were performed, as described in step 3.1, and finally 6 phage clones were screened, numbered as mAb2-B, mAb2-A, mAb2-C, mAb1-A, mAb1-B and mAb1-C, and the affinity detection results are shown in Tables 12 and 13 below.

[0385] Table 12: Binding detection of single point mAb2 affinity maturation positive clones by cell ELISA

[0386] Table 13: Binding detection of single point mAb1 affinity maturation positive clones by cell ELISA

[0387] 5.4 Evaluation of anti-human ITGβ6 fully human affinity matured antibodies

[0388] 5.4.1 Expression of anti-human ITGβ6 fully human affinity matured antibodies

[0389] The sequences of phage clones mAb2-B, mAb2-A, mAb2-C, mAb1-A, mAb1-B and mAb1-C (see Table 1) were provided to Shanghai Baiying Biotechnology Co., Ltd. for gene synthesis, plasmid extraction and expression purification, and antibodies mAb2-B, mAb2-A, mAb2-C, mAb1-A, mAb1-B and mAb1-C were obtained. Among them, the heavy chain constant region of antibodies mAb2-A, mAb2-B and mAb2-C is as shown in SEQ ID NO: 77, and the light chain constant region is as shown in SEQ ID NO: 73; the heavy chain constant region of antibodies mAb1-A, mAb1-B and mAb1-C is as shown in SEQ ID NO: 77, and the light chain constant region is as shown in SEQ ID NO: 74. The heavy chain constant region as shown in SEQ ID NO: 77 has the following substitutions relative to the sequence of the wild-type IgG1 heavy chain constant region: Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering system) in order to reduce the ADCC activity and / or CDC activity of the antibody.

[0390] Example 6: Hydrophilic HIC detection of anti-human ITGβ6 fully human affinity matured antibodies

[0391] Liquid phase HIC detection method: the equipment is Agilent 1260, the analysis column is TSKgel Butyl-NPR, 4.6*100mm, the column temperature is 30°C, the detection wavelength is 280nm, the flow rate is 0.5ml / min, the mobile phase A is 1.5mol / L (NH4)2SO4; the mobile phase B is 25mmol / L Na2HPO4, pH=7.0, 25% IPA. Take an appropriate amount of test product, dilute it with diluent (0.75mol / L (NH4)2SO4) to prepare a 1.0mg / ml solution as the test product solution; take control 1 and control 2 respectively, dilute them with diluent to prepare a 1mg / ml solution as the system suitability solution. Inject about 40μg, analyze the gradient elution: 0-3min, maintain 95% of mobile phase A and 5% of mobile phase B; 3-40min, increase the mobile phase B from 5% to 100%; 40-45min, maintain 95% of mobile phase A and 5% of mobile phase B. After detection, calculate the hydrophobic value of the test product according to the control sample, HIC value=(test product RT-hydrophilic control RT) / (hydrophobic control RT-hydrophilic control RT), RT is the retention time of peak. The results are shown in Table 14.

[0392] Table 14: HIC determination results of anti-human ITGβ6 fully human affinity matured antibodies

[0393] Example 7: Dynamic affinity detection of anti-human ITGβ6 fully human affinity matured antibodies

[0394] Dynamic affinity of anti-human ITGβ6 fully human antibodies to human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 proteins were detected by ForteBio (Pall life sciences). The detailed method was as follows: the tested antibodies were diluted to 5 μg / ml with PBST (0.02% Tween-20), and human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 proteins were gradient diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, 0 nM, then the tested antibodies were captured by Protein A Sensor (Pall life sciences) for 60 s in PBST (0.02% Tween-20) solution, after equilibration for 60 s in PBST buffer, then combined with the four proteins for 90 s respectively, and then dissociated for 180 s, the results were analyzed by global fitting to obtain the affinity constant. The results were shown in Table 15, which showed that the binding activity of mAb2, mAb1 affinity matured antibodies to human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 proteins was significantly enhanced compared with mAb2, mAb1.

