ITGβ6 antibody and use thereof

Abstract

Provided are an ITGβ6 antibody and use thereof. The antibody or an antigen-binding fragment thereof can specifically recognize / bind to ITGβ6, does not bind to ITGβ1, ITGβ3, ITGβ5, ITGβ8, ITG αV, αVβ1, αVβ3, αVβ5, and / or αVβ8, and has good endocytic activity. As the FcγR binding function is removed, the antibody or the antigen-binding fragment thereof has no ADCC activity and / or CDC activity, thus effectively avoiding side reactions caused by ADCC and / or CDC functions. The antibody does not bind to the Fc receptor CD16a, CD32a, or CD64 protein, thereby reducing Fc receptor-mediated non-specific killing and improving drug safety. Moreover, the antibody retains FcRn protein-binding activity, so that the drug half-life is not affected.

Description

An ITGβ6 antibody and its uses Technical Field

[0001] This disclosure relates to the field of biomedicine, specifically to an ITGβ6 antibody and its uses. Background Technology

[0002] The integrin superfamily is a class of transmembrane receptors on the cell surface. They primarily recognize extracellular matrix ligands and cell surface ligands (and also bind some soluble ligands), mediating cell adhesion and cell motility, establishing transmembrane connections with the cytoskeleton, and transmitting bidirectional signals between the intracellular and extracellular environments. Integrins must form heterodimers through the non-covalent binding of their α and β subunits to function. At least 18 α subunits and 8 β subunits have been identified in humans, resulting in 24 combinations, each with different ligand specificities, tissue and cellular distribution, and physiological functions. The integrin β6 (ITGβ6) subunit binds only to the αV subunit. Both αV and β6 are single-transmembrane proteins, composed of an extracellular domain (head + movable leg), a transmembrane segment, and a cytoplasmic tail. αVβ6 is expressed only in certain specific tissue remodeling-related epithelial cells. αVβ6 plays a role in tissue remodeling by binding to multiple ligands, and is involved in various physiological and pathological repair events during embryonic development. Specifically, it includes: (1) binding to extracellular matrix (ECM) molecules such as fibronectin, tendinin-C, and porphyrin to mediate cell adhesion and migration; (2) locally activating transforming growth factor (TGF-β1) to exert anti-inflammatory monitoring, maintain epithelial cell quiescence, and feedback regulate β6 expression. The pathological function of αVβ6 is to promote tumor cell survival, proliferation, invasion, and metastasis by utilizing its tissue remodeling function, and to induce chemotherapy / radiotherapy resistance in tumor cells. It is a poor 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. In the tumor microenvironment, it plays a key role in promoting tumor progression and metastasis.

[0003] Currently, there are no marketed antibody drugs targeting ITGβ6. Therefore, it is urgent and necessary to develop antibodies targeting ITGβ6 with higher specificity, lower toxicity, better clinical efficacy, and more convenient administration methods, which will provide patients with more treatment options. Summary of the Invention

[0004] This disclosure provides an ITGβ6 antibody or its antigen-binding fragment that specifically recognizes / binds to ITGβ6, but does not bind to ITGβ1, ITGβ3, ITGβ5, ITGβ8, ITGαV, αVβ1, αVβ3, αVβ5 and / or αVβ8, exhibiting good endocytic activity. Furthermore, the FcγR binding function of this antibody or its antigen-binding fragment is removed, eliminating ADCC and / or CDC activity, effectively avoiding side effects caused by ADCC and / or CDC function. This antibody does not bind to Fc receptors CD16a, CD32a and CD64 proteins, reducing Fc receptor-mediated non-specific killing and improving drug safety, while retaining FcRn protein binding activity without affecting the drug's half-life.

[0005] According to one aspect of this disclosure, an ITGβ6 antibody or an antigen-binding fragment thereof is provided, said ITGβ6 antibody or antigen-binding fragment comprising:

[0006] a1) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 3; and / or having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or

[0007] a2) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or, having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or

[0008] a3) having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in any of a1) to a2); and / or having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in any of a1) to a2).

[0009] In some implementations, the CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0010] In some embodiments, the ITGβ6 includes human ITGβ6, monkey ITGβ6, and / or mouse ITGβ6; further, human ITGβ6.

[0011] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0012] b1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 30, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 32; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-L2 having the amino acid sequence DAS, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0013] b2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 10, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-L2 having the amino acid sequence DAS, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0014] b3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of b1) to b2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of b1) to b2);

[0015] The CDR is defined according to the IMGT numbering system.

[0016] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0017] c1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 33, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 34, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0018] c2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 15, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 16, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0019] c3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of c1) to c2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of c1) to c2);

[0020] The CDR is defined according to the Kabat numbering system.

[0021] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0022] d1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 36, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 37, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0023] d2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 20, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 21, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0024] d3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of d1) to d2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of d1) to d2);

[0025] The CDR is defined according to the AbM numbering system.

[0026] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0027] e1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 38, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 39, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0028] e2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 22, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 23, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or

[0029] e3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of e1) to e2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of e1) to e2);

[0030] The CDR is defined according to the Chothia numbering system.

[0031] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0032] f1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 4; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 27, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 28, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 29; or

[0033] f2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 25, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 26; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 27, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 28, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 29; or

[0034] f3) VHs including the following three CDRs: CDR-H1, CDR-H2, and CDR-H3 having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in any of f1) to f2); and / or VLs including the following three CDRs: CDR-L1, CDR-L2, and CDR-L3 having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in any of f1) to f2);

[0035] The CDR is defined according to the Contact numbering system.

[0036] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0037] In some embodiments, the heavy chain variable region of the ITGβ6 antibody or its antigen-binding fragment may include the framework region of the heavy chain variable region.

[0038] In some embodiments, the framework region of the heavy chain variable region may include the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the framework region of the heavy chain variable region may include the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably including the framework region of the heavy chain variable region or a mutant thereof derived from human immunoglobulins.

[0039] In some embodiments, the light chain variable region of the ITGβ6 antibody or its antigen-binding fragment may include the framework region of the light chain variable region.

[0040] In some embodiments, the framework region of the light chain variable region may include the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the framework region of the light chain variable region may include the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably including the framework region of the light chain variable region or a mutant thereof derived from human immunoglobulins.

[0041] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0042] g1) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0043] g2) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 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%, or at least 99% sequence identity with it.

[0044] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment may further include a heavy chain constant region and / or a light chain constant region.

[0045] In some embodiments, the heavy chain constant region may include at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the heavy chain constant region may include at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably including at least a portion of the heavy chain constant region or a mutant thereof derived from human immunoglobulins. The mutant of the heavy chain constant region has altered effector functions (e.g., reduced ADCC activity and / or CDC activity) compared to the wild-type heavy chain constant region.

[0046] In some embodiments, the light chain constant region may include at least a portion of a light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, or geese. In some embodiments, the light chain constant region may include at least a portion of a light chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans, preferably including a light chain constant region or a mutant thereof derived from human immunoglobulins.

[0047] In some embodiments, the heavy chain constant region may include a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin, preferably a heavy chain constant region derived from IgG1 immunoglobulin or a mutant thereof.

[0048] In some embodiments, the heavy chain constant region may include the human IgG1 heavy chain constant region or a mutant thereof.

[0049] In some embodiments, the mutant of the human IgG1 heavy chain constant region may include the following substitutions compared to the wild-type sequence from which it originates: L234A, L235A and / or G237A (preferably L234A, L235A and G237A);

[0050] The amino acid positions mentioned above are based on the EU numbering system.

[0051] In some embodiments, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 7 or 9, or an amino acid sequence having 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%, or at least 99% sequence identity with it, or having at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 conserved amino acid substitutions compared to it.

[0052] In some embodiments, the light chain constant region may include light chain constant regions derived from κ and λ immunoglobulins, preferably including light chain constant regions derived from κ immunoglobulins.

[0053] In some embodiments, the light chain constant region may include the amino acid sequence shown in SEQ ID NO: 8 or 51, or an amino acid sequence having 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%, or at least 99% sequence identity with it, or having at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 conserved amino acid substitutions with it.

[0054] In some embodiments, the ITGβ6 antibody or antigen-binding fragment disclosed herein may include post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamate or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0055] In some embodiments of the ITGβ6 antibody or antigen-binding fragment disclosed herein, the heavy chain constant region may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the ITGβ6 antibody or antigen-binding fragment thereof, the N-terminal glutamate or glutamine of the ITGβ6 antibody or antigen-binding fragment thereof may be cyclized to pyroglutamate. In some embodiments of the ITGβ6 antibody or antigen-binding fragment thereof, the N-terminal glutamate or glutamine of the ITGβ6 antibody or antigen-binding fragment thereof may be cyclized to pyroglutamate salt.

[0056] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0057] In some embodiments, the heavy and / or light chains of the ITGβ6 antibody or antigen-binding fragment may include a C-terminal lysine, a lack of a C-terminal lysine, or a lack of a C-terminal glycine-lysine.

[0058] In some embodiments, the heavy and / or light chains of the ITGβ6 antibody or antigen-binding fragment may include N-terminal glutamine or glutamate, N-terminal glutamate or glutamine cyclized to pyroglutamate, or N-terminal glutamate or glutamine cyclized to pyroglutamate. In some embodiments, compositions comprising the ITGβ6 antibody or antigen-binding fragments disclosed herein are provided, wherein various ITGβ6 antibody or antigen-binding fragments may independently comprise C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine, and / or comprise N-terminal glutamine or glutamate, N-terminal glutamate or glutamine cyclized to pyroglutamate, or N-terminal glutamate or glutamine cyclized to pyroglutamate.

