ANTIBODIES AGAINST NECTIN-4 AND ITS CONJUGATES

Abstract

The invention proposes an antibody against Nectin-4 or its antigen-binding fragment, an antibody conjugate containing it, and its use in the manufacture of drugs, particularly for the manufacture of drugs for the treatment and / or prevention of cancer. A mosaic antigen receptor (CAR), immune enforcer cells, nucleic acid molecules, expression vectors, host cells, polyspecific antibodies, fusion proteins and their pharmaceuticals, and a method for detecting the presence or determining the expression levels of Nectin-4 in a sample are also proposed.

Description

Anti-nectin-4 antibody, conjugate containing the antibody and use thereof

[0001] This application claims priority to Chinese Application No. 202010972365.6 filed on September 16, 2020, entitled “Anti-Nectin-4 Antibodies, Conjugates Comprising Said Antibodies and Applications Thereof” and Chinese Application No. 202011624826.7 filed on December 31, 2020, entitled “Anti-Nectin-4 Antibodies, Conjugates Comprising Said Antibodies and Applications Thereof”, the full texts of these applications are incorporated herein by reference in their entirety. Technical Field

[0002] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-nectin-4 antibodies and conjugates containing the same and their applications, especially in the treatment and / or prevention of nectin-4-related diseases. Background Art

[0003] Nectin-4 belongs to the nectin family of proteins, also known as PVRL, and belongs to the immunoglobulin superfamily. Its primary function is to mediate calcium-independent cell adhesion junctions. Nectin family members, including nectin-1, nectin-2, nectin-3, and nectin-4, are approximately 510-550 amino acids in size and are type I transmembrane proteins. Their structure consists of an extracellular segment formed by three Ig-like domains, a transmembrane region, and an intracellular tail. The three Ig-like domains contain one IgV and two IgC domains. The C-terminus of the intracellular region contains a conserved region composed of Glu / Ala-X-Tyr-Val, which binds to the PDZ domain of the afadin protein. Nectin-4 does not share this protein sequence, yet it can still bind to the afadin protein intracellularly.

[0004] Nectin-4 can promote tumor cell survival and proliferation by inhibiting anoikis and activating the PI3K-AKT signaling pathway. Studies have shown that nectin-4 is expressed in gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and esophageal cancer, with stronger expression in non-small cell lung cancer, bladder cancer, breast cancer, and pancreatic cancer. High nectin-4 expression in tumors is closely associated with poor patient prognosis. Therefore, nectin-4 may serve as an ideal target for cancer drug development.

[0005] Enfortumab Vedotin, an ADC targeting nectin-4 (see, for example, WO2012 / 047724), is currently marketed for the treatment of urothelial carcinoma. Enfortumab Vedotin is a fully human anti-nectin-4 antibody conjugated to a VC-MMAE drug with a DAR of 4. Phase III clinical data showed that 4 patients achieved complete remission (CR) and 41 achieved partial remission (PR), with an objective response rate (ORR) of 41%. The median overall survival (OS) was 13.6 months, the median duration of response (DDR) was 5.75 months, and the median progression-free survival (PFS) was 5.4 months. The ORR was 40% for 89 patients who had previously received immune checkpoint inhibitors, 44% for 23 patients who had not received immune checkpoint inhibitors, and 39% for 33 patients with liver metastases. Enfortumab Vedotin has excellent anti-tumor efficacy.

[0006] Nectin-4 is expressed in the placenta and has weak to moderate expression in some normal tissues, such as the skin, esophagus, breast, and bladder. Clinically, the main toxic side effects of enfortumab vedotin are fatigue (54%); grade 3 or higher side effects include anemia (8%), hyponatremia (7%), urinary tract infection (7%), and hyperglycemia (6%). Four patients experienced serious drug-related side effects, including respiratory failure, urinary tract obstruction, diabetic ketoacidosis, and multiple organ failure. This suggests that enfortumab vedotin has a significant toxicity profile.

[0007] Summary of the Invention

[0008] In one general aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to nectin-4, comprising:

[0009] (1a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:3 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:4 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; and / or the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or

[0010] (1b) the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:9 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:10 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; and / or the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or

[0011] (1c) the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:11 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:12 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:7 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof;

[0012] or

[0013] The following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 63 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof;

[0014] or

[0015] (1d) the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 14 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 15 or a variant thereof; and / or the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; or

[0016] (2a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:21 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:22 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof; and / or the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0017] or

[0018] The following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence of SEQ ID NO: 18 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO: 22 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO: 23 or a variant thereof; and / or the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence of SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO: 25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO: 26 or a variant thereof;

[0019] or

[0020] (2b) the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 28 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof; and / or the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof;

[0021] or

[0022] The following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:36 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:28 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof; and / or the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0023] or

[0024] (2c) the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:29 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:30 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0025] or

[0026] The following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:44 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:30 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0027] or

[0028] (2d) the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:31 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:32 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:33 or a variant thereof; and / or the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:34 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:35 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0029] or

[0030] The following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:54 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:32 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:33 or a variant thereof; and / or the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:34 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:35 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof;

[0031] or

[0032] (3a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:39 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:40 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or

[0033] (3b) the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:45 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:46 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or

[0034] (3c) the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:47 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:48 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof;

[0035] or

[0036] The following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:47 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:64 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof;

[0037] or

[0038] (3d) the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:49 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:50 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:51 or a variant thereof; and / or the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:52 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:53 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof;

[0039] The variant has one or more amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0040] In a preferred embodiment, the antibody or antigen-binding fragment further comprises a framework region derived from human or murine immunoglobulin.

[0041] In one aspect, the invention provides an antibody conjugate comprising an antibody of the invention or an antigen-binding fragment thereof and at least one therapeutic agent.

[0042] In another aspect, the present invention provides a chimeric antigen receptor (CAR) and an immune effector cell expressing the CAR.

[0043] In yet another aspect, the present invention also relates to a fusion protein comprising the antibody or antigen-binding fragment thereof of the present invention and another biologically active polypeptide.

[0044] The present invention also relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, multispecific antibody or fusion protein of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0045] In another general aspect, the present invention relates to use of the antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, multispecific antibody, fusion protein or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer.

[0046] The present invention also relates to the use of the antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention in treating and / or preventing cancer.

[0047] In another general aspect, the present invention also provides a method for treating cancer in a subject in need thereof, wherein the cancer is associated with nectin 4. The method comprises administering to the subject a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, antibody conjugate, immune effector cell, multispecific antibody, fusion protein, or pharmaceutical composition of the present invention.

[0048] Preferably, the cancer is selected from gastric cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, urethral cancer, renal pelvis cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, mammary ductal carcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer and esophageal cancer. DETAILED DESCRIPTION

[0049] definition

[0050] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0051] As used herein, "at least one" or "one or more" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0052] The expression "comprising" or its synonyms "including," "containing," and "having" are open-ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, plus any optional elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0053] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring.

[0054] As used herein, "polypeptide" refers to a polymer formed by two or more amino acids linked by peptide bonds. A "protein" can be formed from one or more polypeptides in a covalent or non-covalent manner. Unless otherwise indicated, the terms "polypeptide" and "protein" are used interchangeably herein.

[0055] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen binding site. In this article, the definition of an antibody encompasses an antigen binding fragment. Therefore, the term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen binding fragments. Antibodies can be synthetic (e.g., produced by chemical coupling or biological coupling), enzymatically treated, or recombinantly produced. The antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In one embodiment, the antibody of the present invention is a mouse monoclonal antibody. In another embodiment, the antibody of the present invention is a humanized monoclonal antibody.

[0056] As used herein, "traditional antibodies" or "full-length antibodies" typically comprise four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises a "light chain variable region (VL)" and a "light chain constant region (CL)" from N-terminus to C-terminus. Each heavy chain comprises a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)" from N-terminus to C-terminus. The heavy chain constant region may comprise CH1, CH2, and CH3 from N-terminus to C-terminus. In certain immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may also comprise CH4. The "Fc" fragment refers to a fragment comprising CH2 and CH3, which provides a site for binding to the Fc receptor. The "hinge region" refers to the portion of an antibody that connects the immunoglobulin Fab and Fc fragments. In some cases, the hinge region refers to the portion of a T cell receptor that connects the constant region and the transmembrane domain. Herein, when used for a chimeric antigen receptor, the hinge region may also refer to any functional equivalent. Those skilled in the art can determine the positions of VH, VL, CL, CH1, CH2, CH3 and hinge regions in an antibody based on known algorithms and software. For descriptions of applicable algorithms and software, see, for example, William R. Strohl, Lila M. Strohl, (2012), Antibody structure–function relationships, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp. 37-56.

[0057] Typically, VL and VH each comprise three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and are connected in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. Herein, the CDRs of the light chain variable region (CDRL) may be referred to as CDR-L1, CDR-L2, and CDR-L3, and the CDRs of the heavy chain variable region (CDRH) may be referred to as CDR-H1, CDR-H2, and CDR-H3. It is generally believed that the six CDRs comprising a pair of VH and VL determine the binding specificity of the antigen binding site, but in some cases, other fragments (e.g., single domain antibodies, also known as nanobodies) comprising less than six CDRs (e.g., three, four, or five) also have the ability to bind to antigens. Those skilled in the art can identify CDRs using methods well known in the art, such as using the Kabat, Abm, or Chothia numbering systems. Herein, multiple CDR numbering systems, such as Chothia, Abm, Kabat, and IMGT, may be used for the same variable region. Those skilled in the art will understand that although the CDRs defined by different numbering systems may be different, the CDRs corresponding to the same numbering system represent effective antigen binding sites capable of binding to antigen epitopes. For a description of the CDR numbering system, see, for example, the Kabat numbering system: Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; the Chothia numbering system: Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917; the IMGT numbering system: Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; the Abm numbering system: Martin, ACR and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118.

[0058] In this article, the terms "framework region" and "framework region" can be used interchangeably. As used herein, the terms "framework region", "framework region" or "FR" residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0059] It is generally believed that the "Fv" fragment composed of a VH and a VL through non-covalent interaction is the smallest antigen-binding fragment containing an antigen-binding site. However, a single-domain antibody (VHH, also known as sdAb or nanobody) containing only the heavy chain variable region can also have antigen binding ability. VH and VL can be connected by a peptide linker to obtain a "single-chain Fv (scFv)". By introducing disulfide bonds into Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained respectively. In some embodiments, for example, the antigen-binding fragment in the antibody conjugate, multispecific antibody or CAR of the present invention preferably uses scFv or scdsFv.

[0060] As used herein, "Fab" comprises a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also referred to as Fd). A single-chain "Fab (scFab)" can be obtained by linking the CL and CH1 in "Fab" with a peptide linker. "F(ab')2" essentially comprises two Fab fragments linked by a disulfide bond in the hinge region. "Fab'" is half of F(ab')2, which can be obtained by reducing the disulfide bond in the hinge region of F(ab')2.

[0061] An antibody or antigen-binding fragment may be "monovalent," "bivalent," "trivalent," or "tetravalent," or more, meaning that it has multiple antigen-binding sites (e.g., 1, 2, 3, or 4 or more).

[0062] When the antibody or antigen-binding fragment binds to two or more antigens (e.g., 2, 3, 4, 5, or 6), it can also be referred to as a "multispecific antibody", such as a bispecific antibody, a trispecific antibody, or a tetraspecific antibody, which refers to a multispecific antibody that can bind to 2, 3, or 4 antigens, respectively.

[0063] As used herein, "diabody" refers to an antibody comprising two scFvs, having two antigen-binding sites (divalent), wherein the VH and VL in each scFv are connected by a short peptide linker (about 5-10 amino acid residues), such that the VH and VL chains pair (i.e., the VH of the first scFv and the VL of the second scFv pair, and the VL of the first scFv and the VH of the second scFv pair) to form an antigen-binding site. The diabody can be a bispecific antibody.