[0395] Table 15: Results of dynamic affinity detection of anti-human ITGβ6 fully human antibodies

[0396] Example 8: Cell affinity of anti-human ITGβ6 fully human affinity matured antibodies

[0397] The affinity of anti-human ITGβ6 fully human antibodies to human breast ductal carcinoma cells HCC1954 (Connovate) was detected by flow cytometry (Beckman, model Cytoflex). The adherently growing cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, counted and adjusted to a cell density of 4.0 × 10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension (cell number 2 × 10 5(50ul / well); dilute candidate antibodies and negative control antibody hlgG1 with 1% BSA, start with final concentration 200nM, 3-fold dilution gradient, total 11 concentration points, take 50ul diluted antibody to add to the well plate containing cells, 4 degree incubation for 60min; wash cells with 1% BSA twice, then add 50ul diluted secondary antibody to each well, mix well, 4 degree incubation for 30min; wash cells with 1% BSA twice, then resuspend cells in 200ul 1% BSA, flow cytometry detection. Data processing: export Median PE value, then import data analysis software, calculate EC 50 .

[0398] The HCC1954 tumor cell affinity results of the anti-human ITGβ6 fully human affinity matured antibodies are shown in Figure 3A, Figure 3B and Table 16, respectively. The affinity of mAb2, mAb1 affinity matured antibodies on HCC1954 cells was significantly enhanced compared with mAb2, mAb1.

[0399] Table 16: Results of HCC1954 cell affinity assay of anti-human ITGβ6 fully human affinity matured antibodies

[0400] Example 9: Endocytosis activity detection of anti-human ITGβ6 fully human antibodies

[0401] The endocytosis activity of anti-human ITGβ6 fully human antibodies on human breast ductal carcinoma cells HCC1954 (Connocell) was detected by flow cytometry (Thermo, model Attune NxT). Cells growing adherently were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted, and the cell density was adjusted to 1x10 5 cells / ml, 100ul cell suspension was added to each well of a 96-well plate (the number of cells was 1x10 4After 24h incubation, the 96-well plate was taken out, and the medium was removed. Then, 50 μl of fresh complete medium was added to each well. The test antibody and the negative control antibody hlgG1 were diluted with the complete medium at a final concentration of 8 nM, and 3-fold gradient dilution was performed, resulting in 8 concentration points. The PHrodo red reagent was diluted with the complete medium to 12 μg / ml (the final concentration of PHrodo red was 3 μg / ml). The gradient-diluted test antibody was mixed with the diluted PHrodo reagent at a ratio of 1:1 (30 μl:30 μl), and incubated at room temperature for 30 min in the dark. Then, 50 μl of the test antibody and PHrodo reagent mixture was added to the 96-well plate, and incubated at 37 °C for 24 h in a CO2 incubator. After 24h incubation, the 96-well plate was taken out, and the medium was removed. Then, 100 μl of Trypsin-EDTA (0.25%) was added to each well to digest the cells, and 100 μl of complete medium was added to neutralize the trypsin. After the cells were dispersed by blowing, the FACS machine was used for detection. Data processing: the Median YL1-H value was exported, and then imported into the data analysis software to calculate the EC50 value. 50 As shown in FIGS. 4A, 4B, 4C and Table 17, the endocytosis activity of the mAb2 and mAb1 affinity matured antibodies on HCC1954 cells was significantly enhanced compared with mAb2 and mAb1.

[0402] Table 17: Endocytosis activity of anti-human ITGβ6 fully human affinity matured antibodies

[0403] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details without departing from the spirit and scope of the application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ITGβ6, wherein, The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 62, 63, 61, or 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 4; (b) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 58, 59, 60, or 2; (c) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 5; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 6; or (d) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) and / or CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL), wherein the heavy chain variable region (VH) and / or light chain variable region (VL) comprises at least one mutation, which is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids), compared to the heavy chain variable region and / or light chain variable region of any one of (a)-(c); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the Chothia, AbM, Kabat, or IMGT numbering system. The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