[0059] In some embodiments, the N-terminal glutamine of the heavy chain variable region having a sequence as shown in SEQ ID NO: 3 or 1 or a variant thereof (the variant having 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 with respect to the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions) undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0060] In some embodiments, the N-terminal glutamine of the light chain variable region having the sequence shown in SEQ ID NO: 2 or a variant thereof (the variant having 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 with respect to the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions) undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0061] In some embodiments, the C-terminus of the heavy chain constant region having a sequence as shown in SEQ ID NO: 7 or 9 or a variant thereof (the variant having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 7 or 9, such as up to 15, up to 10, or up to 5 amino acid substitutions; for example, 1, 2, 3, 4, or 5 amino acid substitutions) lacks a lysine residue.

[0062] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment may be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0063] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment comprises:

[0064] q1) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 44, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0065] q2) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0066] q3) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0067] q4) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0068] q5) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0069] q6) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 44, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0070] q7) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0071] q8) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0072] q9) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0073] q10) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0074] q11) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0075] q12) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0076] q13) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or

[0077] q14) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having 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%, or at least 99% sequence identity with it.

[0078] In some embodiments, the antigen-binding fragment of the ITGβ6 antibody may include, but is not limited to, Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv or Fd fragment.

[0079] In some embodiments, the ITGβ6 antibody or antigen-binding fragment thereof disclosed herein is capable of binding ITGβ6 (e.g., human, monkey, or mouse ITGβ6) at an EC50 of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 0.1 nM, or lower.

[0080] In some embodiments, the ITGβ6 antibody or antigen-binding fragment thereof disclosed herein is capable of binding ITGβ6 (e.g., human, monkey, or mouse ITGβ6) at a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or lower.

[0081] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein can induce the internalization (with good endocytic activity) of ITGβ6 (e.g., human, monkey, or mouse ITGβ6).

[0082] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein has a significantly reduced HIC value (compared to 2A2 in CN114828887A).

[0083] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein is capable of specifically binding to human ITGβ6, monkey ITGβ6 or mouse ITGβ6.

[0084] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein is capable of specifically binding to human αVβ6, monkey αVβ6 or mouse αVβ6.

[0085] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein does not bind or substantially does not bind to ITGβ1, ITGβ3, ITGβ5, ITGβ8, ITGαV, αVβ1, αVβ3, αVβ5 and / or αVβ8.

[0086] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein does not have ADCC activity and / or CDC activity.

[0087] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein does not compete with the 2A2 epitope in CN114828887A.

[0088] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment disclosed herein does not bind to Fc receptors CD16a, CD32a and / or CD64 proteins.

[0089] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment of the present disclosure binds to the FcRn protein.

[0090] According to another aspect of this disclosure, a multispecific antibody or antigen-binding fragment thereof is provided, the multispecific antibody or antigen-binding fragment thereof comprising two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the ITGB6 antibody or antigen-binding fragment thereof described above in this disclosure.

[0091] In some embodiments, the individual antigen-binding domains of the multispecific antibody or its antigen-binding fragment retain their respective antigen-binding specificity.

[0092] According to another aspect of this disclosure, a chimeric antigen receptor is provided, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the ITGβ6 antibody or its antigen-binding fragment described above, or the multispecific antibody or its antigen-binding fragment described above.

[0093] According to another aspect of this disclosure, isolated nucleic acid molecules are provided that encode the nucleotide sequences of the aforementioned ITGβ6 antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, or the aforementioned multispecific antibody or its antigen-binding fragment.

[0094] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0095] According to another aspect of this disclosure, a carrier is provided that includes the nucleic acid molecules described above.

[0096] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0097] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0098] According to another aspect of this disclosure, a cell is provided comprising the ITGβ6 antibody or its antigen-binding fragment thereof, the chimeric antigen receptor thereof, the multispecific antibody or its antigen-binding fragment thereof, the nucleic acid molecule thereof, or the vector thereof.

[0099] In some embodiments, the cells do not involve reproductive material.

[0100] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the ITGβ6 antibody or its antigen-binding fragment, chimeric antigen receptor, or multispecific antibody or its antigen-binding fragment as disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0101] In some embodiments, the cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, dendritic cells (DCs), and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above in this disclosure (i.e., modified immune cells).

[0102] According to another aspect of this disclosure, a method for preparing the aforementioned ITGβ6 antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, or the aforementioned multispecific antibody or its antigen-binding fragment is provided. The method includes culturing the aforementioned host cells of this disclosure under conditions allowing expression of the aforementioned ITGβ6 antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, or the aforementioned multispecific antibody or its antigen-binding fragment; and collecting the aforementioned ITGβ6 antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, or the aforementioned multispecific antibody or its antigen-binding fragment from the cultured host cell culture.

[0103] According to another aspect of this disclosure, a conjugate is provided, comprising the aforementioned ITGβ6 antibody or its antigen-binding fragment thereof, or the aforementioned multispecific antibody or its antigen-binding fragment thereof; and a conjugation portion.

[0104] In some implementations, the coupling portion may include, but is not limited to, detectable markers or therapeutic agents.

[0105] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-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 dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the ITGβ6 antibody or its antigen-binding fragment of the present disclosure, or the multispecific antibody or its antigen-binding fragment of the present disclosure, using linkers of different lengths to reduce potential steric hindrance.

[0106] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0107] In some embodiments, the therapeutic agent may include, for example, but not limited to, chemotherapeutic agents, immunosuppressants, and cytotoxic drugs.

[0108] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0109] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising the above-described ITGβ6 antibody or antigen-binding fragment thereof, the above-described chimeric antigen receptor, the above-described multispecific antibody or antigen-binding fragment thereof, the above-described nucleic acid molecule, the above-described carrier, the above-described cell or the above-described conjugate; and a pharmaceutically acceptable carrier.

[0110] In some embodiments, the pharmaceutical composition may also include other pharmaceutically active agents.

[0111] In some embodiments, the other pharmaceutically active agent may be a biologically active drug, such as a drug capable of treating diseases or conditions related to ITGβ6. In some embodiments, the other pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the other pharmaceutically active agent may be selected from, but is not limited to, tumor immunotherapy agents and / or chemotherapy drugs.

[0112] In some embodiments, the tumor immunotherapy agent comprises at least one of the following: monoclonal antibody, immune checkpoint inhibitor, immune cell, oncolytic virus, and tumor vaccine.

[0113] In some embodiments, the target of the monoclonal antibody is selected from one of CD20, HER2, VEGF / VEGFR, EGFR, CD19, FGL1, CD47, CD3, CD30, CD33, CD38, CD52, αVβ3, α5β1, FAP, Tenascin, CEA, EPCAM, PSMA, GAN-GD2, GAN-GD3, GM2, and IGF-IR.

[0114] In some embodiments, the immune checkpoint inhibitor is an inhibitor that acts on negative co-stimulatory (co-inhibitory) molecules of T cells and / or their respective ligands.

[0115] In some embodiments, the negative co-stimulatory (co-inhibitory) molecules acting on T cells and / or their respective ligands are selected from one or more of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2aR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, CD20, CD96, CD73, CD160, STING, CEA, CD47, PVRIG, LAIR1, 2B4, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and DcR3.

[0116] In some embodiments, the inhibitors acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands comprise any one of (b1) to (b3):

[0117] (b1) Antibodies that specifically bind (neutralize) negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands to T cells;

[0118] (b2) Specific binding (neutralization) of ligand proteins or peptides that act on negative costimulatory (co-inhibitory) molecules and / or their respective ligands on T cells;

[0119] (b3) Non-protein compounds that specifically bind (neutralize) to negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands on T cells.