[0064] As used herein, an "antigen-binding fragment" of an antibody refers to a portion of a full-length antibody that is less than the full length but comprises at least a portion of the variable region of the full-length antibody (e.g., comprising one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability to specifically bind to an antigen. An antigen-binding fragment may, for example, include antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinantly produced derivatives. Examples of antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, diabodies, Fd and Fd' fragments, and other fragments (e.g., fragments comprising modifications). An antigen-binding fragment may comprise multiple peptide chains, for example, connected by disulfide bonds and / or by peptide linkers and / or constituted by non-covalent interactions.

[0065] As used herein, the term "monoclonal antibody" refers to a group of highly homogeneous antibodies that are essentially identical to one another except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies typically specifically bind to a single antigenic epitope. The monoclonal antibodies useful in the present invention can be prepared by any method known in the art, such as by producing them from transgenic animals (e.g., transgenic mice), by immortalized B cells (e.g., B cell hybridomas), or by preparing them in bacteria, eukaryotic animals, or plant cells using recombinant DNA methods, or by isolating them from phage antibody libraries. The antibodies or antigen-binding fragments of the present invention are monoclonal antibodies. In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention are humanized monoclonal antibodies.

[0066] A "chimeric antibody" refers to an antibody in which a portion (e.g., CDR, FR, variable region, constant region, or a combination thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining portion is identical or homologous to the corresponding sequence in an antibody derived from another species. As used herein, "chimeric antibody" also encompasses antibodies that contain portions belonging to different antibody types or subclasses. In a specific embodiment, the chimeric antibody has a murine antibody variable region and a human antibody constant region.

[0067] As used herein, "humanized antibody" refers to an antibody containing a non-human antibody and a human antibody sequence. Therefore, a humanized antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. A non-human antibody can be an antibody derived from any non-human species or an antibody (e.g., a chimeric antibody) comprising a portion of a non-human species source. Non-human species can, for example, include mice, rats, rabbits, alpacas, or non-human primates. The technology for obtaining humanized antibodies from non-human antibodies is well known to those skilled in the art. Humanized antibodies can be produced by non-human antibodies (e.g., mouse antibodies or chimeric antibodies), for example, by transplanting the CDR sequences of non-human antibodies (e.g., mouse antibodies) into human antibody framework regions. In some cases, in order to maintain the antigen-binding ability and / or stability of the humanized antibody, key amino acid residues of the non-human antibody (e.g., murine antibody) framework sequence can be retained in the human antibody framework region, i.e., "back mutations" are performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).

[0068] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has a generally accepted definition in the art, which refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, or FASTA software.

[0069] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. The magnitude of "affinity" is usually reported as the equilibrium dissociation constant, K. D or EC 50 .K D The equilibrium association constant (ka) and the equilibrium dissociation constant (kd) can be calculated: D = kd / ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interferometry (e.g., Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay, or flow cytometry.

[0070] In this article, the "specific binding" between an antibody and an antigen refers to the binding between the antibody and the antigen with a high affinity. D A value of at least about 10 -6 to at least about 10 -9M or less, such as at least about 10 -6 , at least about 10 -7 , at least about 10 -8 , at least about 10 -9 M or lower.

[0071] As used herein, an amino acid sequence that is "derived from" or "derived from" a reference amino acid sequence is partially or completely identical or homologous to the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of a human immunoglobulin has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0072] As used herein, a "variant" of a polypeptide or amino acid sequence has one or more amino acid mutations or modifications compared to the polypeptide or amino acid sequence from which it is derived. Amino acid mutations include amino acid substitutions, deletions, or additions. Those skilled in the art will appreciate that amino acids in non-essential regions of a polypeptide or protein can be substituted with suitable conservative amino acids, generally without altering their biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. The following table lists some non-limiting examples of common conservative substitutions of amino acid residues. In some cases, an amino acid substitution is a non-conservative substitution. Those skilled in the art will appreciate that amino acid mutations or modifications can be made to antibodies or antibody fragments to alter their properties, such as by changing the type of antibody glycosylation, altering the ability to form interchain disulfide bonds, or providing reactive groups for the preparation of antibody conjugates. Antibodies or antigen-binding fragments thereof containing such amino acid mutations or modifications are also encompassed within the scope of the antibodies or antigen-binding fragments thereof of the present invention.

[0073] Exemplary conservative substitutions of original residues Preferred substitutions Ala (A) Val, Leu, IleValArg (R) Lys, Gln, AsnLysAsn (N) Gln, His, Asp, Lys, ArgGlnAsp (D) Glu, AsnGluCys (C) Ser, AlaSerGln (Q) Asn, GluAsnGlu (E) Asp, GlnAspGly (G) AlaAlaHis (H) Asn, Gln, Lys, ArgArg

[0074] Ile(I)Leu, Val, Met, Ala, PheLeuLeu(L)Ile, Val, Met, Ala, PheIleLys(K)Arg, Gln, AsnArgMet(M)Leu, Phe, IleLeuPhe(F)Trp, Leu, Val, Ile, Ala,TyrTyrPro(P)AlaAlaSer(S)ThrThrThr(T)Val,SerSerTrp(W)Tyr,PheTyrTyr(Y)Trp,Phe,Thr,SerPheVal(V)Ile,Leu,Met,Phe,AlaLeu

[0075] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, and generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0076] As used herein, "isolated" means that a substance (eg, a nucleic acid molecule or a polypeptide) is separated from its source or environment, ie, does not substantially contain any other components.

[0077] As used herein, a "vector" is a medium for introducing exogenous nucleic acid into a host cell, which, when transformed into an appropriate host cell, is amplified or expressed. The vector typically remains episomal, but can be designed to integrate a gene or part thereof into a chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like.

[0078] As used herein, "expression vector" refers to a vector capable of expressing DNA, which is operably linked to regulatory sequences (such as promoters, ribosome binding sites) that can affect DNA expression. Regulatory sequences can include promoter and terminator sequences, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus, or other vector that, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain free or that are integrated into the host cell genome.

[0079] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A host cell can also be used to express a nucleic acid or a polypeptide encoded by the vector. A host cell can be a eukaryotic cell or a prokaryotic cell.

[0080] As used herein, the term "treatment" refers to the improvement of a disease / symptom, such as alleviating or eliminating the disease / symptom, preventing or slowing the occurrence, progression and / or worsening of the disease / symptom. Therefore, treatment includes prevention, therapy and / or cure.

[0081] "Effective amount" refers to a dose that is sufficient to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods of the disease, or prevent damage or disability caused by the pain of the disease. "Effective amount" refers to the amount required to prevent, cure, improve, block or partially block a disease or symptom. For example, for the treatment of tumors, an "effective amount" of the antibody or its antigen-binding fragment, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, relative to an untreated subject. The efficacy of inhibiting tumor growth can be assessed using conventional tumor animal models in the art, such as spontaneous tumors, induced tumors, and transplanted tumor animal models. Alternatively, the ability to inhibit cell growth can be examined using in vitro detection methods well known in the art. An effective amount of the antibody or its antigen-binding fragment, antibody conjugate, immune effector cell or pharmaceutical composition of the present invention can reduce tumor size, or otherwise alleviate the symptoms of the subject (such as preventing and / or treating metastasis or recurrence). One skilled in the art can determine the effective amount based on factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration, etc. The effective amount can be administered in one or more administrations.

[0082] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0083] As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, dogs, cats, and the like. As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a cancer patient, a human or animal suspected of having cancer, or at risk of developing cancer. As used herein, the terms "subject" and "subject" are used interchangeably.

[0084] Anti-nectin-4 antibody or antigen-binding fragment thereof

[0085] In one general aspect, the present invention provides anti-nectin-4 antibodies or antigen-binding fragments thereof that specifically recognize and bind to nectin-4.

[0086] Nectin-4 can be a protein comprising the full or partial amino acid sequence of human nectin-4 or non-human nectin-4. Preferably, the nectin-4 is human nectin-4. Human nectin-4 can, for example, have the amino acid sequence shown in Uniprot Identification Number (ID): Q96NY8-1. Non-human nectin-4 can, for example, be mammalian nectin-4, such as non-human primate cynomolgus macaque nectin-4, which can, for example, have the amino acid sequence shown in Uniprot Identification Number (ID): L0N6D9. Non-human nectin-4 can also, for example, be rat nectin-4, which can, for example, have the amino acid sequence shown in Uniprot Identification Number (ID): D3ZET1. Non-human nectin-4 can also, for example, be mouse nectin-4, which can, for example, have the amino acid sequence shown in Uniprot Identification Number (ID): Q8R007.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to the IgV domain of Nectin-4. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to the IgV domain of human Nectin-4.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to nectin-4 and blocking its interaction with nectin-1.

[0089] In some embodiments, the anti-nectin-4 antibody comprises a heavy chain variable region (VH) comprising three heavy chain CDRs (CDRHs): CDR-H1, CDR-H2, and CDR-H3. The CDR-H1, CDR-H2, and CDR-H3 are selected from the CDRH group in Table 1.

[0090] Table 1 Heavy chain CDRs of anti-nectin-4 antibodies

[0091]

[0092]

[0093] In some embodiments, the anti-nectin-4 antibody comprises a light chain variable region (VL) comprising three light chain CDRs (CDRLs): CDR-L1, CDR-L2, and CDR-L3. The CDR-L1, CDR-L2, and CDR-L3 are selected from the CDRL group in Table 2.

[0094] Table 2 Light chain CDRs of anti-nectin-4 antibodies

[0095]

[0096] Complementarity determining regions (CDRs)

[0097] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 4 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0098] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof. In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0099] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 12 or 63 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 5 or a variant thereof. In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 14 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 15 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0101] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises:

[0102] (a) the following six heavy and light chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:3 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:4 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:7 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or

[0103] (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence of SEQ ID NO:9 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:10 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:5 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:7 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:8 or a variant thereof; or

[0104] (c) the following six heavy and light chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence of SEQ ID NO: 11 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO: 12 or 63 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO: 5 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO: 7 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO: 8 or a variant thereof; or

[0105] (d) the following six CDRs of the heavy and light chains defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 14 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 15 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 22 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof.

[0107] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 28 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof. In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 29 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 30 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 31 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 32 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 33 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof.

[0110] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises:

[0111] (a) the following six heavy and light chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence of SEQ ID NO:21 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:22 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:23 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:26 or a variant thereof; or

[0112] (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence of SEQ ID NO:27 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:28 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:23 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:26 or a variant thereof; or

[0113] (c) the following six heavy and light chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:29 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:30 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof; or

[0114] (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:31 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:32 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:33 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO:34 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:35 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof.

[0115] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 18 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 22 or a variant thereof, and CDR-H3 comprising the sequence shown in SEQ ID NO: 23 or a variant thereof. In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence shown in SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 25 or a variant thereof, and CDR-L3 comprising the sequence shown in SEQ ID NO: 26 or a variant thereof.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:36 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:28 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:44 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:30 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:23 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:25 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 54 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 32 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 33 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 further comprises the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof.

[0119] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises:

[0120] (a) the following six heavy and light chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence of SEQ ID NO: 18 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO: 22 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO: 23 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO: 24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO: 25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO: 26 or a variant thereof; or

[0121] (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:28 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:23 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:26 or a variant thereof; or

[0122] (c) the following six heavy and light chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence of SEQ ID NO:44 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:30 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:23 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:24 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:25 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:26 or a variant thereof; or

[0123] (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence of SEQ ID NO:54 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:32 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:33 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:34 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:35 or a variant thereof, and CDR-L3 comprising the sequence of SEQ ID NO:26 or a variant thereof.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:39 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:40 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:45 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:46 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:41 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:43 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof.

[0126] In some specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 48 or 64 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 41 or a variant thereof. In other specific embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:49 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:50 or a variant thereof, and CDR-H3 comprising the sequence set forth in SEQ ID NO:51 or a variant thereof. In other embodiments, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO:52 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:53 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof.