2. The antibody or antigen binding fragment thereof specifically binding to ITGβ6 of claim 1, wherein, (a) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 62; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 4; (b) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 63; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 4; (c) CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 61; and, CDR-L1, CDR-L2, and CDR-L3 contained in a light chain variable region (VL) as shown in SEQ ID NO: 4; ​ (d) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 3; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO: 4; (e) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO: 58; (f) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO: 59; (g) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO: 60; (h) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 1; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO: 2; or (i) CDR-H1, CDR-H2, and CDR-H3 contained in the VH region as depicted in SEQ ID NO: 5; and, CDR-L1, CDR-L2, and CDR-L3 contained in the VL region as depicted in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof; (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof; (1 b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 13 or a variant thereof; CDR-H2 of SEQ ID NO: 14 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or (1 c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof; CDR-H2 of SEQ ID NO: 40 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 48 or a variant thereof; CDR-H2 of SEQ ID NO: 49 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof; (2b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 46 or a variant thereof; CDR-H2 of SEQ ID NO: 47 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or (2c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 39 or a variant thereof; CDR-H2 of SEQ ID NO: 40 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof. (2c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 50 or a variant thereof; CDR-H2 of SEQ ID NO: 51 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof; or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 31 or a variant thereof; CDR-H2 of SEQ ID NO: 32 or a variant thereof; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 29 or a variant thereof; CDR-L2 of SEQ ID NO: 30 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof; (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 15 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 16 or a variant thereof; CDR-L2 of SEQ ID NO: 17 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or (3c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 44 or a variant thereof; CDR-H2 of SEQ ID NO: 45 or a variant thereof; CDR-H3 of SEQ ID NO: 41 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42 or a variant thereof; CDR-L2 of SEQ ID NO: 43 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof; or, (4) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 20 or a variant thereof; CDR-H2 of SEQ ID NO: 21 or a variant thereof; CDR-H3 of SEQ ID NO: 70, 72, 68, or 22 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 23 or a variant thereof; CDR-L2 of SEQ ID NO: 24 or a variant thereof; CDR-L3 of SEQ ID NO: 25 or a variant thereof; (4b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 7 or a variant thereof; CDR-H2 of SEQ ID NO: 8 or a variant thereof; CDR-H3 of SEQ ID NO: 9 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 10 or a variant thereof; CDR-L2 of SEQ ID NO: 11 or a variant thereof; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12 or a variant thereof; or (4c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof; CDR-H2 of SEQ ID NO: 34 or a variant thereof; CDR-H3 of SEQ ID NO: 35 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 36 or a variant thereof; CDR-L2 of SEQ ID NO: 37 or a variant thereof; CDR-L3 of SEQ ID NO: 38 or a variant thereof; wherein the variant of any one of (1a), (1b), (1c), (2a), (2b), (2c), (3a), (3b), (3c), (4a), (4b), (4c) has one or several (e.g., 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 1 or a variant thereof; CDR-H2 of SEQ ID NO: 2 or a variant thereof; CDR-H3 of SEQ ID NO: 3 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 4 or a variant thereof; CDR-L2 of SEQ ID NO: 5 or a variant thereof; CDR-L3 of SEQ ID NO: 6 or a variant thereof; (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 26; CDR-H2 of SEQ ID NO: 27; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; CDR-L3 of SEQ ID NO: 25; (1b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 13; CDR-H2 of SEQ ID NO: 14; CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or (1c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 39; CDR-H2 of SEQ ID NO: 40; CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; CDR-L3 of SEQ ID NO: 38; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 48; CDR-H2 of SEQ ID NO: 49; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; CDR-L3 of SEQ ID NO: 25; (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 46; CDR-H2 of SEQ ID NO: 47; CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or (2c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 50; CDR-H2 of SEQ ID NO: 51; CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; CDR-L3 of SEQ ID NO: 38; or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) wherein the CDRs are defined by the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 31; CDR-H2 of SEQ ID NO: 32; CDR-H3 of SEQ ID NO: 69, 71, 67, or 28; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 29; CDR-L2 of SEQ ID NO: 30; CDR-L3 of SEQ ID NO: 25; (3b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 18; CDR-H2 of SEQ ID NO: 19; CDR-H3 of SEQ ID NO: 15; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 16; CDR-L2 of SEQ ID NO: 17; CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or (3c) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 50; CDR-H2 of SEQ ID NO: 51; CDR-H3 of SEQ ID NO: 41; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; CDR-L3 of SEQ ID NO:

38. (3c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 44; CDR-H2 of SEQ ID NO: 45; and CDR-H3 of SEQ ID NO: 41; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 42; CDR-L2 of SEQ ID NO: 43; and CDR-L3 of SEQ ID NO: 38; or, (4) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 20; CDR-H2 of SEQ ID NO: 21; and CDR-H3 of SEQ ID NO: 70, 72, 68, or 22; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 23; CDR-L2 of SEQ ID NO: 24; and CDR-L3 of SEQ ID NO: 25; (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 10; CDR-L2 of SEQ ID NO: 11; and CDR-L3 of SEQ ID NO: 64, 65, 66, or 12; or (4c) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 33; CDR-H2 of SEQ ID NO: 34; and CDR-H3 of SEQ ID NO: 35; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 36; CDR-L2 of SEQ ID NO: 37; and CDR-L3 of SEQ ID NO:

38.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, the antibody or antigen binding fragment thereof comprises: (a) a VH comprising a sequence as set forth in SEQ ID NO: 62, 63, 61, or 3, or a variant thereof, and / or a VL comprising a sequence as set forth in SEQ ID NO: 4, or a variant thereof; (b) a VH comprising a sequence as set forth in SEQ ID NO: 1, or a variant thereof, and / or a VL comprising a sequence as set forth in SEQ ID NO: 58, 59, 60, or 2, or a variant thereof; (c) a VH comprising a sequence as set forth in SEQ ID NO: 5, or a variant thereof, and / or a VL comprising a sequence as set forth in SEQ ID NO: 6, or a variant thereof; wherein said variant has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived or comprises a substitution, deletion, or addition of one or a few amino acids (e.g., 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived; preferably, said substitution is a conservative substitution.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein, The antibody or antigen-binding fragment thereof comprises: (a) a VH comprising a sequence as set forth in SEQ ID NO: 62, and a VL comprising a sequence as set forth in SEQ ID NO: 4; (b) a VH comprising a sequence as set forth in SEQ ID NO: 63, and a VL comprising a sequence as set forth in SEQ ID NO: 4; (c) a VH comprising a sequence as set forth in SEQ ID NO: 61, and a VL comprising a sequence as set forth in SEQ ID NO: 4; (d) a VH comprising a sequence as set forth in SEQ ID NO: 3, and a VL comprising a sequence as set forth in SEQ ID NO: 4; (e) a VH comprising a sequence as set forth in SEQ ID NO: 1, and a VL comprising a sequence as set forth in SEQ ID NO: 58; (f) a VH comprising a sequence as set forth in SEQ ID NO: 1, and a VL comprising a sequence as set forth in SEQ ID NO: 59; (g) a VH comprising a sequence as set forth in SEQ ID NO: 1, and a VL comprising a sequence as set forth in SEQ ID NO: 60; (h) a VH comprising a sequence as set forth in SEQ ID NO: 1, and a VL comprising a sequence as set forth in SEQ ID NO: 2; or (i) a VH comprising a sequence as set forth in SEQ ID NO: 5, and a VL comprising a sequence as set forth in SEQ ID NO:

6.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, The antibody or antigen-binding fragment thereof is a murine, chimeric, or humanized antibody.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, The antibody or antigen-binding fragment thereof further comprises a constant region from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4); preferably, the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or a mutated or chemically modified Fc region having an altered effector function as compared to a wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of a human IgGl heavy chain constant region having the following substitutions as compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala, and Gly237Ala (positions according to the EU numbering system); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises or is derived from a light chain constant region from a human immunoglobulin (e.g. kappa or lambda); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 76 or a variant thereof having up to 20 conservative substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g. 1, 2, 3, 4 or 5 amino acid conservative substitutions) compared to SEQ ID NO: 76; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 76 or a variant thereof having up to 20 conservative substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g. 1, 2, 3, 4 or 5 amino acid conservative substitutions) compared to SEQ ID NO: 76; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 76 or a variant thereof having up to 20 conservative substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g. 1, 2, 3, 4 or 5 amino acid conservative substitutions) compared to SEQ ID NO: 76; More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 76 or 77 and a light chain constant region (CL) as set forth in SEQ ID NO: 73 or 74.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein, The heavy chain constant region (CH) as set forth in SEQ ID NO: 76 or 77 or a variant thereof lacks a C-terminal lysine.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 62 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 74; (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 62 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 74; (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 62 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 74; (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 62 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 74; (5) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 58 and a light chain constant region (CL) as set forth in SEQ ID NO: 73; (6) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 59 and a light chain constant region (CL) as set forth in SEQ ID NO: 73; (7) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 60 and a light chain constant region (CL) as set forth in SEQ ID NO: 73; (8) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 73; or (9) a heavy chain comprising a VH as set forth in SEQ ID NO: 5 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 77, and, a light chain comprising a VL as set forth in SEQ ID NO: 6 and a light chain constant region (CL) as set forth in SEQ ID NO:

74.