[0120] In some embodiments, the inhibitor of the ligand of the T-cell negative co-stimulatory (co-inhibitory) molecule is selected from: CTLA-4 inhibitors (e.g., ipilimumab, tremelimumab, AAGEN-1884, ATOR-1015, MGD019 (PD-1 / CTLA-4 bispecific antibody)), PD-1 inhibitors (e.g., nivolumab, tislelizumab (BGB-A317), spartazumab). (spartalizumab), MEDI0680, PDR001, MGA012 (retifanlimab), sintilimab, toripalimab, cemiplimab, MGD019 (PD-1 / CTLA-4 bispecific antibody), MGD013 (tebotelimab, PD-1 / LAG-3 bispecific antibody), PD-L1 inhibitors (e.g., atezolizumab). ab), camrelizumab, durvalumab, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105), PD-L2 inhibitors, B7-1 inhibitors, B7-2 inhibitors, B7-H3 inhibitors (e.g., enoblituzumab, MGD009, MGC018), B7- H4 inhibitors, B7-H6 inhibitors, A2aR inhibitors (CPI-444, PBF509), IDO inhibitors (e.g., GDC0919 (navoximod), epadostat, indoximid, BMS986205), TIM-3 inhibitors (e.g., TSR022 (TIM-3 monoclonal antibody), MBG453 (TIM-3 monoclonal antibody)), BTLA inhibitors, VISTA inhibitors, TIGIT inhibitors (e.g., BMS-986207, AB...). 154, COM902 (CGEN-15137), OMP-313M32), LAG-3 inhibitors (e.g., BMS 986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-70l (LAG-5250), IMP321, TSR-033, LAG525, BI754111, FS-118, MGD013 (tebotelimab,PD-1 / LAG-3 bispecific antibodies), CD40 inhibitors (e.g., BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9 or dacetuzumab (huS2C6, PRO 64553, RG3636, SGN 14, SGN-40), CD20 inhibitors (e.g., rituximab (RITUXAN; IDEC-102; IDEC-C2B8), ABP) 798, ofatumumab or obinutuzumab), CD96 inhibitors, CD73 inhibitors (e.g., MEDI9447 (oleclumab)), CD160 inhibitors (e.g., BY55), STING inhibitors, CEA inhibitors (e.g., cergutuzumabamunaleukin (RG7813, RO-6895882) or RG7802 (RO6958688)), CD47 inhibitors (e.g., HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM), P VRIG inhibitors (e.g., COM701 (CGEN-15029)), LAIR1 inhibitors, 2B4 inhibitors, KIR inhibitors (e.g., lirilumab (1-7F9, BMS-986015, IPH2101, IPH4102), CEACAM1 inhibitors (e.g., CM-24 (MK-6018)), GARP inhibitors (e.g., ARGX-115), PS inhibitors, CSF1R inhibitors (e.g., pexidartinib, LY3022855, FPA008, BLZ945), CD94 / NKG2A inhibitors, TDO inhibitors, TNFR inhibitors, and DCR3 inhibitors, one or more of these.

[0121] In some embodiments, the immune cells comprise at least one of: chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), T cell receptor chimeric T cells (TCR-T), tumor-infiltrating immune cells (TILs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells.

[0122] In some embodiments, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus; and further comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus.

[0123] In some embodiments, the tumor vaccine comprises at least one of dendritic cell (DC) vaccines, nucleic acid vaccines, and peptide vaccines.

[0124] In some embodiments, the other pharmaceutically active agents include: ITGβ6 inhibitors, TROP2 inhibitors, B7H3 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met inhibitors, VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0125] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment, chimeric antigen receptor, multispecific antibody or its antigen-binding fragment, nucleic acid molecule, carrier, cell or conjugate, and the other pharmaceutically active agents are provided as independent components or as mixed components.

[0126] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0127] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0128] According to another aspect of this disclosure, a diagnostic or therapeutic kit is provided, the kit comprising the ITGβ6 antibody or antigen-binding fragment thereof disclosed herein, the chimeric antigen receptor thereof, the multispecific antibody or antigen-binding fragment thereof, the nucleic acid molecule thereof, the vector thereof, the cell thereof, the conjugate thereof, or the pharmaceutical composition thereof.

[0129] In an optional implementation, the kit may also include instructions and / or a delivery device.

[0130] In some implementations, the kit can be used to diagnose diseases or conditions associated with ITGβ6.

[0131] In some embodiments, the kit can be used to prevent or treat diseases or conditions associated with ITGβ6.

[0132] According to another aspect of this disclosure, the use of the above-described ITGβ6 antibody or antigen-binding fragment thereof, the chimeric antigen receptor, the multispecific antibody or antigen-binding fragment thereof, the nucleic acid molecule, the carrier, the cell, the conjugate, or the pharmaceutical composition thereof in the preparation of a medicament for inhibiting cell (e.g., cells expressing ITGβ6 protein, e.g., tumor cells expressing ITGβ6, e.g., tumor cells overexpressing ITGβ6) proliferation, or for preventing or treating diseases or conditions related to ITGβ6.

[0133] In some embodiments, the ITGβ6 antibody or its antigen-binding fragment, chimeric antigen receptor, multispecific antibody or its antigen-binding fragment, nucleic acid molecule, carrier, cell, conjugate or pharmaceutical composition is administered in combination with other pharmaceutically active agents, for example, simultaneously, separately or sequentially.

[0134] In some embodiments, the other pharmaceutically active agent is a biologically active drug, such as a drug capable of treating diseases or conditions associated with ITGβ6.

[0135] In some embodiments, the other pharmaceutically active agent is a drug with antitumor activity.

[0136] In some embodiments, the other pharmaceutically active agents may be selected from, but are not limited to, tumor immunotherapy agents and / or chemotherapy drugs.

[0137] In some embodiments, the tumor immunotherapy agent comprises at least one of the following: monoclonal antibody, immune checkpoint inhibitor, immune cell, oncolytic virus, and tumor vaccine (i.e., the above-mentioned tumor immunotherapy agent).

[0138] In some embodiments, the other pharmaceutically active agents include: ITGβ6 inhibitors, TROP2 inhibitors, B7H3 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met inhibitors, VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0139] According to another aspect of this disclosure, a method for inhibiting the proliferation of cells (e.g., cells expressing ITGβ6 protein, such as tumor cells expressing ITGβ6, such as tumor cells overexpressing ITGβ6) is provided, the method comprising contacting the cells with the aforementioned ITGβ6 antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned multispecific antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned cells, the aforementioned conjugate, or the aforementioned pharmaceutical composition.

[0140] In some embodiments, the cells may be in vitro. In some embodiments, the cells may be in vivo in a subject, wherein the subject suffers from a disease or condition related to ITGβ6.

[0141] According to another aspect of this disclosure, a method for preventing or treating diseases or conditions related to ITGβ6 is provided, the method comprising administering to a subject in need an effective amount of the aforementioned ITGB6 antibody or antigen-binding fragment thereof, the aforementioned chimeric antigen receptor, the aforementioned multispecific antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned cell, the aforementioned conjugate, or the aforementioned pharmaceutical composition.

[0142] In some embodiments, the method may further include administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0143] In some embodiments, the second therapy may be applied simultaneously, separately, or sequentially with the ITGβ6 antibody or its antigen-binding fragment described herein, the chimeric antigen receptor, the multispecific antibody or its antigen-binding fragment, the nucleic acid molecule, the carrier, the cell, the conjugate, or the pharmaceutical composition described herein.

[0144] According to another aspect of this disclosure, a method for detecting the presence or level of ITGβ6 in a sample is provided. The method includes contacting the sample with the aforementioned ITGβ6 antibody or antigen-binding fragment of this disclosure, or the aforementioned multispecific antibody or antigen-binding fragment of this disclosure, under conditions allowing the formation of a complex with ITGβ6, and detecting the formation of the complex. In such an embodiment, the method can be used to diagnose diseases or conditions related to ITGβ6. In such an embodiment, the method may include the steps of: detecting the presence or level of ITGβ6 in a sample from a subject and comparing it with a reference value (e.g., from a healthy subject); wherein an increase in the expression level compared to the reference value indicates that the subject has a disease or condition related to ITGβ6.

[0145] According to another aspect of this disclosure, the use of the above-described ITGβ6 antibody or its antigen-binding fragment or the above-described multispecific antibody or its antigen-binding fragment in the preparation of a detection kit is provided, the kit being used to detect the presence or level of ITGβ6 in a sample, or to diagnose diseases or conditions related to ITGβ6.

[0146] In this disclosure, the ITGβ6-related disease or condition can be 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 bile duct cancer, or any combination thereof. Attached Figure Description

[0147] Figure 1: Results of the binding activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) with CHOS-human / monkey / mouse ITGβ6 cells.

[0148] Figure 2: Results of the binding activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) with CHOS-human / monkey ITGβ6 cells.

[0149] Figure 3A: Results of the binding activity test between NC(hIgG1) and human ITGβ6 protein.

[0150] Figure 3B: Results of the binding activity test between Ab01 and human ITGβ6 protein.

[0151] Figure 3C: Results of the binding activity test between Ab02 and human ITGβ6 protein.

[0152] Figure 3D: Results of the binding activity test between NC(hIgG1) and monkey αVβ6 protein.

[0153] Figure 3E: Results of the binding activity test between Ab01 and monkey αVβ6 protein.

[0154] Figure 3F: Results of the binding activity test between Ab02 and monkey αVβ6 protein.

[0155] Figure 4A: Results of endocytic activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) on BT20 cells.

[0156] Figure 4B: Results of endocytic activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) on HCC70 cells.

[0157] Figure 4C: Results of endocytic activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) on HCC1954 cells.

[0158] Figure 4D: Results of endocytic activity assay of fully human anti-human ITGβ6 antibodies (Ab01, Ab02) on BxPC-3 cells.

[0159] Figure 5: Results of the binding activity assay between the fully human anti-human ITGβ6 antibody (Ab04) and CHOS-human ITGβ6 cells.

[0160] Figure 6A: Results of endocytic activity assay of HCC70 cells by the fully human anti-ITGB6 antibody (Ab04).

[0161] Figure 6B: Results of endocytic activity assay of HCC1954 cells by the fully human anti-human ITGβ6 antibody (Ab04).

[0162] Figure 6C: Results of endocytic activity assay of BXPC-3 cells by the fully human anti-human ITGβ6 antibody (Ab04).

[0163] Figure 7: Results of the detection test for competition between anti-human ITGB6 fully human antibodies (Ab03, Ab04) and the 2A2 epitope.