[0128] In a specific embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises:

[0129] (a) the following six heavy and light chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence of SEQ ID NO:39 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:40 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:8 or a variant thereof; or

[0130] (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence of SEQ ID NO:45 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:46 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:8 or a variant thereof; or

[0131] (c) the following six heavy and light chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence of SEQ ID NO:47 or a variant thereof, CDR-H2 comprising the sequence of SEQ ID NO:48 or 64 or a variant thereof, CDR-H3 comprising the sequence of SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence of SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence of SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence of SEQ ID NO:8 or a variant thereof; or

[0132] (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO:49 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:50 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO:51 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO:52 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:53 or a variant thereof, and CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof.

[0133] In the embodiments of the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0134] Framework region (FR)

[0135] In some embodiments, the VH further comprises a heavy chain framework region (FR). In other embodiments, the VL further comprises a light chain framework region (FR). The heavy chain framework region and / or the light chain framework region can be each independently derived from the framework region of any species immunoglobulin. Preferably, the heavy chain framework region and the light chain framework region are each independently derived from the heavy chain framework region and the light chain framework region of a human or mouse immunoglobulin. In one embodiment, the heavy chain framework region and the light chain framework region each independently comprise the amino acid sequence of the heavy chain framework region and the light chain framework region derived from a mouse immunoglobulin, the heavy chain framework region and the light chain framework region of a human immunoglobulin, or a combination thereof. In one embodiment, the heavy chain framework region and the light chain framework region each independently comprise the amino acid sequence of the heavy chain framework region and the light chain framework region derived from a human germline antibody.

[0136] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain framework region derived from a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a light chain framework region derived from a human immunoglobulin. Therefore, in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is humanized. In such embodiments, the heavy chain framework region and / or light chain framework region of the antibody or antigen-binding fragment thereof of the present invention may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region and / or light chain framework region may comprise one or more amino acid return mutations, in which corresponding mouse amino acid residues are present.

[0137] In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VH having a sequence as shown in SEQ ID NO: 1 or a variant thereof. In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VL having a sequence as shown in SEQ ID NO: 2 or a variant thereof.

[0138] In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 1 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 2 or a variant thereof.

[0139] In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VH having a sequence as shown in SEQ ID NO: 55 or a variant thereof. In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VL having a sequence as shown in SEQ ID NO: 56 or a variant thereof.

[0140] In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 55 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 56 or a variant thereof.

[0141] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof. In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VL having a sequence as shown in SEQ ID NO: 20 or a variant thereof.

[0142] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 20 or a variant thereof.

[0143] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 57 or a variant thereof. In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VL having a sequence as shown in SEQ ID NO: 58 or a variant thereof.

[0144] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 57 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 58 or a variant thereof.

[0145] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VH having a sequence as shown in SEQ ID NO: 37 or a variant thereof. In yet another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VL having a sequence as shown in SEQ ID NO: 38 or a variant thereof.

[0146] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 37 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 38 or a variant thereof.

[0147] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VH having a sequence as shown in SEQ ID NO: 59 or a variant thereof. In yet another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises a VL having a sequence as shown in SEQ ID NO: 60 or a variant thereof.

[0148] In another preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to nectin-4 comprises: a VH having a sequence as shown in SEQ ID NO: 59 or a variant thereof, and a VL having a sequence as shown in SEQ ID NO: 60 or a variant thereof.

[0149] In specific embodiments, the antibody or antigen-binding fragment thereof comprises:

[0150] (1) a VH having a sequence as shown in SEQ ID NO: 1 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 2 or a variant thereof; or

[0151] (2) a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 20 or a variant thereof; or

[0152] (3) a VH having a sequence as shown in SEQ ID NO: 37 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 38 or a variant thereof; or

[0153] (4) a VH having a sequence as shown in SEQ ID NO: 55 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 56 or a variant thereof; or

[0154] (5) a VH having a sequence as shown in SEQ ID NO: 57 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 58 or a variant thereof; or

[0155] (6) A VH having a sequence as shown in SEQ ID NO: 59 or a variant thereof and / or a VL having a sequence as shown in SEQ ID NO: 60 or a variant thereof.

[0156] In embodiments as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0157] constant region

[0158] In some embodiments, the anti-nectin-4 antibody further comprises a heavy chain constant region. In another embodiment, the anti-nectin-4 antibody further comprises a light chain constant region. The heavy chain constant region and the light chain constant region can each be independently derived from the heavy chain constant region and the light chain constant region of an immunoglobulin of any species. In a preferred embodiment, the heavy chain constant region and the light chain constant region are each independently derived from the heavy chain constant region or a variant thereof and the light chain constant region or a variant thereof of a murine immunoglobulin. In a more preferred embodiment, the heavy chain constant region and the light chain constant region are each independently derived from the heavy chain constant region or a variant thereof and the light chain constant region or a variant thereof of a human immunoglobulin. The variant has one or more amino acid substitutions, deletions, or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions, or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids).

[0159] The heavy chain constant region may comprise an amino acid sequence selected from at least a portion of a hinge region, CH1, CH2, CH3, or a combination thereof. In one embodiment, the heavy chain constant region comprises CH1. In one embodiment, the heavy chain constant region comprises CH1-hinge region-CH2. In one embodiment, the heavy chain constant region comprises CH1-hinge region-CH2-CH3. In some embodiments, the heavy chain constant region further comprises CH4. The heavy chain constant region may be derived from a heavy chain constant region of an immunoglobulin of any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b), or a combination thereof. Preferably, the heavy chain constant region is derived from an IgG (e.g., IgG1, IgG2, IgG3, IgG4) heavy chain constant region. In a preferred embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region.

[0160] The light chain constant region can be derived from a lambda light chain or a kappa light chain constant region. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region.

[0161] In a specific embodiment, the heavy chain constant region comprises the sequence of SEQ ID NO: 61 or a variant thereof. In a specific embodiment, the light chain constant region comprises the sequence of SEQ ID NO: 62 or a variant thereof. In these embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids) compared to the sequence from which it is derived.

[0162] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein:

[0163] (a) the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 1 or a variant thereof and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61 or a variant thereof, and the light chain comprises: a VL having a sequence as shown in SEQ ID NO: 2 or a variant thereof and a light chain constant region having a sequence as shown in SEQ ID NO: 62 or a variant thereof; or (b) the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61 or a variant thereof, and the light chain comprises: a VL having a sequence as shown in SEQ ID NO: 20 or a variant thereof and a light chain constant region having a sequence as shown in SEQ ID NO: 62 or a variant thereof; or (c) the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 37 or a variant thereof and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61 or a variant thereof, and the light chain comprises: a VL having a sequence as shown in SEQ ID NO: 38 or a variant thereof and a light chain constant region having a sequence as shown in SEQ ID NO: 62 or a variant thereof; or (d) the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61 or a variant thereof NO:55 or a variant thereof and a VH having a sequence shown in SEQ ID NO:61 or a variant thereof, and the light chain comprises: a VL having a sequence shown in SEQ ID NO:56 or a variant thereof and a light chain constant region having a sequence shown in SEQ ID NO:62 or a variant thereof; or (e) the heavy chain comprises: a VH having a sequence shown in SEQ ID NO:57 or a variant thereof and a heavy chain constant region having a sequence shown in SEQ ID NO:61 or a variant thereof, and the light chain comprises: a VL having a sequence shown in SEQ ID NO:58 or a variant thereof and a light chain constant region having a sequence shown in SEQ ID NO:62 or a variant thereof; or (f) the heavy chain comprises: a VH having a sequence shown in SEQ ID NO:59 or a variant thereof and a heavy chain constant region having a sequence shown in SEQ ID NO:61 or a variant thereof, and the light chain comprises: a VL having a sequence shown in SEQ ID NO:60 or a variant thereof and a light chain constant region having a sequence shown in SEQ ID NO:62 or a variant thereof.

[0164] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

[0165] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, or humanized antibodies.

[0166] Mouse antibodies

[0167] In one embodiment, the anti-nectin-4 antibody of the present invention is a murine antibody comprising a VH and a VL, wherein the VH comprises the heavy chain CDRs and heavy chain framework regions described above, and the VL comprises the light chain CDRs and light chain framework regions described above, wherein the heavy chain framework region and the light chain framework region each independently comprise an amino acid sequence derived from a murine immunoglobulin framework region. In one embodiment, the anti-nectin-4 antibody of the present invention is a monoclonal antibody produced from a mouse hybridoma cell.

[0168] In a specific embodiment, the murine antibody comprises a VH comprising CDR-H1, CDR-H2, CDR-H3 as described in Group a of Table 1, and a heavy chain framework region, wherein the heavy chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VH having a sequence as set forth in SEQ ID NO: 1 or a variant thereof. In another specific embodiment, the murine antibody further comprises a VL comprising CDR-L1, CDR-L2, CDR-L3 as described in Group a of Table 2, and a light chain framework region, wherein the light chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VL having a sequence as set forth in SEQ ID NO: 2 or a variant thereof.

[0169] In another specific embodiment, the murine antibody comprises a VH comprising CDR-H1, CDR-H2, CDR-H3 as described in Group b of Table 1, and a heavy chain framework region, wherein the heavy chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VH having a sequence as set forth in SEQ ID NO: 19 or a variant thereof. In yet another specific embodiment, the murine antibody further comprises a VL comprising CDR-L1, CDR-L2, CDR-L3 as described in Group b of Table 2, and a light chain framework region, wherein the light chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VL having a sequence as set forth in SEQ ID NO: 20 or a variant thereof.

[0170] In another specific embodiment, the murine antibody comprises a VH comprising CDR-H1, CDR-H2, CDR-H3 as described in Group c of Table 1, and a heavy chain framework region, wherein the heavy chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VH having a sequence as set forth in SEQ ID NO: 37 or a variant thereof. In another specific embodiment, the murine antibody further comprises a VL comprising CDR-L1, CDR-L2, CDR-L3 as described in Group c of Table 2, and a light chain framework region, wherein the light chain framework region comprises an amino acid sequence derived from a murine immunoglobulin framework region. In a preferred embodiment, the murine antibody comprises a VL having a sequence as set forth in SEQ ID NO: 38 or a variant thereof.

[0171] In embodiments as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0172] In one embodiment, the murine antibody comprises a VH having a sequence as shown in SEQ ID NO: 1 and a VL having a sequence as shown in SEQ ID NO: 2.

[0173] In another specific embodiment, the murine antibody comprises a VH having the sequence set forth in SEQ ID NO:19 and a VL having the sequence set forth in SEQ ID NO:20.

[0174] In another specific embodiment, the murine antibody comprises a VH having a sequence as shown in SEQ ID NO:37 and a VL having a sequence as shown in SEQ ID NO:38.

[0175] chimeric antibodies

[0176] In one embodiment, the anti-nectin-4 antibody of the present invention is a chimeric antibody. In some embodiments, the chimeric antibody comprises the heavy chain CDRs and / or light chain CDRs of the murine antibody described above. In some embodiments, the chimeric antibody comprises the heavy chain variable region and / or light chain variable region of the murine antibody described above. In some embodiments, the chimeric antibody comprises the heavy chain variable region and light chain variable region of the murine antibody described above, as well as a heavy chain constant region and / or light chain constant region, wherein the heavy chain constant region is derived from the heavy chain constant region of a non-murine immunoglobulin and / or the light chain constant region is derived from the light chain constant region of a non-murine immunoglobulin. In a preferred embodiment, the heavy chain constant region is derived from the heavy chain constant region of a human immunoglobulin and / or the light chain constant region is derived from the light chain constant region of a human immunoglobulin. In a preferred embodiment, the heavy chain constant region is derived from the heavy chain constant region of a human immunoglobulin, and the light chain constant region is derived from the light chain constant region of a human immunoglobulin.