11. The antibody or antigen-binding fragment thereof of claim 5, 6, or 10, wherein, The N-terminal glutamine of the VH comprising a sequence as set forth in any one of SEQ ID NOs: 62, 63, 61, 3, 1, 5, or a variant thereof, is subjected to cyclization to form pyroglutamic acid or pyroglutamate.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein, The antibody or antigen binding fragment thereof comprises: (1) a heavy chain having a sequence as set forth in SEQ ID NO: 78 and a light chain having a sequence as set forth in SEQ ID NO: 79; (2) a heavy chain having a sequence as set forth in SEQ ID NO: 80 and a light chain having a sequence as set forth in SEQ ID NO: 79; (3) a heavy chain having a sequence as set forth in SEQ ID NO: 87 and a light chain having a sequence as set forth in SEQ ID NO: 79; (4) a heavy chain having a sequence as set forth in SEQ ID NO: 88 and a light chain having a sequence as set forth in SEQ ID NO: 79; (5) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 89; (6) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 90; (7) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 91; (8) a heavy chain having a sequence as set forth in SEQ ID NO: 93 and a light chain having a sequence as set forth in SEQ ID NO: 92; or (9) a heavy chain having a sequence as set forth in SEQ ID NO: 94 and a light chain having a sequence as set forth in SEQ ID NO:

95.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein, the N-terminal glutamine of the heavy chain having a sequence as set forth in any one of SEQ ID NOs: 78, 80, 87, 88, 93, 94 is cyclized to form pyroglutamic acid or pyroglutamate; and / or the C-terminal lysine of the heavy chain having a sequence as set forth in any one of SEQ ID NOs: 78, 80, 87, 88, 93, 94 is lacking.

14. The antibody or antigen-binding fragment thereof of claim 13, wherein, the antibody comprises: (1) a heavy chain having a sequence as set forth in SEQ ID NO: 81 and a light chain having a sequence as set forth in SEQ ID NO: 79; (2) a heavy chain having a sequence as set forth in SEQ ID NO: 82 and a light chain having a sequence as set forth in SEQ ID NO: 79; (3) a heavy chain having a sequence as set forth in SEQ ID NO: 83 and a light chain having a sequence as set forth in SEQ ID NO: 79; (4) a heavy chain having a sequence as set forth in SEQ ID NO: 84 and a light chain having a sequence as set forth in SEQ ID NO: 79; (5) a heavy chain having a sequence as set forth in SEQ ID NO: 85 and a light chain having a sequence as set forth in SEQ ID NO: 79; or (6) a heavy chain having a sequence as set forth in SEQ ID NO: 86 and a light chain having a sequence as set forth in SEQ ID NO:

79.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein, the antibody or antigen-binding fragment thereof is selected from the group consisting of a ScFv, a Fab, a Fab', a Fab'-SH, a F(ab')2, an Fv fragment, a disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein, the antibody or antigen-binding fragment thereof carries a label; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof has one or more features selected from the group consisting of: (1) specifically binds human or monkey ITGβ6, e.g., as determined by ELISA or flow cytometry; (2) specifically binds human or monkey αVβ6, e.g., as determined by ForteBio; (3) does not bind αVβ1, αVβ3, αVβ5, ITGβ8, and ITGαV, e.g., as determined by flow cytometry; (4) has reduced or eliminated ADCC activity; (5) has reduced CDC activity; (6) induces ITGβ6 internalization, e.g., as determined by flow cytometry; (7) inhibits cell (such as tumor cell) proliferation; and / or (8) inhibits tumor growth.

18. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof, of any one of claims 1-17.

19. The isolated nucleic acid molecule of claim 18, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein, the nucleic acid molecule encoding an antibody heavy chain variable region has a sequence selected from the group consisting of: (a) a nucleotide sequence as set forth in SEQ ID NO: 54, 52, or 56, or (b) a sequence substantially identical to the nucleotide sequence recited in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity to the nucleotide sequence recited in (a), or a sequence having one or more nucleotide substitutions), or (c) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence recited in (a); and / or, the nucleic acid molecule encoding an antibody light chain variable region has a sequence selected from the group consisting of: (d) a nucleotide sequence as set forth in SEQ ID NO: 55, 53, or 57, or (e) a sequence substantially identical to the nucleotide sequence recited in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99%, or more sequence identity to the nucleotide sequence recited in (d), or a sequence having one or more nucleotide substitutions), or (f) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence recited in (d).