[0164] Figure 8: ADCC activity test results of the fully human anti-ITGB6 antibody (Ab04).

[0165] Figure 9: CDC activity test results of the fully human anti-ITGB6 antibody (Ab04). Detailed Implementation

[0166] abbreviations

[0167] CDR: Complementarity-determining region in the variable region of immunoglobulins.

[0168] FR: Antibody framework region: Amino acid residues in the antibody variable region other than CDR residues.

[0169] VH: Antibody heavy chain variable region.

[0170] VL: Variable region of antibody light chain.

[0171] IgG: Immunoglobulin G.

[0172] IMGT: Based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the numbering system of (IMGT), see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0173] Kabat: An immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0174] Chothia: An immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

[0175] AbM: The CDR definition method is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272).

[0176] Contact: The definition of CDR is derived from Martin's related research (Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)).

[0177] mAb: Monoclonal antibody.

[0178] EC 50 : The concentration that produces 50% efficacy or is combined with.

[0179] IC 50 : produces a concentration that inhibits 50%.

[0180] ELISA: Enzyme-linked immunosorbent assay.

[0181] PCR: Polymerase chain reaction.

[0182] HRP: Horseradish peroxidase.

[0183] KD: Equilibrium dissociation constant.

[0184] Ka : Binding rate constant.

[0185] Kd: Dissociation rate constant.

[0186] ADCC: Antibody-dependent cell-mediated cytotoxicity.

[0187] CDC: Complement-dependent cytotoxicity.

[0188] FACS: Flow Cytometry Technology.

[0189] CDR-H1: Complementarity-determining region 1 in the variable region of the immunoglobulin heavy chain.

[0190] CDR-H2: Complementarity-determining region 2 in the variable region of immunoglobulin heavy chain.

[0191] CDR-H3: Complementarity-determining region 3 in the variable region of the immunoglobulin heavy chain.

[0192] CDR-L1: Complementarity-determining region 1 in the variable region of the immunoglobulin light chain.

[0193] CDR-L2: Complementarity-determining region 2 in the variable region of the immunoglobulin light chain.

[0194] CDR-L3: Complementarity-determining region 3 in the variable region of the immunoglobulin light chain.

[0195] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0196] definition

[0197] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0198] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0199] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0200] Those skilled in the art will understand that when a range of values ​​is listed herein, it is intended to indicate that any intermediate value or range of values ​​within the listed range is also within the scope of this disclosure.

[0201] As used in this article, "antibody" refers to a globulin produced by the immune system in response to antigen stimulation, resulting from the proliferation and differentiation of B lymphocytes into plasma cells. Antibodies specifically bind to corresponding antigens and mediate immune effects. They are primarily found in serum and body fluids and are important immune molecules mediating humoral immunity. The term "antibody" can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0202] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0203] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0204] The terms “full-length antibody” and “intact antibody” used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0205] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0206] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0207] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0208] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0209] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of an antibody may further contain all or part of the constant regions of the heavy or light chain.

[0210] The CDR of the antibody or antigen-binding fragment thereof disclosed herein can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof disclosed herein is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0211] As used in this paper, the term "variable" refers to the fact that certain segments of the variable region are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0212] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus allowing for further subclassing of the same class of Ig. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0213] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0214] As used herein, the term "Fc domain" or "Fc region" is used to define a C-terminal region of an antibody heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. An IgG Fc region contains an IgG CH2 domain and an IgG CH3 domain. The CH2 domain as used herein may be a native sequence CH2 domain or a variant CH2 domain. The CH3 region as used herein may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may contain one or more mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIII) and / or complement. It is presumed that reducing or eliminating binding to Fc receptor γ will reduce or eliminate antibody-mediated ADCC. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate antibody-mediated CDC. Mutations that reduce or eliminate the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art. These mutations include the so-called "LALA mutation," which involves replacing leucine residues at positions 1.3 and 1.2 of the IMGT site in the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, it is also known to generate α-glycosylated antibodies by mutating asparagine (N) at position 84.4 of the IMGT site in the CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), thereby mutating conserved N-chain glycosylation sites to reduce IgG1 effector function. As another alternative, complement activation (C1q binding) and ADCC are known to be reduced by mutating proline at position 114 of the IMGT site in the CH2 domain to alanine or glycine (P114A or P114G). These mutations can be combined to produce antibody molecules with further reduced or no ADCC or CDC activity.

[0215] "Regions equivalent to the Fc region of immunoglobulins" include variants of the naturally occurring alleles of the immunoglobulin Fc region, as well as modified variants capable of producing substitutions, additions, or deletions that substantially do not diminish the immunoglobulin-mediated effector functions (such as antibody-dependent cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the immunoglobulin Fc region without substantially losing biological function. Such variants can be selected according to general rules known in the art to minimize the impact on activity.

[0216] As used in this article, the term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0217] The term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages), causing these cytotoxic effector cells to specifically bind to the antigen-attached target cells and then kill the target cells by secreting cytotoxins.

[0218] The term "complement-dependent cytotoxicity (CDC)" refers to cytotoxic effects involving complement, which involve the binding of specific antibodies to corresponding antigens on the cell membrane surface to form complexes that activate the classical complement pathway. The resulting membrane attack complex exerts a lytic effect on the target cell.

[0219] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0220] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomic antibodies, trisomic antibodies, tetrasomic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0221] As used herein, the term "antigen-binding domain" or "antigen-binding site" refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0222] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformational configuration composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this disclosure can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0223] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (Kd and K, respectively). a The ratio of ). In some embodiments, the dissociation constant (K) of the molecule bound to the antigen. d) is ≤100nM, ≤10nM, <1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (e.g., 10 -7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0224] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA that has been separated from its native environment. In this disclosure, recombinant polynucleotides encoding antibodies or antigen-binding fragments thereof contained in vectors are also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their native chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this disclosure, in positive and negative strand forms, and in double strand forms. Isolated polynucleotides or nucleic acids of this disclosure further include synthetically generated molecules of this type. Additionally, polynucleotides or nucleic acids may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0225] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors of this disclosure comprise expression cassettes. Expression vectors can be transcribed into large amounts of stable mRNA. Once the expression vector is within the target cell, cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of this disclosure comprises an expression cassette containing a polynucleotide sequence encoding an antibody of this disclosure or an antigen-binding fragment thereof. The term "expression cassette" of this disclosure refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of this disclosure contains a polynucleotide sequence encoding an antibody of this disclosure or an antigen-binding fragment thereof.

[0226] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen-binding molecule disclosed herein. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0227] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC comprises a binding protein, a drug, and a connector linking the binding protein to the drug.

[0228] As used herein, the term "linker" or "connector" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. Domains in the antibodies or antigen-binding fragments of this disclosure may be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. In some embodiments, all linkers present in the antibodies or antigen-binding fragments of this disclosure have the same amino acid sequence. In other embodiments, at least two linkers present in the antibodies or antigen-binding fragments of this disclosure have different amino acid sequences. Linkers should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains they are linked to fold correctly and are properly presented to perform their biological functions. In various embodiments, linkers have a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker may be selected from (Glycine, glutamine, and / or serine residues). n S) m (G) n Or (EAAAK) n , where n and m are each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5.

[0229] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a receptor having desired antigen specificity and signal transduction domains to propagate intracellular signals upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, naïve T cells depend on antigen-presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages can be engineered to express, for example, chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating the tumor and killing target cancer cells. CARs may include antigen-binding domains, transmembrane domains, and intracellular domains. The antigen-binding domain binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens binding to the antigen-binding domain of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0230] As used herein, the term "modified immune cell" refers to an immune cell that has been genetically modified to express a CAR. In some embodiments, the immune cell is a T cell or a cell derived therefrom. In some embodiments, the immune cell is a natural killer (NK) cell or a cell derived therefrom. In some embodiments, the immune cell is a B cell or a cell derived therefrom. In some embodiments, the immune cell is a monocyte or a macrophage, or a cell derived therefrom.

[0231] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0232] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0233] As used herein, the terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human.

[0234] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments of the present disclosure, along with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0235] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0236] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of this disclosure or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, to a patient having one or more ITGβ6-related diseases or symptoms, the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms, or to inhibit the development of such symptoms to any clinically measurable extent.

[0237] As used herein, the term "prevention" refers to delaying, suppressing, or preventing the onset of ITGβ6-related diseases in mammals where the initiation of cancer or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, for example, through genetic screening or other methods. The term also includes treating mammals with precancerous lesions to halt the progression of the precancerous lesions to malignancy or to induce their regression.

[0238] As used herein, the term "detectable marker" encompasses a marker that can be detected directly or indirectly, either attached to the antibody or its antigen-binding fragment, or present independently in the kit. Suitable markers include, but are not limited to, molecules detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Suitable markers include, but are not limited to, fluorescent dyes (e.g., GFT and its variants, FITC, TRITC, fluorescein, and rhodamine), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase), molecules containing radionuclides (i.e., radioisotopes), chemiluminescent molecules, electrochemiluminescent molecules, biotin, digoxin / digoxigenin ligand, or haptens, and other entities that are or can be detectable. The antibodies or their antigen-binding fragments in this disclosure are attached to a "detectable marker," thus being detectably marked.