[0177] In some embodiments, the chimeric antibody comprises CDR-H1, CDR-H2, and CDR-H3 of the murine antibody described in Group a of Table 1. In some embodiments, the chimeric antibody further comprises CDR-L1, CDR-L2, and CDR-L3 of the murine antibody described in Group a of Table 2.

[0178] In one embodiment, the chimeric antibody comprises a VH having a sequence as shown in SEQ ID NO: 1 or a variant thereof. In one embodiment, the chimeric antibody further comprises a VL having a sequence as shown in SEQ ID NO: 2 or a variant thereof.

[0179] In another embodiment, the chimeric antibody comprises a VH having a sequence as shown in SEQ ID NO: 19 or a variant thereof. In one embodiment, the chimeric antibody further comprises a VL having a sequence as shown in SEQ ID NO: 20 or a variant thereof.

[0180] In another specific embodiment, the chimeric antibody comprises a VH having a sequence as shown in SEQ ID NO: 37 or a variant thereof. In a specific embodiment, the chimeric antibody further comprises a VL having a sequence as shown in SEQ ID NO: 38 or a variant thereof.

[0181] In embodiments as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0182] In some embodiments, the chimeric antibody described above further comprises a heavy chain constant region and / or a light chain constant region. In a preferred embodiment, the heavy chain constant region is derived from a heavy chain constant region of a human immunoglobulin, and the light chain constant region is derived from a light chain constant region of a human immunoglobulin. In one embodiment, the heavy chain constant region is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region. In one embodiment, the light chain constant region is derived from a human λ or κ light chain constant region. In one embodiment, the light chain constant region is derived from a human κ light chain constant region. In a specific embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 61 or a variant thereof. In a specific embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof. In these embodiments, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids) compared to the sequence from which it is derived.

[0183] In a specific embodiment, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 1 and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61, and the light chain comprises: a VL having a sequence as shown in SEQ ID NO: 2 and a light chain constant region having a sequence as shown in SEQ ID NO: 62.

[0184] In another specific embodiment, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having the sequence shown in SEQ ID NO: 19 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: a VL having the sequence shown in SEQ ID NO: 20 and a light chain constant region having the sequence shown in SEQ ID NO: 62.

[0185] In another specific embodiment, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having a sequence as shown in SEQ ID NO: 37 and a heavy chain constant region having a sequence as shown in SEQ ID NO: 61, and the light chain comprises: a VL having a sequence as shown in SEQ ID NO: 38 and a light chain constant region having a sequence as shown in SEQ ID NO: 62.

[0186] Humanized antibodies

[0187] In one embodiment, the anti-nectin-4 antibody of the present invention is a humanized antibody comprising a VH and a VL, wherein the VH comprises the heavy chain CDRs and heavy chain framework region described above, and the VL comprises the light chain CDRs and light chain framework region described above, wherein the heavy chain framework region and the light chain framework region each independently comprise an amino acid sequence derived from a heavy chain framework region and a light chain framework region of a human immunoglobulin. In such embodiments, the heavy chain framework region and / or the light chain framework region may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region and / or the light chain framework region may comprise one or more amino acid back mutations, wherein the back mutations have corresponding mouse amino acid residues. Humanized antibodies can be produced from non-human antibodies (e.g., mouse antibodies or chimeric antibodies). In one embodiment, humanized antibodies are prepared from anti-nectin-4 mouse antibodies of the present invention as described in the Examples of the present invention.

[0188] In one embodiment, the humanized antibody comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, CDR-H3 or a variant thereof of the murine antibody as described above. In one embodiment, the humanized antibody further comprises CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, CDR-L3 or a variant thereof of the murine antibody as described above.

[0189] In a specific embodiment, the humanized antibody comprises a VH comprising a CDR-H1 or variant thereof, a CDR-H2 or variant thereof, a CDR-H3 or variant thereof of a murine antibody as described above, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL comprising a CDR-L1 or variant thereof, a CDR-L2 or variant thereof, a CDR-L3 or variant thereof of a murine antibody as described above, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0190] In one embodiment, the humanized antibody comprises a VH, as defined by the Chothia numbering system, comprising a CDR-H1 as shown in SEQ ID NO: 3, a CDR-H2 as shown in SEQ ID NO: 4, a CDR-H3 as shown in SEQ ID NO: 5, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the Chothia numbering system, comprising a CDR-L1 as shown in SEQ ID NO: 6, a CDR-L2 as shown in SEQ ID NO: 7, a CDR-L3 as shown in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0191] In one embodiment, the humanized antibody comprises a VH, as defined by the Abm numbering system, comprising CDR-H1 as set forth in SEQ ID NO: 9, CDR-H2 as set forth in SEQ ID NO: 10, CDR-H3 as set forth in SEQ ID NO: 5, and a heavy chain framework region comprising an amino acid sequence derived from a heavy chain framework region of a human immunoglobulin. In another embodiment, the humanized antibody further comprises a VL, as defined by the Abm numbering system, comprising CDR-L1 as set forth in SEQ ID NO: 6, CDR-L2 as set forth in SEQ ID NO: 7, CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a light chain framework region of a human immunoglobulin.

[0192] In a specific embodiment, the humanized antibody comprises a VH, as defined by the Kabat numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 11, a CDR-H2 as set forth in SEQ ID NO: 12 or 63, a CDR-H3 as set forth in SEQ ID NO: 5, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL, as defined by the Kabat numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 6, a CDR-L2 as set forth in SEQ ID NO: 7, a CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0193] In one embodiment, the humanized antibody comprises a VH, as defined by the IMGT numbering system, comprising CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14, CDR-H3 as set forth in SEQ ID NO: 15, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the IMGT numbering system, comprising CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17, CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0194] In one embodiment, the humanized antibody comprises a VL having a sequence as shown in SEQ ID NO: 56 or a variant thereof. In one embodiment, the humanized antibody comprises a VH having a sequence as shown in SEQ ID NO: 55 or a variant thereof.

[0195] In one embodiment, the humanized antibody comprises a VH, as defined by the Chothia numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 21 or 18, a CDR-H2 as set forth in SEQ ID NO: 22, a CDR-H3 as set forth in SEQ ID NO: 23, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the Chothia numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 24, a CDR-L2 as set forth in SEQ ID NO: 25, a CDR-L3 as set forth in SEQ ID NO: 26, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0196] In a specific embodiment, the humanized antibody comprises a VH, as defined by the Abm numbering system, comprising CDR-H1 as set forth in SEQ ID NO: 27 or 36, CDR-H2 as set forth in SEQ ID NO: 28, CDR-H3 as set forth in SEQ ID NO: 23, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL, as defined by the Abm numbering system, comprising CDR-L1 as set forth in SEQ ID NO: 24, CDR-L2 as set forth in SEQ ID NO: 25, CDR-L3 as set forth in SEQ ID NO: 26, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0197] In a specific embodiment, the humanized antibody comprises a VH, as defined by the Kabat numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 29 or 44, a CDR-H2 as set forth in SEQ ID NO: 30, a CDR-H3 as set forth in SEQ ID NO: 23, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL, as defined by the Kabat numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 24, a CDR-L2 as set forth in SEQ ID NO: 25, a CDR-L3 as set forth in SEQ ID NO: 26, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0198] In a specific embodiment, the humanized antibody comprises a VH, as defined by the IMGT numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 31 or 54, a CDR-H2 as set forth in SEQ ID NO: 32, a CDR-H3 as set forth in SEQ ID NO: 33, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL, as defined by the IMGT numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 34, a CDR-L2 as set forth in SEQ ID NO: 35, a CDR-L3 as set forth in SEQ ID NO: 26, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0199] In one embodiment, the humanized antibody comprises a VL having a sequence as shown in SEQ ID NO: 58 or a variant thereof. In one embodiment, the humanized antibody comprises a VH having a sequence as shown in SEQ ID NO: 57 or a variant thereof.

[0200] In one embodiment, the humanized antibody comprises a VH, as defined by the Chothia numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 39, a CDR-H2 as set forth in SEQ ID NO: 40, a CDR-H3 as set forth in SEQ ID NO: 41, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the Chothia numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 42, a CDR-L2 as set forth in SEQ ID NO: 43, a CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0201] In one embodiment, the humanized antibody comprises a VH, as defined by the Abm numbering system, comprising CDR-H1 as set forth in SEQ ID NO: 45, CDR-H2 as set forth in SEQ ID NO: 46, CDR-H3 as set forth in SEQ ID NO: 41, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the Abm numbering system, comprising CDR-L1 as set forth in SEQ ID NO: 42, CDR-L2 as set forth in SEQ ID NO: 43, CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0202] In a specific embodiment, the humanized antibody comprises a VH, as defined by the Kabat numbering system, comprising a CDR-H1 as set forth in SEQ ID NO: 47, a CDR-H2 as set forth in SEQ ID NO: 48 or 64, a CDR-H3 as set forth in SEQ ID NO: 41, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another specific embodiment, the humanized antibody further comprises a VL, as defined by the Kabat numbering system, comprising a CDR-L1 as set forth in SEQ ID NO: 42, a CDR-L2 as set forth in SEQ ID NO: 43, a CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0203] In one embodiment, the humanized antibody comprises a VH, as defined by the IMGT numbering system, comprising CDR-H1 as set forth in SEQ ID NO: 49, CDR-H2 as set forth in SEQ ID NO: 50, CDR-H3 as set forth in SEQ ID NO: 51, and a heavy chain framework region comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In another embodiment, the humanized antibody further comprises a VL, as defined by the IMGT numbering system, comprising CDR-L1 as set forth in SEQ ID NO: 52, CDR-L2 as set forth in SEQ ID NO: 53, CDR-L3 as set forth in SEQ ID NO: 8, and a light chain framework region comprising an amino acid sequence derived from a human immunoglobulin light chain framework region.

[0204] In one embodiment, the humanized antibody comprises a VL having a sequence as shown in SEQ ID NO: 60 or a variant thereof. In one embodiment, the humanized antibody comprises a VH having a sequence as shown in SEQ ID NO: 59 or a variant thereof.

[0205] In embodiments as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0206] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein, compared to the heavy chain variable region and / or light chain variable region described in the aforementioned humanized antibody, at least one CDR and / or framework region comprises an amino acid mutation, wherein the amino acid mutation is a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof). Preferably, the substitution is a conservative substitution.

[0207] In some embodiments, the anti-nectin-4 humanized antibody further comprises a heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region. In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 61 or a variant thereof.

[0208] In some embodiments, the anti-nectin-4 humanized antibody further comprises a light chain constant region. In one embodiment, the light chain constant region is derived from a human lambda or kappa light chain constant region. In one embodiment, the light chain constant region is derived from a human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof.

[0209] In embodiments as described above, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived. Alternatively, the variant has up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids) compared to the sequence from which it is derived.

[0210] In a specific embodiment, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having the sequence shown in SEQ ID NO: 55 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: a VL having the sequence shown in SEQ ID NO: 56 and a light chain constant region having the sequence shown in SEQ ID NO: 62.

[0211] In a specific embodiment, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having the sequence shown in SEQ ID NO: 57 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: a VL having the sequence shown in SEQ ID NO: 58 and a light chain constant region having the sequence shown in SEQ ID NO: 62.

[0212] In a specific embodiment, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: a VH having the sequence shown in SEQ ID NO: 59 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: a VL having the sequence shown in SEQ ID NO: 60 and a light chain constant region having the sequence shown in SEQ ID NO: 62.