20. The isolated nucleic acid molecule of claim 19, wherein: (1) the nucleic acid molecule encoding an antibody heavy chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 54, and / or the nucleic acid molecule encoding an antibody light chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 55; (2) the nucleic acid molecule encoding an antibody heavy chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 52, and / or the nucleic acid molecule encoding an antibody light chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 53; or (3) the nucleic acid molecule encoding an antibody heavy chain variable region has a nucleotide sequence as set forth in SEQ ID NO: 56, and / or the nucleic acid molecule encoding an antibody light chain variable region has a nucleotide sequence as set forth in SEQ ID NO:

57.

21. A vector comprising the nucleic acid molecule of any one of claims 18-20; preferably, the vector is a cloning vector or an expression vector.

22. A host cell comprising the nucleic acid molecule of any one of claims 18-20 or the vector of claim 21.

23. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-17, comprising culturing the host cell of claim 22 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

24. The method of claim 23, wherein the host cell is a Chinese hamster ovary cell.

25. The antibody or antigen-binding fragment thereof obtainable by the method of claim 23 or 24.

26. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17 and a conjugating moiety linked thereto; Preferably, the conjugating moiety is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin, or a radionuclide).

27. A multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof of any one of claims 1-17 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed to another target; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

28. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof (e.g., ScFv) of any one of claims 1-17, a transmembrane domain, and one or more intracellular T cell signaling domains.

29. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen-binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: an ITGβ6 inhibitor, a TROP2 inhibitor, a B7H3 inhibitor, a PTK7 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof; Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as a mixed component.

30. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen-binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition of claim 29, and optionally instructions for use and / or a device for administration.

31. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen-binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition of claim 29, for the manufacture of a medicament for the treatment and / or adjuvant treatment of a tumor. Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination, e.g. simultaneously, separately or sequentially, with a further pharmaceutically active agent. Preferably, the further pharmaceutically active agent is a drug having anti-tumor activity. Preferably, the further pharmaceutically active agent is selected from the group consisting of an ITGβ6 inhibitor, a TROP2 inhibitor, a B7H3 inhibitor, a PTK7 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic drug or any combination thereof.

32. The use of claim 31, wherein the tumor is an ITGβ6-positive tumor. Preferably, the tumor is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or cholangiocarcinoma.

33. A method of inhibiting cell proliferation, comprising contacting the cell with the antibody or antigen-binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen-binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition of claim 29; Preferably, the cell is an ITGβ6-expressing cell, e.g. a tumor cell. Preferably, the tumor cell overexpresses ITGβ6.

34. A method for treating and / or adjuvant treatment of a tumor in a subject, said method comprising administering to a subject in need thereof an effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing said chimeric antigen receptor, or the pharmaceutical composition of claim 29.

35. The method of claim 34, further comprising administering to said subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; Optionally, said second therapy can be applied simultaneously, separately or sequentially to the method of claim 34.

36. The method of claim 34 or 35, wherein said tumor is an ITGβ6-positive tumor; Preferably, said tumor is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or cholangiocarcinoma, or any combination thereof.

37. A method of detecting the presence or level of ITGβ6 in a sample, comprising contacting said sample with the antibody or antigen binding fragment thereof of any one of claims 1-17 under conditions allowing the formation of a complex between said antibody or antigen binding fragment thereof and ITGβ6, and detecting the formation of said complex; Preferably, said method is for diagnosing a tumor, such as an ITGβ6-positive tumor, such as colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or cholangiocarcinoma, or any combination thereof; Preferably, said method comprises detecting the expression level of ITGβ6 in a test sample from a subject, and comparing the expression level to a reference value, wherein an increased expression level compared to the reference value is indicative of a tumor.

38. Use of the antibody or antigen binding fragment thereof of any one of claims 1-17, or the isolated nucleic acid molecule of any one of claims 18-20, or the vector of claim 21, or the host cell of claim 22, or the antibody or antigen binding fragment thereof of claim 25, or the conjugate of claim 26, or the multispecific antibody of claim 27, in the manufacture of a diagnostic kit for detecting the presence or level of ITGβ6 in a sample and / or diagnosing a tumor; Preferably, said tumor is an ITGβ6-positive tumor. Preferably, said tumor is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, skin cancer, head and neck cancer, oral cancer, or biliary duct cancer or any combination thereof.

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