[0239] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0240] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is b12seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). BL2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0241] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0242] As used in this article, "antibody-mediated internalization" refers to the phenomenon where antibodies cross the cell membrane after binding to cell surface antigens. Internalization includes antibody-mediated receptor internalization.

[0243] The present disclosure is now described with reference to the following embodiments, which are intended to illustrate the present disclosure (and not limit the present disclosure).

[0244] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this disclosure are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed in this disclosure.

[0245] Sequence information

[0246] Information about the sequences involved in this disclosure is described in Table 1 below.

[0247] Table 1. Sequences involved in this disclosure.

[0248] Example 1: Preparation of ITGβ6 antigen and control antibody and construction of cell lines

[0249] Human ITGαV & ITGβ6 (αVβ6), monkey αVβ6, rat αVβ6, and mouse αVβ6 proteins were purchased from AcroBiosyste MS. The ITGβ6 control antibody 2A2 was derived from patent CN114828887A. Gene synthesis, plasmid extraction, and expression purification were performed to obtain control antibody 2A2 (its variable region sequence is shown in Table 1; the heavy chain constant region is the hIgG1 heavy chain constant region with the amino acid sequence shown in SEQ ID NO: 7, and the light chain constant region is the hKappa light chain constant region with the amino acid sequence shown in SEQ ID NO: 8). The negative control antibody hIgG1 is an anti-chicken lysozyme antibody. Its variable region sequence is derived from patent CA2309763A1. Its heavy chain variable region is fused with the human IgG1 heavy chain constant region (SEQ ID NO: 7), and its light chain variable region is fused with the human Kappa light chain constant region (SEQ ID NO: 8). Gene synthesis, plasmid extraction, and expression purification were also performed to obtain the negative control antibody hIgG1.

[0250] Human ITGβ6 (Uniprot: P18564), monkey ITGβ6 (Uniprot: A0A2K5TZ36), rat ITGβ6 (Uniprot: Q6AYF4), mouse ITGβ6 (Uniprot: Q9Z0T9), human ITGαV (Uniprot: P06756), monkey ITGαV (Uniprot: A0A2K5WCD3), rat ITGαV (Uniprot: F1LZX9), mouse ITGαV (Uniprot: P43406), human ITGβ1 (Uniprot: P05556), human ITGβ3 The full-length sequences of human ITGβ5 (Uniprot: P05106), human ITGβ5 (Uniprot: P18084), and human ITGβ8 (Uniprot: P26012) were synthesized at GenScript and constructed into the pLVX vector. After plasmid extraction, the virus was packaged and used to construct overexpression cell lines CHOS-human ITGβ6, CHOS-monkey ITGβ6, CHOS-rat ITGβ6, CHOS-mouse ITGβ6, CHOS-human αVβ6, CHOS-monkey αVβ6, CHOS-rat αVβ6, CHOS-mouse αVβ6, CHOS-human ITGαV, CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, and CHOS-human ITGβ8. The sequences of human ITGβ6 (Uniprot: P18564) from positions 22 to 709, monkey ITGβ6 (Uniprot: A0A2K5TZ36) from positions 22 to 709, and mouse ITGβ6 (Uniprot: Q9Z0T9) from positions 22 to 708 were synthesized at GenScript and constructed into a vector containing a V5-tag. These were then used to construct three cell lines: CHO-S-hITGB6(22-709)(T)-v5, CHO-S-fasITGB6(22-709)(T)-v5, and CHO-S-mITGB6(22-708)(T)-v5.

[0251] Example 2: Preparation of anti-human ITGβ6 antibody

[0252] Human ITGβ6 protein (Kactus, Cat: ITG-HM 1B6) and mouse ITGβ6 protein (Kactus, Cat: ITG-MM1B6) were emulsified with adjuvants and used for RenLite immunization. TM Mice (Biocytogram, complete human heavy chain variable region combined with common light chain in-situ substitution, mouse description in PCT / CN2021 / 097652, the full text of which is incorporated herein by reference). Antibody immune responses were monitored by antigen-specific immunoassay.

[0253] Once the expected immune response is achieved, antigen-specific immune cells are isolated from the immunized mice to further obtain anti-ITGβ6 antibodies or the light chain variable region and heavy chain variable region sequences of anti-ITGβ6 antibodies. In this embodiment, single-cell technology (using...) The Optofluidic System (Berkeley Lights Inc.) screened plasma cells that secreted antigen-specific monoclonal antibodies, and obtained the variable region sequences of the antibodies through reverse transcription and PCR sequencing. The obtained variable region sequences were cloned into vectors containing sequences encoding the human IgG1 heavy chain constant region (amino acid sequence as shown in SEQ ID NO: 7, hIgG1 heavy chain constant region) and vectors containing sequences encoding the human Kappa light chain constant region (amino acid sequence as shown in SEQ ID NO: 8, hKappa light chain constant region) for antibody expression, yielding antibodies Ab01 and Ab02 (their variable region sequences and CDR sequences are shown in Table 1). The binding affinity of the expressed antibodies to ITGβ6 was verified using FACS. The results, as shown in Figure 1, indicate that Ab01 and Ab02 can bind to CHOS-human / monkey / mouse ITGβ6 cells.

[0254] Example 3: Preliminary evaluation of anti-human ITGβ6 antibody

[0255] 3.1 Species Crossover of Anti-human ITGB6 Antibodies

[0256] CHOS-human ITGβ6 cells or CHOS-monkey ITGβ6 cells were used at a dose of 1×10 5 Cells / wells were transferred to 96-well plates at their respective densities. Anti-ITGβ6 antibody was added to each well and incubated at 4°C for 30 minutes. After antibody incubation, the plates were washed twice with PBS, and 50 μL of Alexa antibody diluted 1:5000 was added to each well except for the NC (hIgG1) wells. 647AffiniPure TM F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch Laboratories, Inc., 109-606-170), incubated at 4°C for 15 min, followed by flow cytometry analysis. Human IgG1 (hIgG1) was used as an isotype control. The results are shown in Figure 2, indicating that Ab01 and Ab02 can bind to CHOS-human / monkey ITGβ6 cells.

[0257] 3.2 Binding affinity of anti-ITGβ6 antibody

[0258] Fortebio, equipped with a Protein A sensor, validated the binding affinity of the anti-ITGβ6 antibody to His-tagged human ITGβ6 protein (human ITGβ6-His, Kactus, Cat: ITG-HM1B6) and monkey ITGαV & ITGβ6 protein (monkey ITGαV & ITGβ6-His, Kactus, Cat: ITG-RM1V6).

[0259] Purified anti-ITGβ6 antibody was captured and detected on a Protein A sensor. Antibody was added to a final concentration of 5 μg / mL and then bound to recombinant human ITGβ6-His or monkey ITGαV & ITGβ6-His at a final concentration of 200 nM. Binding and dissociation times were set to 200 seconds and 400 seconds, respectively. For the isotype control (hIgG1), an antibody targeting an unrelated target protein was used.

[0260] The data were fitted to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110), and the kinetic binding rate (k) was obtained. on ) and dissociation rate (k off Affinity is determined by the entropy of the kinetic rate constant (KD = k). off / k on The results, as shown in Figures 3A-3F, indicate that Ab01 and Ab02 can bind to human ITGβ6 and monkey ITGαV & ITGβ6 proteins.

[0261] 3.3 Endocytotic activity of anti-ITGβ6 antibody

[0262] Add 50 μl of anti-ITGβ6 antibody (final concentration 10 μg / mL or 2.5 μg / mL) and 50 μl of pHAb-AffiniPure Fab goat anti-human IgG secondary antibody (Promega, G9845) to a 96-well plate and incubate in the dark for 15 minutes. Then add 100 μl of BT20 cells, HCC70 cells, HCC1954 cells, or BxPC-3 cells (cell density approximately 10,000 cells per well). After incubation at 37°C for 6 hours, centrifuge and wash cells with FACS buffer. Flow cytometry is used to detect MFI, and the antibody endocytosis rate (antibody MFI / control antibody MFI) is calculated. Human IgG1 (hIgG1) is used as the isotype control. After treatment with 2.5 μg / mL Isotype control or Ab01 and Ab02 for 6 hours, flow cytometry detected fluorescence signals in BT20 and HCC70 cells, as shown in Figures 4A and 4B. Ab01 and Ab02 exhibited good endocytic activity. After treatment with 10 μg / mL Isotype control (hIgG1), positive control, or Ab01 and Ab02 for 6 hours, flow cytometry detected fluorescence signals in HCC1954 and BxPC-3 cells. The positive control PC was 2A2, as shown in Figures 4C and 4D. This further demonstrates that Ab01 and Ab02 possess good endocytic activity.

[0263] Example 4: Evaluation of fully human anti-human ITGβ6 antibodies with ADCC and CDC functions removed

[0264] 4.1 Expression of fully human anti-human ITGβ6 antibody

[0265] To reduce the non-specific binding of the antibody constant region to FcγR in the human body, the variable region sequences of Ab01 and Ab02 were fused with the antibody constant region modified to remove FcγR binding. Based on the full-length light and heavy chain sequences, gene synthesis, plasmid extraction, expression, and purification were performed to obtain antibodies Ab03 and Ab04 that do not bind to FcγR. (The only difference between antibodies Ab03 and Ab04 and Ab01 and Ab02 is that the heavy chain constant region (the hIgG1 heavy chain constant region with the amino acid sequence shown in SEQ ID NO: 7) of Ab01 and Ab02 was replaced with the mutant hIgG1 heavy chain constant region (without FcγR binding) with the amino acid sequence shown in SEQ ID NO: 9.)