[0213] The anti-nectin-4 antibodies or antigen-binding fragments thereof of the present invention can be prepared and produced using methods known in the art. Such methods may include, for example, preparing and isolating antibodies or antigen-binding fragments from phage display libraries, yeast display libraries, or immortalized B cells (e.g., mouse B cell hybridomas or EBV-immortalized B cells). Methods for immunizing animals can also be used, such as immunizing animals (e.g., mice) with antigens or DNA encoding the antigens, and isolating antibody-expressing B cells from the immunized animals. Preferably, the antibody-expressing B cells are immortalized, for example, to prepare hybridomas or EBV-immortalized B cells. Host cells can also be transformed with nucleic acid molecules or expression vectors encoding the antibodies or antigen-binding fragments thereof of the present invention, and then expressing the anti-nectin-4 or antigen-binding fragments thereof of the present invention from the host cells. In some embodiments, the anti-nectin-4 antibodies of the present invention are obtained from immunized animals, such as mice immunized with human nectin-4 protein.

[0214] Multispecific antibodies

[0215] In one aspect, the present invention provides a multispecific antibody comprising the anti-nectin-4 antibody or antigen-binding fragment thereof of the present invention and at least one other antibody or antigen-binding fragment thereof or antibody analog, such that the multispecific antibody can bind to nectin-4 and at least one other antigen.

[0216] In one embodiment, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. In one embodiment, the multispecific antibody is a bispecific diabody comprising a first scFv (or scdsFv) and a second scFv (or scdsFv), wherein the first scFv (or scdsFv) comprises a VH of the present invention and a VL of another antibody connected by a short peptide linker, and the second scFv (or scdsFv) comprises a VL of the present invention and a VH of the other antibody connected by a short peptide linker, wherein the other antibody binds to an antigen other than nectin-4, and the short peptide linker comprises 5-10 amino acid residues.

[0217] In one embodiment, the other antigen is a cell surface protein, receptor, or receptor subunit. The cell surface protein can be, for example, an immune cell receptor. The immune cell can be, for example, an immune effector cell, such as a natural killer (NK) cell, a natural killer T (NKT) cell, or a cytotoxic T (CTL) cell. In a preferred embodiment, the bispecific antibody can simultaneously bind to nectin-4 and PD-1.

[0218] Multispecific antibodies are multivalent antibodies, for example, they can be bivalent, trivalent, tetravalent or more.

[0219] In a preferred embodiment, the multispecific antibody comprises an antigen-binding fragment selected from the group consisting of scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2.

[0220] The multispecific antibody may further comprise an antibody heavy chain constant region and / or a light chain constant region. The heavy chain constant region may comprise an amino acid sequence selected from at least a portion of a hinge region, CH1, CH2, CH3, or a combination thereof. Preferably, the heavy chain constant region comprises CH1.

[0221] Methods for constructing multispecific antibodies using antibodies or antibody fragments of interest are well known to those skilled in the art (see, e.g., International Patent Application Publication No. WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121:210; Traunecker et al., (1991) EMBO, 10:3655-3659).

[0222] Multispecific antibodies can be produced and isolated using various techniques known in the art. For example, nucleic acid molecules encoding multispecific antibodies can be obtained using recombinant DNA technology, optionally cloned into expression vectors, and then transformed into host cells with the nucleic acid molecules or expression vectors. The transformed host cells are cultured under appropriate conditions to allow expression of the nucleic acid or expression vector, and finally, the multispecific antibodies are isolated and purified from the host cells or culture medium.

[0223] Fusion protein

[0224] In yet another aspect, the present invention provides a fusion protein comprising the antibody or antigen-binding fragment thereof of the present invention and another biologically active polypeptide.

[0225] In preferred embodiments, the additional biologically active polypeptide is a polypeptide or protein having therapeutic activity, binding activity, or enzymatic activity. Non-limiting examples of biologically active polypeptides may include, but are not limited to, protein toxins (e.g., diphtheria toxin, ricin), enzymes (e.g., urease, horseradish peroxidase), and cytokines.

[0226] Nucleic acids, vectors and host cells

[0227] In another aspect, the present invention provides a nucleic acid molecule comprising a polynucleotide sequence encoding an anti-nectin-4 antibody or antigen-binding fragment of the present invention. The nucleic acid can be obtained using methods known in the art, for example, by isolation from a phage display library, a yeast display library, an immunized animal, an immortalized cell (e.g., a mouse B cell hybridoma, an EBV-mediated immortalized B cell), or chemical synthesis. The nucleic acid molecule of the present invention can be codon-optimized for the host cell used for expression. The nucleic acid molecule can be isolated.

[0228] In yet another aspect, the present invention also provides a vector comprising the nucleic acid molecule. In one embodiment, the nucleic acid molecule of the present invention is prepared as a recombinant nucleic acid. In one embodiment, the nucleic acid molecule of the present invention is cloned into an expression vector. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid comprising the nucleic acid of the present invention can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (e.g., polymerase chain reaction (PCR) technology), etc. (see Sambrook, J., E. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also comprise a polynucleotide sequence encoding a polypeptide or protein that can facilitate detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistidine tags (His6) or glutathione S-transferase (GSH) tags), polypeptides comprising protease cleavage sites, and reporter proteins (e.g., fluorescent proteins). The nucleic acid molecules of the present invention can be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacteria, yeast or mammalian cells. In other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or a phagemid vector.

[0229] The present invention also provides a host cell comprising at least one nucleic acid or vector as described above. In preferred embodiments, the host cell herein is used to express an anti-nectin-4 antibody or antigen-binding fragment thereof of the present invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0230] Bacteria (e.g., E. coli BL21 (DE3)) are particularly advantageous for expressing smaller antigen-binding fragments, such as Fv, scFv, Fab, and Fab' fragments. In some embodiments, nucleic acid molecules encoding the heavy chain variable region and the light chain variable region are cloned into a single vector and introduced into BL21 (DE3). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0231] The present invention also provides a method for producing the anti-nectin-4 antibody or antigen-binding fragment of the present invention in a host cell, wherein the method comprises the following steps:

[0232] (I) transforming a host cell with at least one nucleic acid or expression vector described herein,

[0233] (II) culturing the transformed host cell under appropriate conditions to allow expression of the nucleic acid or expression vector, and

[0234] (III) Isolating and purifying the antibody or antigen-binding fragment of the present invention from the host cells or culture medium.

[0235] In some embodiments, a single vector is used that contains a polynucleotide sequence encoding a heavy chain and a light chain. In some embodiments, two vectors are used, one of which encodes the light chain of the antibody and the other encodes the heavy chain of the antibody. In some embodiments, the host cell further comprises a chaperone plasmid that can help improve the solubility, stability, and / or folding of the antibody or antibody fragment. Techniques for separating and purifying antibodies from host cells are well known to those skilled in the art.

[0236] Antibody conjugates

[0237] The present invention also provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof of the present invention conjugated to at least one therapeutic agent.Antibody drug conjugate (ADC) is a typical antibody conjugate, wherein the therapeutic agent may be, for example, a cytotoxic agent.

[0238] In some preferred embodiments, the antibody conjugates of the present invention are capable of targeting nectin-4-positive target cells and tracking or killing the target cells. In some more preferred embodiments, the antibody conjugates of the present invention selectively kill nectin-4-positive cancer cells. In some embodiments, the cancer cells are selected from gastric cancer cells, breast cancer cells, bladder cancer cells, lung cancer cells, liver cancer cells, colon cancer cells, rectal cancer cells, head and neck cancer cells, and ovarian cancer cells.

[0239] The therapeutic agent can be selected from cytotoxic agents, therapeutic antibodies (e.g., antibodies or antigen-binding fragments thereof that specifically bind to another antigen), radioactive isotopes, oligonucleotides and their analogs (e.g., interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxin, ricin toxin), and enzymes (e.g., urease). Cytotoxic agents refer to substances that inhibit or reduce the activity, function, and / or kill cells. Examples of cytotoxic agents can include, but are not limited to, maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatin, cryptophycin, vinca alkaloids (e.g., vinblastine, vincristine), colchicines, dolastatins, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g., mitomycin, actinocycline),

[0013] Examples of the present invention include, but are not limited to, chloramphenicol, chlortetracycline ...

[0240] The radioisotope may be selected from, for example 212 Bi, 213 Bi, 131 I. 125 I. 111 In, 177 Lu, 186 Re、 188 Re、 153 Sm, 90 Y. Antibodies labeled with radioactive isotopes are also called radioimmunoconjugates.

[0241] In one embodiment, the therapeutic agent is a molecule with anti-tumor biological activity. Molecules with anti-tumor biological activity include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In another preferred embodiment, the therapeutic agent is a radioisotope.

[0242] In one embodiment, the therapeutic agent is

[0243] The therapeutic agent can be conjugated to the antibody or antigen-binding fragment of the present invention via a linker using any technique known in the art. The linker can contain an active group for covalent conjugation, such as an amine, hydroxylamine, maleimido, carboxyl, phenyl, thiol, sulfhydryl, or hydroxyl. The linker can be cleavable or non-cleavable. Cleavable linkers include enzyme-cleavable linkers (e.g., peptides containing protease cleavage sites), pH-sensitive linkers (e.g., hydrazone linkers), or reducible linkers (e.g., disulfide bonds).

[0244] In one embodiment, the linker comprises an active group selected from an amine, a hydroxylamine, a maleimido, a carboxyl, a phenyl, a thiol, a sulfhydryl, and a hydroxyl group. In one embodiment, the linker is a chemical bond. In one embodiment, the linker comprises an amino acid or a peptide consisting of 2-10 amino acids. The amino acid can be a natural or unnatural amino acid.

[0245] In a preferred embodiment, the linker is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, One of 1 and 2 represents the position where the linker is connected to the antibody or antigen-binding fragment thereof of the present invention, and the other represents the position where the linker is connected to the therapeutic agent.

[0246] In some embodiments, the therapeutic agent and linker are conjugated to form an intermediate prior to conjugation to the antibody or antigen-binding fragment of the invention. In some embodiments, the intermediate is attached via a thiol bond formed with a sulfhydryl group on the antibody or antigen-binding fragment of the invention. The structure and preparation of such intermediates, as well as methods for preparing antibody conjugates using them, are described, for example, in International Patent Application Publication No. WO2019 / 114666, the relevant contents of which are incorporated herein by reference in their entirety.

[0247] In one embodiment, the antibody conjugate has the structure shown in formula (I),

[0248] A-(LD) γ

[0249] (Ⅰ);

[0250] wherein A is an antibody or antigen-binding fragment thereof of the present invention;

[0251] L is a linker; preferably, L comprises an amino acid or a peptide consisting of 2-10 amino acids; further preferably, L is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, One of 1 and 2 indicates the position where L is connected to A, and the other indicates the position where L is connected to D;

[0252] D is a therapeutic agent; preferably, D is selected from cytotoxic agents, therapeutic antibodies, radioisotopes, oligonucleotides and analogs thereof, bioactive peptides, protein toxins and enzymes; more preferably, D is a molecule with anti-tumor biological activity; further preferably, D is

[0253] γ represents the number of (LD) moieties linked to A, which is an integer of 1-10.

[0254] In one embodiment, the (LD) moiety has the structure of formula (1) and is linked to the compound having the structure of formula (1) by forming a thioether bond with a sulfhydryl group on the antibody or antigen-binding fragment thereof of the present invention.

[0255]

[0256] The antibody conjugate of the present invention has the structure of formula (2):

[0257]

[0258] wherein A is an antibody or antigen-binding fragment of the present invention, and γ refers to the number of (LD) moieties linked to A via thiol bonds with the sulfhydryl groups of A, which is an integer from 1 to 10. In a specific embodiment, A is an anti-human nectin-4 humanized monoclonal antibody of the present invention.

[0259] In another aspect, the present invention provides a composition comprising an antibody conjugate of the present invention. In one embodiment, the molar ratio (DAR value) of the therapeutic agent to the anti-nectin-4 antibody or antigen-binding fragment thereof in the composition is a decimal or integer between 1 and 10, such as a decimal or integer between 1 and 8, such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.79, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4. 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0, 7.03, 7.1, 7.12, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0260] CAR and CAR-expressing immune effector cells

[0261] The antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptors (CARs). Therefore, in another aspect, the present invention also provides a chimeric antigen receptor (CAR).