[0266] 4.2 Detection of hydrophilicity HIC and isoelectric point (PI) of anti-human ITGB6 fully human antibody

[0267] The isoelectric point (PI) of candidate antibodies was determined using isoelectric focusing. The method is briefly described below: The analysis was performed using the Maurice isoelectric focusing system from ProteinSimple, combined with its capillary cartridge. The antibody was diluted with water, and the detection system utilized a pH gradient formed by a 4% final concentration of the amphoteric electrolyte 3-10 (GE).

[0268] Liquid chromatography-HIC detection method: The equipment was an Agilent 1260, the analytical column was a TSKgel Butyl-NPR, 4.6*100mm, the column temperature was 30℃, the detection wavelength was 280nm, the flow rate was 0.5ml / min, the mobile phase A was 1.5mol / L (NH4)2SO4; the mobile phase B was 25mmol / L Na2HPO4, pH=7.0, 25% isopropanol. Take an appropriate amount of the test sample and dilute it with diluent (0.75mol / L (NH4)2SO4) to prepare a 1.0mg / ml solution as the test solution; take hydrophobic and hydrophilic controls respectively and dilute them with diluent to prepare 1mg / ml solutions as system suitability solutions. Inject approximately 40 μg of sample and perform analytical gradient elution: 0-3 min, maintain mobile phase A at 95% and mobile phase B at 5%; 3-40 min, increase mobile phase B from 5% to 100%; 40-45 min, maintain mobile phase A at 95% and mobile phase B at 5%. After detection, calculate the hydrophobicity value of the test sample based on the control sample. The HIC value is calculated as: (Test sample RT - Hydrophilic control RT) / (Hydrophobic control RT - Hydrophilic control RT), where RT is the peak retention time. The isoelectric point and HIC determination results of the antibodies are shown in Table 2. The hydrophilicity of Ab03 and Ab04 is superior to that of the control antibody 2A2, and their isoelectric points are within the normal range.

[0269] Table 2: Isoelectric point and HIC assay results of fully human anti-human ITGβ6 antibody

[0270] 4.3 Detection of protein binding of fully human anti-human ITGβ6 antibody

[0271] To detect the affinity of the fully human anti-human ITGβ6 antibody for human αVβ6, monkey αVβ6, rat αVβ6, and mouse αVβ6 proteins, the proteins were diluted to 1 μg / ml with CBS coating buffer, and 100 μl / well was coated onto each well of a 96-well microplate. The plates were incubated overnight at 4°C. The plates were washed once with PBS + 0.05% Tween-20, pH 7.4 (PBST), and the plates were dried. Then, 100 μl of 2% BSA was added to each well, and the plates were incubated at 37°C for 2 hours before removing the blocking buffer. The human antibody and control antibody were serially diluted with 2% BSA, starting at 200 nM, with 3-fold gradients and 11 concentration points. 100 μl of each antibody was added to each well of the microplate, and the plates were incubated at 37°C for 2 hours. The plates were washed three times with PBST, and after drying, 100 μl of each antibody was added to each well. HRP goat anti-human IgG (H+L) (Jackson) was incubated at 37°C for 1 hour. The plate was washed 5 times with PBST, and after drying, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well. After reacting at room temperature for 5 min, 50 μl of 2M H2SO4 was added to each well to terminate the reaction. The absorbance at OD450 nm was read using a microplate reader. Nonlinear curve fitting was performed to calculate EC50. 50 The results are shown in Table 3. Ab04 has a good binding affinity for human αVβ6, can bind to monkey αVβ6 and mouse αVβ6, but does not bind to rat αVβ6.

[0272] Table 3: Binding results of fully human anti-human ITGβ6 antibody with αVβ6 protein from different species

[0273] 4.4 Dynamic affinity assay for fully human anti-ITGB6 antibodies

[0274] The dynamic affinity of the fully human anti-human ITGβ6 antibody to human αVβ6, monkey αVβ6, rat αVβ6 and mouse αVβ6 proteins was detected using ForteBio (Pall Life Sciences). The specific method is as follows: The antibody to be tested was diluted to 5 μg / ml with PBST (0.02% Tween-20). Human αVβ6, monkey αVβ6, rat αVβ6, and mouse αVβ6 proteins were serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM, respectively. The antibody was then captured for 60 s in PBST (0.02% Tween-20) solution using a Protein A Sensor (Pall Life Sciences). After equilibration in PBST for 60 s, the antibody was bound to each of the four proteins for 90 s, followed by dissociation for 180 s. Based on the measured results, global fitting was used to analyze the results and obtain the affinity constants. The results are shown in Table 4. The results show that Ab04 can bind to human αVβ6, monkey αVβ6, and mouse αVβ6 proteins, but not to rat αVβ6.

[0275] Table 4: Dynamic affinity assay results of fully human anti-human ITGβ6 antibody

[0276] 4.5 Detection of cell affinity, species cross-reference, and specificity of fully human anti-human ITGβ6 antibody

[0277] The affinity of the fully human anti-human ITGβ6 antibody for human breast ductal carcinoma cell line HCC1954 (Kangnuoqin Biotechnology) and human esophageal carcinoma cell line TE-4 (Nanjing Kebai) was determined using a flow cytometer (Beckman, model Cytoflex). The affinity of the fully human anti-human ITGβ6 antibody for CHOS-human ITGβ6 and CHOS-human αVβ6 was also determined. The species cross-species analysis of the fully human anti-human ITGβ6 antibody was also performed, including cross-species interactions with CHOS-monkey ITGβ6 and CHOS-monkey αVβ6. Affinity to ITGβ6, CHOS-rat ITGβ6, CHOS-rat αVβ6, CHOS-mouse ITGβ6, and CHOS-mouse αVβ6; Detection of the specific binding of fully human anti-human ITGβ6 antibodies to ITGβ6: Affinity with CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8, and CHOS-human ITGαV. The affinity test method is the same for all cells, as detailed below:

[0278] Adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, and the cell density was counted and adjusted to 4.0 × 10⁻⁶. 6 / ml, washed twice with RPMI 1640 + 1% FBS, resuspended in RPMI 1640 + 1% FBS solution, and 50 μl of cell suspension was added to each well of a 96-well conical plate (cell count 2 × 10⁶). 5 (cells / well); Dilute candidate antibody and negative control antibody hIgG1 with RPMI 1640 + 1% FBS, starting at a final concentration of 200 nM, and serially dilute 3-fold for a total of 11 concentration points. Add 50 μl of the diluted antibody to a conical plate containing cells and incubate at 4°C for 60 min; wash cells twice with RPMI 1640 + 1% FBS, then add 50 μl of the diluted secondary antibody (dilution factor 1:200, Jackson Immuno Research, CAT #109-115-170) to each well, mix well, and incubate at 4°C for 30 min; wash cells twice with RPMI 1640 + 1% FBS, then resuspend the cells in 200 μl of RPMI 1640 + 1% FBS for flow cytometry analysis. Data processing: Export Median PE or numerical values, then import into data analysis software to calculate EC50. 50 .

[0279] The affinity results of the fully human anti-human ITGβ6 antibody for HCC1954 and TE-4 tumor cells are shown in Table 5. Ab03 and Ab04 have high affinity for tumor cells.

[0280] Table 5: Results of Affinity Measurement of Fully Human Anti-ITGβ6 Antibody with Tumor Cells

[0281] The affinity of the fully human anti-human ITGβ6 antibody for CHOS-human ITGβ6 and CHOS-human αVβ6 cells is shown in Figure 5 and Tables 6-1 and 6-2. Ab04 has a high cell affinity for CHOS-human ITGβ6 and CHOS-human αVβ6.

[0282] Table 6-1: Affinity assay results of fully human anti-human ITGβ6 antibody with CHOS-human ITGβ6 cells

[0283] Table 6-2: Results of Affinity assay of fully human anti-human ITGβ6 antibody with CHOS-human αVβ6 cells

[0284] The affinity results of the fully human anti-human ITGβ6 antibody for CHOS-monkey ITGβ6, CHOS-monkey αVβ6, CHOS-rat ITGβ6, CHOS-rat αVβ6, CHOS-mouse ITGβ6 and CHOS-mouse αVβ6 cells are shown in Tables 7-1 and 7-2. The results show that Ab04 can bind to CHOS-monkey ITGβ6, CHOS-monkey αVβ6, CHOS-mouse ITGβ6 and CHOS-mouse aVβ6 cells, but does not bind to CHOS-rat ITGβ6 and CHOS-rat αVβ6 cells.

[0285] Table 7-1: Results of Cell Species Cross-Assessment for Fully Human Anti-human ITGβ6 Anti-ITGβ6 Antibody

[0286] Table 7-2: Results of cross-assay of anti-human ITGβ6 fully human antibody against cell species 2

[0287] The affinity results of the fully human anti-human ITGβ6 antibody for CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8, and CHOS-human ITGαV cells are shown in Table 8. The results show that Ab03 and Ab04 do not bind to CHOS-human αVβ1, CHOS-human αVβ3, CHOS-human αVβ5, CHOS-human ITGβ8, and CHOS-human ITGαV cells, indicating that Ab03 and Ab04 have specificity for binding to ITGβ6.