[0262] As used herein, the term "chimeric antigen receptor (CAR)" is an artificial receptor that specifically recognizes and binds to a target (e.g., an antigen expressed on a cancer cell surface), such as by comprising an antigen binding fragment that specifically binds to an antigen on a cancer cell surface, and can confer target specificity to cells (e.g., immune effector cells) expressing CAR on the surface. Preferably, CAR recognition of the target results in activation and / or amplification of immune effector cells expressing CAR.

[0263] In some embodiments, the chimeric antigen receptor comprises an antibody that specifically binds to nectin-4 or an antigen-binding fragment thereof (e.g., scFv) of the present invention, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the present invention also provides a nucleic acid molecule, which may comprise a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof (e.g., ScFv) of the present invention. In certain embodiments, the nucleic acid molecule of the present invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. The nucleic acid molecule may be isolated.

[0264] On the other hand, the present invention also provides an immune effector cell, which is genetically modified to express the CAR of the present invention on the surface. Preferably, the immune effector cell is selected from T lymphocytes (such as cytotoxic T cells (CTL)), Natural killer cells (NK) and natural killer T cells (NKT). Immune effector cells target Nectin-4 positive diseased cells (for example, Nectin-4 positive cancer cells) and are activated to start effector functions, for example, causing the death of Nectin-4 positive cancer cells.

[0265] In another aspect, the present invention also relates to a nucleic acid molecule encoding the CAR, an expression vector comprising the nucleic acid molecule, and an immune cell (eg, CTL) expressing the CAR.

[0266] Pharmaceutical composition

[0267] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment, antibody conjugate, composition, CAR-expressing immune effector cell, multispecific antibody or fusion protein of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0268] The pharmaceutical compositions provided herein may be in a variety of dosage forms, including but not limited to solid, semisolid, liquid, powder, or lyophilized forms.

[0269] Depending on the dosage form, pharmaceutically acceptable carriers and / or excipients may include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, structure-forming excipients, preservatives, antioxidants (e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraethylene glycol, PEG-10 ... Acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, emollients, solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., non-ionic surfactants such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents, and emulsifiers.

[0270] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).For the composition comprising antibody or its Fab or antibody conjugate, preferred dosage form can be generally for example injection, lyophilized powder.For the composition comprising antibody or its Fab or antibody conjugate, suitable carrier and / or excipient can be selected from buffer (such as citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agent (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactant (such as polysorbate 80, polysorbate 20, poloxamer) or its combination.

[0271] Pharmaceutical compositions are generally sterile and stable under preparation and storage conditions. By mixing the active substance (e.g., antibody or antibody conjugate of the present invention) into a suitable solvent in the amount required, and adding one or a combination thereof of the carriers and / or excipients listed above as needed, followed by aseptic microfiltration, sterile injection can be prepared. Typically, dispersants are prepared by incorporating the active substance into a sterile carrier and / or excipient containing a basic dispersion medium and other required components listed above. For the preparation of sterile powders for sterile injection, preferred preparation methods are vacuum drying and freeze drying (lyophilization), which obtain a powder of the active ingredient plus any additional required components from its pre-sterile filtered solution.

[0272] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, for example, by systemic or topical administration. The route of administration includes, but is not limited to, parenteral (for example, intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous or intracavitary), topical, epidural or mucosal (for example, intranasal, oral, vaginal, rectal, sublingual or topical). It will be understood by those skilled in the art that exact dosage will depend on various factors, such as the metabolic kinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a specific compound, therapeutic purposes, route of administration and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors known to the medical field. Methods of administration can be, for example, injection or infusion.

[0273] As a general guide, the dosage range of the antibody conjugates of the present invention can be about 0.0001 to 100 mg / kg, more generally 0.01 to 20 mg / kg subject body weight. For example, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight or 20 mg / kg body weight, or within the range of 1-20 mg / kg. Exemplary treatment regimens require weekly administration, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or an initial dosing interval slightly shorter than the later dosing interval lengthened. The mode of administration can be intravenous drip.

[0274] Nectin-4 related diseases

[0275] The antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention, or drugs prepared therefrom, can be used to treat and / or prevent diseases. The diseases are associated with Nectin-4. Preferably, the diseases are associated with abnormal expression of Nectin-4. The term "abnormal expression" refers to excessively high or low protein expression levels in a sample compared to a normal sample (or a standard sample, such as a sample from a subject not suffering from a disease associated with abnormal expression of Nectin-4).

[0276] Preferably, the disease is characterized by high expression of Nectin-4. For example, Nectin-4 is highly expressed in tissues (e.g., bladder) of subjects suffering from or suspected of suffering from a disease (e.g., bladder cancer), while Nectin-4 is not expressed or is expressed at low levels in the tissues of subjects not suffering from the disease.

[0277] Preferably, the disease is cancer. Preferably, the cancer is selected from gastric cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, urethral cancer, renal pelvis cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, mammary ductal carcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer and esophageal cancer.

[0278] Cancer treatment can be assessed by, for example, but not limited to, the degree of tumor regression, change in tumor weight or size, time to progression, survival time, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity, etc.

[0279] Diagnostic and therapeutic uses

[0280] The anti-nectin-4 antibodies, or antigen-binding fragments thereof, or antibody conjugates thereof of the present invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with nectin-4. For example, the anti-nectin-4 antibodies, or antigen-binding fragments thereof, or antibody conjugates provided herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic assays or imaging assays.

[0281] In some embodiments, to detect the expression level of nectin-4 in a subject and / or track cells expressing nectin-4, the antibodies or antigen-binding fragments of the present invention can be conjugated with at least one label to obtain a labeled antibody. Such labeled antibodies are also included within the scope of the antibodies or antigen-binding fragments of the present invention. Preferably, the label is selected from an enzyme (e.g., horseradish peroxidase), a fluorescent dye, a radioactive isotope, biotin, and colloidal gold. Techniques for conjugating labels to antibodies are well known to those skilled in the art.

[0282] In one aspect, the present invention provides a method for detecting the presence of nectin-4 in a sample or determining its expression level, comprising:

[0283] (a) contacting a sample with the antibody or antigen-binding fragment of the present invention or a labeled antibody;

[0284] (b) detecting the formation of an immune complex between the antibody or antigen-binding fragment and nectin-4 in the sample or determining the amount of the immune complex to detect the presence of nectin-4 in the sample or determine the expression level of nectin-4 in the sample.

[0285] The present invention also provides a method for diagnosing a disease associated with nectin-4 in a subject, comprising:

[0286] (a) obtaining a sample from a subject;

[0287] (b) detecting the expression level of nectin-4 in the sample using the antibody or antigen-binding fragment of the present invention or the labeled antibody;

[0288] (c) The expression level of nectin-4 is compared with the expression level of a reference protein, wherein an increase or decrease in the expression level of nectin-4 compared with the expression level of the reference protein indicates abnormal expression of nectin-4.

[0289] The reference protein can be nectin-4 in a standard sample, which can be, for example, a sample from a subject who does not suffer from a disease associated with abnormal nectin-4 expression. The sample can be in any form, such as cells, tissues, or body fluids. The above detection can be performed using techniques known in the art, including but not limited to Western blotting, flow cytometry, immunohistochemistry (IHC), and ELISA.

[0290] The present invention also relates to the use of the antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention in treating and / or preventing diseases.

[0291] The present invention also relates to the use of the antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins, or pharmaceutical compositions of the present invention in the preparation of medicaments for treating and / or preventing a disease. In one embodiment, the disease is a cancer as described herein.

[0292] The antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention can be co-administered with at least one or more therapeutic agents described herein. The mode of co-administration is not limited. For example, the above therapeutic agents can all be administered at once or separately. When administered separately (using different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

[0293] Treatment

[0294] The present invention also provides a method for treating a disease in a subject in need thereof, wherein the disease is associated with nectin-4. In one embodiment, the disease is a cancer as described herein.

[0295] The method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, antibody conjugate, composition, immune effector cell, multispecific antibody, fusion protein or pharmaceutical composition of the present invention.

[0296] In certain embodiments, the methods of the present invention further comprise administering a treatment regimen selected from the group consisting of surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibition, and palliative care.

[0297] In certain embodiments, the method of the present invention further includes further administering one or more therapeutic agents selected from the following: chemotherapeutics, radioisotopes, immune checkpoint inhibitors, antibodies targeting other antigens and other anti-tumor drugs. Chemotherapeutics can include, for example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, microtubule inhibitors. Other anti-tumor drugs can include, for example, angiogenesis inhibitors, deacetylase (HDAC) inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib, Marizomib, Oprozomib) and tyrosine kinase inhibitors (e.g., BTK inhibitors). Examples of chemotherapeutic agents include, but are not limited to, cyclophosphamide, ifosfamide, melphalan, busulfan, mechlorethamine, chlorambucil, cypermethrin, carmustine (BCNU), lomustine (CCNU), cisplatin (DDP), carboplatin (CBP), oxaliplatin (OXA), methotrexate (MTX), 6-mercaptopurine (6-MP), 5-fluorouracil (5-FU), cytarabine, gemcitabine, vinblastine, vincristine, vindesine, camptothecin, irinotecan, topotecan, rubitecan, etoposide, teniposide, paclitaxel, taxanes, docetaxel, liposomal paclitaxel, dactinomycin, idarubicin, doxorubicin, epirubicin, mitomycin, bleomycin, doxorubicin, epirubicin. Immune checkpoint inhibitors include, but are not limited to, antibodies or antigen-binding fragments thereof that specifically bind to PD-1, PD-L1, CTLA4, LAG-3, TIM-3, or VISTA. Radioisotopes may include, for example 212 Bi, 213 Bi, 131 I. 125 I. 111 In, 177 Lu, 186 Re、 188 Re、 153 Sm, 90 Y.

[0298] In some embodiments, the antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention are used in combination with chemotherapeutic agents. In one embodiment, the chemotherapeutic agent is selected from cisplatin, oxaliplatin, gemcitabine, cyclophosphamide, 5-fluorouracil and combinations thereof. In other embodiments, the antibody conjugates of the present invention are used in combination with immune checkpoint inhibitors. In one embodiment, the immune checkpoint inhibitors are selected from anti-PD-L1 antibodies or antigen-binding fragments thereof, anti-PD-1 antibodies or antigen-binding fragments thereof and combinations thereof. In yet other embodiments, the antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions of the present invention are used in combination with radioactive isotopes.

[0299] Reagent test kit

[0300] The present invention also provides a test kit comprising an antibody or antigen-binding fragment, antibody conjugate, labeled antibody, multispecific antibody, fusion protein, pharmaceutical composition or immune effector cell of the present invention, and instructions for use. The test kit may also comprise a suitable container. In certain embodiments, the test kit also comprises a device for administration. Typically, the test kit also comprises a label for indicating the intended use and / or method of use of the test kit contents. The term "label" includes any written or recorded material provided on or with the test kit or otherwise provided with the test kit. Beneficial effects

[0301] In one specific aspect, the present invention provides anti-nectin-4 antibodies or antigen-binding fragments thereof, particularly humanized anti-nectin-4 monoclonal antibodies. The antibodies of the present invention have high affinity for nectin-4: In the Examples, the binding affinity of the antibodies of the present invention for nectin-4 was tested using various methods, such as ELISA, biofilm interferometry, and flow cytometry. The results showed that the anti-nectin-4 antibodies of the present invention have high affinity for both in vitro purified nectin-4 and cell surface nectin-4. Surprisingly, the dynamic affinity of humanized monoclonal antibodies 31HZ, 56HZ, and 74HZ for human nectin-4 and cynomolgus monkey nectin-4 was superior to that of the prior art, Enfortumab, indicating that the antibodies of the present invention have higher affinity for nectin-4 in terms of in vivo binding activity; this is also confirmed by the higher endocytic activity of antibodies 31HZ, 56HZ, and 74HZ. Furthermore, the antibodies of the present invention exhibit high specificity for nectin-4: 31HZ, 56HZ, and 74HZ all specifically recognize human nectin-4, but not nectin-1, nectin-2, or nectin-3. Furthermore, the antibodies of the present invention block the binding of nectin-4 to nectin-1, and the competitive binding abilities of 31HZ, 56HZ, and 74HZ for nectin-4 are comparable to those of enfortumab.