[0288] Table 8: Affinity assay results of fully human anti-human ITGβ6 antibody with CHOS overexpressing cells.

[0289] 4.6 Detection of endocytic activity of fully human anti-ITGB6 antibody

[0290] The endocytic activity of the fully human anti-human ITGβ6 antibody against human breast ductal carcinoma cells HCC70 (Nanjing Kebai), human breast ductal carcinoma cells HCC1954 (ATCC), and human orthotopic pancreatic adenocarcinoma cells BxPC-3 (ATCC) was detected using an Incucyte instrument. Target cells were collected and resuspended in complete culture medium, adjusting the cell density to 5000 cells per 100 μl. 100 μl of the cell suspension was transferred to a 96-well plate and incubated overnight in a cell culture incubator (37℃ / 5% CO2). Working solutions for the test / control and the labeling reagent (Sartorius, CAT#4722) (4×) were prepared using experimental buffer. The final concentration of the test / control was 10 nM, and the final concentration of the labeling reagent was 30 nM. The working solutions of the test sample / control and the labeling reagent were mixed at a molar ratio of 1:3 and a volume ratio of 1:1, and incubated in a cell culture incubator (37℃ / 5% CO2) for 15 minutes to ensure complete coupling. 100 μl of the coupling mixture was transferred to the corresponding wells of a 96-well experimental plate, and the plate was incubated in a cell culture incubator (37℃ / 5% CO2) for 48 h. Images were taken at specified time points using an Incucyte instrument matching the detection wavelength. The raw experimental data and results were transmitted via... The Live-Cell Analysis System was used to analyze and export the data. Further analysis was performed on the raw data, and the area under the curve (AUC) was calculated. The results are shown in Figures 6A, 6B, and 6C and Table 9. The endocytic activity of Ab04 on the three cells was stronger than that of 2A2.

[0291] Table 9: Assay of endocytic activity of fully human anti-human ITGβ6 antibody

[0292] 4.7 Antigen-binding epitope analysis of fully human anti-human ITGβ6 antibody

[0293] Human αVβ6 protein was diluted to 1 μg / ml with CBS coating buffer, and 100 μl / well was coated onto a 96-well microplate and incubated overnight at 4°C. The microplate was washed once with PBST (0.2% Tween-20), drained, and then 100 μl of 2% BSA was added to each well. After incubation at 37°C for 2 hours, the blocking buffer was removed. The antibody was serially diluted with 2% BSA (starting at a final concentration of 200 nM, with 3-fold serial dilutions, 8 concentration points). Biotin-2A2 was diluted to 2000 ng / ml with 2% BSA. The diluted antibody was added to the corresponding well at a rate of 50 μl / well, and the diluted biotin-2A2 was added to the 96-well microplate at a rate of 50 μl / well. The plates were incubated at 37°C for 2 hours, resulting in a final concentration of 1000 ng / ml for biotin-2A2. After washing the plate three times with PBST and draining it, horseradish peroxidase-conjugated streptavidin secondary antibody was diluted 1:10000 with 2% BSA, and 100 μl / well was added to the corresponding well. The plate was incubated at 37°C for 1 hour. After washing the plate five times with PBST (0.2% Tween-20) and draining it, 100 μl of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well. After reacting at room temperature for 15 min, 50 μl of 2M H2SO4 was added to each well to terminate the chromogenic reaction. The absorbance at OD450 nm was read using a microplate reader. The raw data were imported into data analysis software for nonlinear curve fitting. The results are shown in Figure 7. Ab03 and Ab04 do not compete with the 2A2 epitope.

[0294] 4.8 Detection of ADCC activity of fully human anti-human ITGB6 antibody

[0295] In the presence of effector cells Jurkat-NFAT / luciferase-CD16a (engineered Jurkat cells stably expressing CD16a and a luciferase reporter gene regulated by the NFAT response element, obtained through lentiviral packaging, infection, and pressure selection), antibodies, and target cells, luciferase expression is activated. Using Bio-lite reagent (Vazyme Biotech) as a substrate, a chemiluminescent signal is emitted. The specific method is as follows: Jurkat-NFAT / luciferase-CD16a and CHOS-human αVβ6 cells are collected by centrifugation, resuspended in RPMI 1640 + 1% FBS medium, and the density is adjusted to 2.0 × 10⁻⁶ cells. 6 / ml and 1.0×10 6Add 50 μl / well to each well of a 96-well plate; dilute the antibody with RPMI 1640 + 1% FBS medium (starting at a final concentration of 66.67 nM, 3-fold dilution, 11 concentration points), and add 50 μl / well to each well of the 96-well plate. Incubate at 37°C for 5 h; add 20 μl of Bio-lite reagent, vortex to mix, and read the chemiluminescence signal value using a microplate reader to calculate EC50. 50 The results are shown in Figure 8 and Table 10; Ab04 does not exhibit ADCC activity.

[0296] Table 10: ADCC activity of fully human anti-human ITGβ6 antibody

[0297] Note: In Table 10, " / " indicates that it does not have ADCC activity.

[0298] 4.9 CDC Activity Assay for Fully Human Anti-human ITGB6 Antibody

[0299] To detect the CDC (complement-dependent cytotoxicity) effect and its strength of the fully human ITGβ6 antibody, target cells (CHOS-human αVβ6) were collected and counted, and the cell density was adjusted to 2.5 × 10⁶ cells using DMEM basal medium. 5 Add 40 μl of cell suspension per well to a 96-well plate (cell count 1 × 10⁶ cells / ml). 4 (100% complement concentration per well); Take one bottle of lyophilized guinea pig serum complement (Bersee, CAT#BM361Y), add 1 ml of DMEM basal medium to reconstitute, at this point the complement concentration is 100%. Take 950 μl of 100% complement and add it to 950 μl of DMEM basal medium and mix well, at this point the complement concentration is 50%. Add 20 μl of 50% guinea pig serum complement to each well, so that the final complement concentration in the well is 10%. Add 40 μl of antibody dilution buffer to each well, the final antibody concentration is: 666.67 nM starting, 3-fold dilution, 11 concentrations. Place the 96-well plate in a 37℃ CO2 incubator for 2 h. After incubation, add 20 μl of Cell Counting luminescent substrate solution to each well of the 96-well plate, shake to mix, and use a microplate reader to read the chemiluminescence signal value and calculate EC. 50 The results are shown in Figure 9 and Table 11; Ab04 did not exhibit CDC activity.

[0300] Table 11: CDC activity of fully human anti-human ITGβ6 antibody

[0301] Note: In Table 11, " / " indicates that it does not have CDC activity.

[0302] 4.10 Detection of Fc receptor binding activity of fully human anti-human ITGB6 antibody

[0303] The dynamic affinity of the fully human anti-human ITGβ6 antibody to human Fc receptor proteins FcRn (acro,cat#FCM-H8286), CD16a (acro,cat#CDA-H82E9), CD32a (acro,cat#CDA-H82E6), and CD64 (acro,cat#FCA-H82E8) was detected using ForteBio (Pall Life Sciences). The specific method is as follows: Biotinylated human Fc receptor proteins FcRn, CD16a, CD32a, or CD64 were diluted to 2 μg / ml using PBST solution. The biotinylated proteins to be tested were captured using an SA Sensor (Pall Life Sciences). The antibody to be tested was diluted to an initial concentration of 5000 nM using PBST and then diluted 2-fold to 7 concentration points. Binding and dissociation were performed in 1:1 mode with global fitting. The results were analyzed to obtain the binding rate, dissociation rate, and affinity constant. The results are shown in Table 12. Ab04 does not bind to Fc receptor CD16a, CD32a, and CD64 proteins, which can reduce Fc receptor-mediated non-specific killing and improve drug safety. At the same time, Ab04 retains the FcRn protein binding activity and does not affect the drug's half-life.

[0304] Table 12: Results of Fc receptor binding activity of fully human anti-human ITGβ6 antibody

[0305] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. An ITGβ6 antibody or an antigen-binding fragment thereof, wherein the ITGβ6 antibody or the antigen-binding fragment thereof comprises: a1) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 3; and / or having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or a2) having CDR-H1, CDR-H2, and CDR-H3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or, having CDR-L1, CDR-L2, and CDR-L3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or a3) having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in any of a1) to a2); and / or having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in any of a1) to a2).