[0302] In another specific aspect, the present invention also provides an anti-nectin-4 antibody conjugate that can target nectin-4-positive cells. The antibody conjugate of the present invention can target nectin-4-positive cells with high affinity and high specificity, which is very beneficial for treating cancer or in vivo diagnosis because high affinity and specificity can reduce off-target toxic effects and reduce side effects. The examples also confirm this point. The ADC of the present invention has strong killing activity against various tumor cell lines that endogenously express human nectin-4, such as human breast ductal carcinoma cells, human gastric cancer cells, and human breast cancer cells. Furthermore, in a mouse non-small cell lung cancer xenograft model, the ADC of the present invention exhibited significant tumor inhibitory activity and had good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0303] Figure 1A shows the flow cytometry results of the A549-Nectin-4 stable cell line.

[0304] Figure 1B shows the flow cytometry results of the T24-Nectin-4 stable cell line.

[0305] FIG2 shows the affinity test results of anti-human nectin-4 mouse antibody.

[0306] FIG3A shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 31HZ and human nectin-4 protein.

[0307] FIG3B shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 74HZ and human nectin-4 protein.

[0308] FIG3C shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 56HZ and human nectin-4 protein.

[0309] FIG3D shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 31HZ and cynomolgus monkey nectin-4 protein.

[0310] FIG3E shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 74HZ and cynomolgus monkey nectin-4 protein.

[0311] FIG3F shows the affinity ELISA test results of anti-human nectin-4 humanized monoclonal antibody 56HZ and cynomolgus monkey nectin-4 protein.

[0312] FIG4A shows the flow cytometric detection results of the affinity of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ to T47D cells.

[0313] FIG4B shows the flow cytometric detection results of affinity between anti-human nectin-4 humanized monoclonal antibody 56HZ and NCI-N87 cells.

[0314] FIG5A shows the results of endocytic activity detection of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ on T47D cells.

[0315] FIG5B shows the results of endocytic activity detection of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ in MDA-MB-468 cells.

[0316] FIG5C shows the results of endocytic activity detection of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ on A549-nectin-4 cells.

[0317] FIG5D shows the results of endocytic activity detection of anti-human nectin-4 humanized monoclonal antibody 56HZ on T24-nectin-4 cells.

[0318] FIG6A shows the detection results of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ recognizing the human nectin-4 domain.

[0319] FIG6B shows the detection results of the anti-human nectin-4 humanized monoclonal antibody 56HZ recognizing the human nectin-4 domain.

[0320] FIG7A shows the results of competitive activity assay of anti-human nectin-4 humanized monoclonal antibodies 31HZ and 74HZ.

[0321] FIG7B shows the results of the competition activity test of the anti-human nectin-4 humanized monoclonal antibody 56HZ.

[0322] FIG8A shows the specific detection results of the anti-human nectin-4 humanized monoclonal antibody 31HZ.

[0323] FIG8B shows the specificity detection results of the anti-human nectin-4 humanized monoclonal antibody 74HZ.

[0324] FIG8C shows the specific detection results of the anti-human nectin-4 humanized monoclonal antibody 56HZ.

[0325] FIG9A shows the killing results of anti-human Nectin-4 ADC 31HZ-TL001 and 74HZ-TL001 on A549-Nectin-4 cells.

[0326] FIG9B shows the killing results of anti-human nectin-4 ADC 31HZ-TL001 and 74HZ-TL001 on A549 cells.

[0327] FIG9C shows the killing results of anti-human Nectin-4 ADC 31HZ-TL001 and 56HZ-TL001 on T24-Nectin-4 cells.

[0328] FIG9D shows the killing results of anti-human Nectin-4 ADC 31HZ-TL001 and 56HZ-TL001 on T24 cells.

[0329] FIG10A shows the killing results of anti-human nectin-4 ADC 31HZ-TL001 and 74HZ-TL001 on T47D cells.

[0330] FIG10B shows the killing results of anti-human nectin-4 ADC 31HZ-TL001 and 74HZ-TL001 on MDA-MB-468 cells.

[0331] FIG10C shows the killing results of anti-human nectin-4 ADC 31HZ-TL001 and 56HZ-TL001 on NCI-N87 cells.

[0332] FIG11 shows the changes in tumor volume over time in each group of mice in the NOD / SCID mouse subcutaneous NCI-H322M cell transplanted tumor model.

[0333] FIG12 shows the changes in body weight of mice in each group over time in the NOD / SCID mouse subcutaneous NCI-H322M cell transplantation tumor model.

[0334] Figure 13 shows that NOD / SCID mice subcutaneously Changes in tumor volume over time in each group of mice in the BL9200 PDX gastric cancer model.

[0335] Figure 14 shows that NOD / SCID mice subcutaneously Changes in body weight over time in each group of mice in the BL9200 PDX gastric cancer model.

[0336] Example

[0337] The following examples are intended to illustrate the present invention only and should not be construed as limiting the present invention in any way. Experimental methods in the following examples where no specific conditions are specified were performed according to conventional methods and conditions or according to the product specifications.

[0338] Example 1 Preparation of humanized monoclonal antibody against human nectin-4

[0339] 1.1 Construction of human nectin-4 overexpressing cell line

[0340] To verify the specificity and function of human nectin-4 antibody, the complete coding sequence of human nectin-4 (Uniprot ID: Q96NY8-1) (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) was cloned into the lentiviral vector pLVX-IRES-puro, and the virus was prepared using the lentiviral packaging system described in the literature (Mohammadi Z et al., Mol Biotechnol. 2015 Sep; 57(9): 793-800). After the obtained viruses were obtained, A549 non-small cell lung cancer cells (ATCC) and T24 human bladder transitional cell carcinoma cells (ATCC) were infected, respectively. After puromycin selection and monoclonal selection, monoclonal A549-Nectin-4 stable cell lines and T24-Nectin-4 stable cell lines overexpressing human nectin-4 were obtained.

[0341] 1.2 Detection of human nectin-4 overexpressing cell lines

[0342] The A549-Nectin-4 and T24-Nectin-4 stable cell lines were characterized by flow cytometry (Beckman, CytoFlex flow cytometer; detection antibody Enfortumab, sequence from International Patent Application Publication No. WO2012 / 047724) to confirm that the cells correctly expressed human nectin-4. As shown in Figures 1A and 1B, the flow cytometry results showed that both A549-Nectin-4 and T24-Nectin-4 had a high positive rate (nearly 100%), indicating that A549-Nectin-4 and T24-Nectin-4 are stable cell lines with good homogeneity and can be used for subsequent experiments.

[0343] 1.3 Preparation of anti-human nectin-4 mouse monoclonal antibody

[0344] Wild-type mice were immunized with protein to obtain anti-human nectin-4 mouse monoclonal antibodies. Two Babl / c and two C57 mice, each aged 5-6 weeks, were immunized with human nectin-4 protein (Sino Biological, Cat: 19771-H08H) emulsified in complete Freund's adjuvant (CFA) as a subcutaneous immunization. Subsequently, nectin-4 protein emulsified in incomplete Freund's adjuvant (IFA) was used for intraperitoneal boosts of 25 μg every two weeks. After three boosts, 5×10 T47D tumor cells endogenously expressing nectin-4 were used. 6 Cells (a type of human breast ductal carcinoma cell) were finally boosted intraperitoneally, and spleen cells were harvested 3-5 days later for hybridoma fusion. Mouse spleen cells were fused with the Sp2 / 0 (ATCC, Cat#CRL-1581) mouse myeloma cell line using a standard fusion process using PEG fusion. HAT pressure screening was then performed, and ELISA screening was performed 10-14 days later to obtain approximately 200 positive clones that bind to the nectin-4 protein. Further screening by flow cytometry yielded three positive hybridoma clones that could recognize T47D tumor cells for subcloning. Finally, subclone sorting was performed by limiting dilution to obtain single clones, which were numbered: 31#, 56#, and 74#.

[0345] 1.4 Affinity detection of anti-human nectin-4 mouse antibody

[0346] The affinity of candidate molecules to human nectin-4 was tested by ELISA. Hybridoma monoclonals 31#, 56#, and 74# were cultured in serum-free culture to obtain 3-10 mL of supernatant, which was then purified using Protein A (MabSelect SuRe, GE) to obtain mouse antibodies.

[0347] ELISA was used to determine the affinity of mouse monoclonal antibodies isolated and purified from the culture supernatants of hybridoma cell lines 31#, 56#, and 74#. Human nectin-4 (Sino Biological, Cat: 19771-H08H) was diluted to 1 μg / mL in carbonate (CBS) buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C for 16-20 hours. The CBS buffer was aspirated from the 96-well plate, and the plate was washed once with PBST (pH 7.4, PBS containing 0.05% Tween 20). Then, 200 μL / well of 2% PBST in skim milk powder (blocking buffer) was added and incubated at room temperature for 1 hour. The blocking buffer was removed, and the plate was washed three times with PBST buffer. The anti-nectin-4 mouse antibody to be tested was diluted to the appropriate concentration in PBST / 2% skim milk powder and added to 100 μL / well. The plate was incubated at room temperature for 1.5 hours. Remove the reaction mixture, wash the plate three times with PBST, and then add 50 μL / well of HRP-conjugated goat anti-mouse IgG secondary antibody (purchased from The Jackson Laboratory) diluted in PBST / 1% skim milk powder (1:5000) and incubate at room temperature for 1 hour. Wash the plate three times with PBST, then add 100 μL / well of TMB and incubate at room temperature for 5-10 minutes. Stop the color development by adding 50 μL / well of 0.2 M sulfuric acid. Detect absorbance (OD) at dual wavelengths of 450 / 620 nm using a microplate reader.

[0348] The test results are shown in Figure 2 and Table 3. All three anti-human nectin-4 mouse antibodies can bind to human nectin-4 protein with high affinity. 50 Between 30 and 70 pM.

[0349] Table 3 Anti-human Nectin-4 affinity detection

[0350] Clone number EC 50 (pM)31#54.9556#36.3374#69.39

[0351] 1.5 Variable region amplification of anti-nectin-4 mouse monoclonal antibody

[0352] Hybridoma cells were cultured to about 8,000-10,000 cells, and the cells were lysed and the first-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher Sci. Cat#18080-200). VH and VK genes were amplified by PCR from the cDNA using primers. The PCR products were purified using a DNA purification kit (Qiagen, Cat#28104) and ligated to the PUC57 vector. Approximately 5 clones were picked for sequencing for each ligation reaction. The sequence was analyzed using Vector NTI 11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The variable region sequence and CDR sequence of the anti-human nectin-4 mouse antibody were then obtained using IMGT and abYsis as shown in Table 4 below.

[0353] Table 4 Anti-human Nectin-4 mouse antibody variable region and CDR amino acid sequences

[0354]

[0355] Note: The numbers in the VH, VL, CDR-H1, CDRH2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 columns represent their corresponding SEQ ID NOs. For example, the VH of antibody 31# corresponds to SEQ ID NO: 1, and the VL corresponds to SEQ ID NO: 2.

[0356] 1.6 Humanization of anti-human nectin-4 mouse antibody

[0357] Murine antibodies 31#, 56#, and 74# were humanized using the CDR-grafting antibody humanization method. Briefly, humanization involves the following steps: The amino acid sequences of the mouse monoclonal antibody are compared with those of the human germline antibody to identify sequences with high homology and superior physicochemical properties as the human germline framework sequences; HLA-DR affinity is analyzed and examined to select human germline framework sequences with low affinity; and the six CDRs of the mouse antibody are then transplanted onto the selected heavy and light chain framework sequences.