2. The ITGβ6 antibody or its antigen-binding fragment according to claim 1, characterized in that, The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system; Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: b1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 30, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 32; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-L2 having the amino acid sequence DAS, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or b2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 10, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 11, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 12; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-L2 having the amino acid sequence DAS, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or b3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of b1) to b2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of b1) to b2); The CDR is defined according to the IMGT numbering system; Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: c1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 33, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 34, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or c2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 15, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 16, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or c3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of c1) to c2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of c1) to c2); The CDR is defined according to the Kabat numbering system; Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: d1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 36, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 37, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or d2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 20, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 21, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or d3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of d1) to d2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of d1) to d2); The CDR is defined according to the AbM numbering system; Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: e1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 38, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 39, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 35; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or e2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 22, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 23, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 18, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 14; or e3) VH including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-H1, CDR-H2 and CDR-H3 as shown in any of e1) to e2); and / or VL including the following three CDRs: having one or more amino acid substitutions, deletions or additions compared to CDR-L1, CDR-L2 and CDR-L3 as shown in any of e1) to e2); The CDR is defined according to the Chothia numbering system; Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: f1) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 41, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 4; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 27, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 28, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 29; or f2) A VH comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 25, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 26; and / or, a VL comprising the following three CDRs: CDR-L1 having the amino acid sequence shown in SEQ ID NO: 27, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 28, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 29; or f3) VHs including the following three CDRs: CDR-H1, CDR-H2, and CDR-H3 having one or more amino acid substitutions, deletions, or additions compared to CDR-H1, CDR-H2, and CDR-H3 as shown in any of f1) to f2); and / or VLs including the following three CDRs: CDR-L1, CDR-L2, and CDR-L3 having one or more amino acid substitutions, deletions, or additions compared to CDR-L1, CDR-L2, and CDR-L3 as shown in any of f1) to f2); The CDR is defined according to the Contact numbering system.

3. The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 2, characterized in that, The heavy chain variable region of the ITGβ6 antibody or its antigen-binding fragment also includes the framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes the framework region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the heavy chain variable region or a mutant thereof derived from human immunoglobulins. Preferably, the light chain variable region of the ITGβ6 antibody or its antigen-binding fragment further includes the framework region of the light chain variable region; Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes the framework region of the light chain variable region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even further includes the framework region of the light chain variable region or a mutant thereof derived from human immunoglobulins.

4. The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The ITGβ6 antibody or its antigen-binding fragment includes: g1) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 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%, or at least 99% sequence identity with it; or g2) Heavy chain variable region (VH), comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 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%, or at least 99% sequence identity with it; and / or, light chain variable region (VL), comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 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%, or at least 99% sequence identity with it.

5. The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 4, characterized in that, The ITGβ6 antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even more specifically includes the heavy chain constant region or a mutant thereof derived from human immunoglobulins. Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; further includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, rats, lemurs, macaques, chimpanzees, or humans; and even more particularly includes the light chain constant region or a mutant thereof derived from human immunoglobulins. Preferably, the heavy chain constant region includes the heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin or a mutant thereof; Preferably, the heavy chain constant region includes the human IgG1 heavy chain constant region or a mutant thereof; More preferably, the mutants of the human IgG1 heavy chain constant region include those with the following substitutions compared to their derived wild-type sequence: L234A, L235A, and / or G237A. The amino acid positions mentioned above are based on the EU numbering system. Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins; Preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 7 or 9, or an amino acid sequence having at least 80% sequence identity with it, or having up to 20 conserved amino acid substitutions compared to it; Preferably, the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 8 or 51, or an amino acid sequence having at least 80% sequence identity with it, or having at most 20 conserved substitutions of amino acids with it.

6. The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 5, characterized in that, The ITGβ6 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

7. The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 6, characterized in that, The antigen-binding fragment of the ITGβ6 antibody is a Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, single-chain Fv (scFv), dsFv, or Fd fragment; and / or The heavy chain and / or light chain of the antibody or its antigen-binding fragment includes a C-terminal lysine residue, lacks a C-terminal lysine residue, or lacks a C-terminal glycine-lysine residue; and / or The N-terminus of the heavy chain and / or light chain of the antibody or its antigen-binding fragment includes glutamine or glutamate, or the N-terminal glutamate or glutamine is cyclized into pyroglutamic acid or pyroglutamate salt. Preferably, the ITGβ6 antibody or its antigen-binding fragment comprises: q1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 44, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q2) The heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence having at least 80% sequence identity with it; and / or, the light chain, comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q3) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q4) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q5) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q6) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 44, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q7) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q8) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q9) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q10) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q11) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q12) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity with it; or q13) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 80% sequence identity with it; and / or a light chain comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it; or q14) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence having at least 80% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 50, or an amino acid sequence having at least 80% sequence identity with it.

8. A multispecific antibody or an antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the ITGβ6 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7.

9. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the ITGβ6 antibody or its antigen-binding fragment as described in any one of claims 1 to 7, or the multispecific antibody or its antigen-binding fragment as described in claim 8.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the ITGβ6 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment as claimed in claim 8, or the chimeric antigen receptor as claimed in claim 9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. Cells comprising the ITGβ6 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment as claimed in claim 8, the chimeric antigen receptor as claimed in claim 9, the nucleic acid molecule as claimed in claim 10, or the vector as claimed in claim 11.

13. A method for preparing the ITGB6 antibody or its antigen-binding fragment according to any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment according to claim 8, or the chimeric antigen receptor according to claim 9, the method comprising: The cells of claim 12 are cultured, and the ITGβ6 antibody or its antigen-binding fragment, the chimeric antigen receptor, or the multispecific antibody or its antigen-binding fragment are collected from the cultured cell culture.

14. A conjugate comprising the ITGβ6 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 7 or the multispecific antibody or its antigen-binding fragment as claimed in claim 8; and a conjugation portion.

15. The coupling according to claim 14, characterized in that, The coupling portion is a detectable marker or therapeutic agent. Preferably, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin. Preferably, the therapeutic agent includes a chemotherapeutic agent, an immunosuppressant, and / or a cytotoxic drug.

16. A pharmaceutical composition comprising: The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment according to claim 8, the chimeric antigen receptor according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the cell according to claim 12, or the conjugate according to any one of claims 14 to 15; and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, characterized in that, The pharmaceutical composition also includes other pharmaceutically active agents. Preferably, the other pharmaceutically active agents include drugs capable of treating diseases or conditions associated with ITGβ6. Preferably, the other pharmaceutically active agent is a drug with antitumor activity. Preferably, the other pharmaceutically active agents include tumor immunotherapy agents and / or chemotherapy drugs. Preferably, the tumor immunotherapy agent comprises at least one of the following: monoclonal antibody, immune checkpoint inhibitor, immune cells, oncolytic virus, and tumor vaccine. Preferably, the other pharmaceutically active agents include: ITGβ6 inhibitors, TROP2 inhibitors, B7H3 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met inhibitors, VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

18. Diagnostic or therapeutic reagent kits, comprising: The ITGβ6 antibody or its antigen-binding fragment according to any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment according to claim 8, the chimeric antigen receptor according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the cell according to claim 12, the conjugate according to any one of claims 14 to 15, or the pharmaceutical composition according to any one of claims 16 to 17; and optionally, a description and / or a delivery device.

19. Use of the ITGB6 antibody or its antigen-binding fragment according to any one of claims 1 to 7, the multispecific antibody or its antigen-binding fragment according to claim 8, the chimeric antigen receptor according to claim 9, the nucleic acid molecule according to claim 10, the vector according to claim 11, the cell according to claim 12, the conjugate according to any one of claims 14 to 15, or the pharmaceutical composition according to any one of claims 16 to 17 in at least one of (1) to (5): (1) Diagnose diseases or conditions related to ITGβ6; (2) Prevention or treatment of diseases or conditions related to ITGβ6; (3) Inhibits cell proliferation; (4) Detect the presence or level of ITGβ6 in the sample; (5) Prepare a product, said product being used in at least one of (1)-(4); Preferably, the cells are cells expressing ITGβ6, more preferably tumor cells expressing ITGβ6, and even more preferably tumor cells overexpressing ITGβ6. Preferably, the disease or condition associated with ITGβ6 is a tumor. Preferably, the ITGβ6-related disease or condition is an ITGβ6-positive tumor. Preferably, the diseases or conditions associated with ITGβ6 include 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, bile duct cancer, or any combination thereof.

20. A method for preventing or treating diseases or conditions associated with ITGβ6, the method comprising administering to a subject in need an effective amount of an ITGβ6 antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 7, a multispecific antibody or antigen-binding fragment thereof as claimed in claim 8, a chimeric antigen receptor as claimed in claim 9, a nucleic acid molecule as claimed in claim 10, a carrier as claimed in claim 11, a cell as claimed in claim 12, a conjugate as claimed in any one of claims 14 to 15, or a pharmaceutical composition as claimed in any one of claims 16 to 17.

21. The method according to claim 20, characterized in that, The method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. Preferably, the disease or condition associated with ITGβ6 is a tumor. Preferably, the ITGβ6-related disease or condition is an ITGβ6-positive tumor. Preferably, the diseases or conditions associated with ITGβ6 include 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, bile duct cancer, or any combination thereof.

22. A method for detecting the presence or level of ITGβ6 in a sample, the method comprising the following steps: The sample is brought into contact with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the multispecific antibody or antigen-binding fragment thereof according to claim 8, so as to allow the antibody or antigen-binding fragment thereof to form a complex with ITGβ6, and the formation of the complex is detected.

23. The method according to claim 22, characterized in that, The method is used to diagnose diseases or conditions related to ITGβ6. Preferably, the method includes the following steps: detecting the presence or level of ITGβ6 in a sample from a subject and comparing it with a reference value; wherein an increase in the expression level compared to the reference value indicates that the subject has an ITGβ6-related disease or condition. Preferably, the disease or condition associated with ITGβ6 is a tumor. Preferably, the ITGβ6-related disease or condition is an ITGβ6-positive tumor. Preferably, the diseases or conditions associated with ITGβ6 include 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, bile duct cancer, or any combination thereof.

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