[0358] Computer simulations and molecular docking were then used to analyze the variable region and its surrounding framework amino acid sequences, examining their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, the key amino acids in the mouse antibody's amino acid sequence that interact with the nectin-4 protein and maintain its spatial structure were identified. These mouse amino acids were then retained in the transplanted antibody. Specifically, a series of back mutations were performed on the amino acid residues in the FR region of the humanized template to ensure that the humanized antibody retained the antigen-binding ability of the mouse antibody as much as possible.

[0359] According to the above method, humanized antibodies were constructed based on the CDRs of murine antibodies 31#, 56#, and 74#, and were named 31HZ, 56HZ, and 74HZ, respectively; wherein the heavy chain constant region of each antibody was a human IgG1 heavy chain constant region (SEQ ID NO: 61), and the light chain constant region of each antibody was a human Kappa light chain constant region (SEQ ID NO: 62). The amino acid sequences of the light chain CDRs of the constructed humanized antibodies 31HZ, 56HZ, and 74HZ were consistent with those of the light chain CDRs of 31#, 56#, and 74#, respectively. The amino acid sequences of the heavy chain CDRs of the constructed humanized antibodies 31HZ, 56HZ, and 74HZ are shown in 5.1:

[0360] Table 5.1 Heavy chain CDR amino acid sequences of anti-human nectin-4 humanized antibodies

[0361]

[0362] The amino acid sequences of the variable and constant regions of the three further humanized antibodies 31HZ, 56HZ, and 74HZ are shown in Table 5.2:

[0363] Table 5.2 Amino acid sequences of variable and constant regions of humanized anti-human nectin-4 antibodies

[0364]

[0365] 1.7 Preparation of humanized anti-human nectin-4 antibody

[0366] The amino acid sequences of the heavy and light chains of the humanized anti-human nectin-4 antibody were synthesized and ligated into plasmid pTT5 (commissioned to Nanjing GenScript Biotechnology Co., Ltd.) using codon optimization. The corresponding plasmids for the heavy and light chains were simultaneously transfected into CHO-E cells, and the supernatant was purified using protein A (MabSelect SuRe, GE) to obtain the humanized anti-human nectin-4 antibody.

[0367] Example 2 Affinity detection of anti-human nectin-4 humanized monoclonal antibody

[0368] 2.1 Affinity Detection of Anti-Human Nectin-4 Humanized Monoclonal Antibody and Nectin-4 Proteins from Different Species

[0369] The affinity of anti-human nectin-4 humanized monoclonal antibody to nectin-4 proteins from different species was detected by ELISA. The specific steps are as follows: dilute human nectin-4 or cynomolgus monkey nectin-4 antigen to 1 μg / mL with CBS coating solution (weigh 0.32g Na2CO3, 0.59g NaHCO3, dissolve in deionized water, and adjust the volume to 200mL), and coat 100μL per well in the ELISA plate and incubate at 4°C overnight; the next day, discard the liquid in the wells and wash once with 300μL PBS; add 100μL PBS (containing 2% BSA, purchased from BOVOGEN, product number BSAS 1.0) and block at 37°C for 2 hours; dilute 31HZ, 56HZ, 74HZ and Enfortumab antibodies (starting at 1500ng / mL, 3-fold dilution, 11 concentration points) with PBS (containing 2% BSA), take 100μL and add it to the corresponding wells, incubate at 37°C for 2 hours; 300μL Wash three times with PBST; dilute HRP-labeled goat anti-human secondary antibody (purchased from Jackson, 109-035-00) at a ratio of 1:10,000 with PBS (containing 2% BSA), take 100 μL and add it to the corresponding wells, incubate at 37°C for 1 hour; wash five times with 300 μL PBST; add 100 μL TMB color development solution (purchased from Huzhou Yingchuang, TMB-S-004) to the corresponding wells, and develop at room temperature for 5-20 minutes; add 50 μL 2N H2SO4 to stop the reaction, read the OD450nm on a microplate reader (purchased from MD, SpectraMax M2) and import it into Graphpad Prism for curve fitting.

[0370] The experimental results are shown in Figures 3A-3F and Table 6. The affinity of 31HZ to human nectin-4 protein and cynomolgus monkey nectin-4 protein is better than that of Enfortumab; the affinity of 56HZ and 74HZ to human nectin-4 protein and cynomolgus monkey nectin-4 protein is basically equivalent to that of Enfortumab.

[0371] Table 6 Affinity test of anti-human nectin-4 humanized monoclonal antibody and nectin-4 protein from different species

[0372]

[0373] 2.2 Dynamic affinity testing of anti-human nectin-4 humanized monoclonal antibody and nectin-4 proteins from different species

[0374] Using Octet Fortebio The dynamic affinity of a humanized anti-human nectin-4 monoclonal antibody was determined. The main steps involved immobilizing the anti-human nectin-4 monoclonal antibody (at a concentration of 5 μg / mL) onto a ProA sensor. The antibody then bound to human or cynomolgus monkey nectin-4 (gradiently diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM). After sufficient reaction, the solid-phase conjugate was dissociated in PBST buffer. After completion of the experiment, the results were analyzed using Data Analysis 11.0 software, using a 1:1 model and global fitting, to determine the affinity constants for the antibody and antigen.

[0375] The experimental results are shown in Table 7. The dynamic affinity of 31HZ, 56HZ and 74HZ to human Nectin-4 protein and cynomolgus monkey Nectin-4 protein is better than that of Enfortumab.

[0376] Table 7 Dynamic affinity test between anti-human nectin-4 humanized monoclonal antibody and nectin-4 protein from different species

[0377]

[0378] 2.3 Detection of affinity between humanized anti-human nectin-4 monoclonal antibody and human nectin-4 on tumor cell membrane

[0379] T47D cells were used to detect the affinity of 31HZ and 74HZ to human nectin-4 on the cell membrane. The specific steps were as follows: digest T47D cells, resuspend them by centrifugation, and wash them twice with PBS; resuspend the cells in PBS (containing 1% BSA) and press 3×10 5 Cells were plated into 96-well pointed-bottom plates with a volume of 50 μl per well. 50 μL of the antibody to be tested was added to each well, starting at a final concentration of 15 μg / mL and diluted 4-fold for a total of 10 concentration points. Human IgG was used as a negative control. The cells were mixed and reacted at 4°C in the dark for 1 hour. The cells were washed 3 times with PBS, and FITC-labeled anti-human Fc secondary antibody (Brand: BioLegend, Catalog No.: 409322) was added and incubated at 4°C in the dark for 0.5 hour. The cells were washed 3 times with PBS and detected by flow cytometry (Flow cytometer Brand: Beckman, Model: Cytoflex).

[0380] The same method was used to detect the affinity of 56HZ to human nectin-4 on the membrane surface of NCI-N87 cells (a human gastric cancer cell, ATCC). The antibody was initially diluted 3-fold at 3.33 μg / mL and 10 concentration points were used.

[0381] The experimental results are shown in Figures 4A, 4B and Table 8. The affinity of 31HZ and 56HZ for Nectin-4 on the cell membrane is comparable to that of Enfortumab.

[0382] Table 8 Affinity detection of anti-human nectin-4 humanized monoclonal antibody and human nectin-4 on tumor cell membrane

[0383]

[0384] Note: / : Not tested.

[0385] Example 3 Detection of endocytic activity of humanized anti-human nectin-4 monoclonal antibody

[0386] Flow cytometry was used to detect the endocytic activity of humanized monoclonal antibodies against human nectin-4. The specific steps were as follows: T47D, MDA-MB-468 (a human breast cancer cell line, ATCC), or A549-nectin-4 cells were trypsinized and counted, and the cells were resuspended in PBS (containing 1% BSA) and the cell density was adjusted to 3×10 6 / mL; 100 μL of resuspended cells were added, and the test antibodies 31HZ, 56HZ, and 74HZ were added at a final concentration of 10 μg / mL, with human IgG as a negative control, and incubated on ice for 1 hour; after incubation, the cells were washed three times with pre-cooled PBS, resuspended in cell culture medium and divided into two parts, one part was placed in a 37°C cell culture incubator and incubated for 1, 2, and 4 hours (endocytosis group), and the other part was continued to incubate on ice for 1, 2, and 4 hours (affinity group); after incubation, the cells were washed three times with pre-cooled PBS, resuspended with 50 μL PBS (containing 1% BSA), anti-human fluorescent secondary antibody (BioLegend) was added, and incubated at 4°C for half an hour; after incubation, the cells were washed three times with pre-cooled PBS, and then the fluorescence signal on the cells (expressed as mean fluorescence intensity (MFI)) was detected by flow cytometry (Beckman) and the formula was: endocytosis ratio (%) = [1-(MFI 37℃抗体组 -MFI 37℃对照组 ) / (MFI 冰上抗体组 –MFI 冰上对照组 )]×100% to calculate the antibody internalization rate.

[0387] The same method was used to detect the endocytic activity of 56HZ in T24-Nectin-4 cells.

[0388] The experimental results are shown in Figures 5A-5D. The endocytic activity of 31HZ, 56HZ and 74HZ is better than that of Enfortumab. Example 4 Detection of humanized monoclonal antibody binding to human nectin-4 domain

[0389] Human nectin-4 contains one IgV domain and two IgC domains. To detect the domains bound by humanized monoclonal antibodies against human nectin-4, an A549 stable cell line A549-Nectin-4-IgV overexpressing the nectin-4 IgV domain and an A549 stable cell line A549-Nectin-4-IgCC overexpressing the two IgC domains of nectin-4 were constructed. The domains bound by the antibodies to be tested were detected by flow cytometry. The specific steps are as follows: A549-Nectin-4-IgV and A549-Nectin-4-IgCC cells were digested, resuspended by centrifugation, and washed twice with PBS; the cells were resuspended in PBS (containing 1% BSA) and plated into 96-well conical bottom plates at 300,000 cells / well with a volume of 50 μl per well; 50 μL of the antibody to be tested was added to each well at a final concentration of 40 μg / mL, and human IgG was used as a negative control; the cells were mixed and reacted at 4°C in the dark for 1 hour; the cells were washed three times with PBS, FITC-labeled anti-human Fc secondary antibody (Brand: BioLegend, Catalog No.: 409322) was added, and the cells were incubated at 4°C in the dark for 0.5 hour; the cells were washed three times with PBS, and the cells were detected by flow cytometry (Flow cytometer Brand: Beckman, Model: Cytoflex).

[0390] The experimental results are shown in Figures 6A and 6B. 31HZ, 56HZ, 74HZ and Enfortumab only bind to the IgV domain of human nectin-4, but not the IgC domain.

[0391] Example 5 Competitive Activity Detection of Anti-Human Nectin-4 Humanized Monoclonal Antibody

[0392] Human nectin-4 is linked to another nectin-4 molecule or nectin-1 molecule through the IgV domain. Since 31HZ, 56HZ, and 74HZ all bind to the IgV domain of nectin-4, the competitive activity of the three was detected by competitive ELISA. The specific steps are as follows: dilute the nectin-4 protein to 2μg / mL with CBS coating solution, 100μL per well, incubate at 37℃ for two hours; 300μL Wash once with PBST, add PBS (2% BSA), 100 μL per well, incubate at 37°C for 2 hours; dilute biotin-labeled Nectin-1-hFc to 20 μg / mL with PBS (2% BSA); dilute the antibody to be detected with PBS (2% BSA), 31HZ and 74HZ starting at 3.73 μg / mL, 3-fold dilution, 9 concentration points; 56HZ starting at 1.25 μg / mL, 2-fold dilution, 10 concentration points; mix the above two diluted solutions in equal volumes, add 100 μL / well to the corresponding wells, incubate at room temperature for 2 hours; wash three times with 300 μL PBST; dilute HRP-streptavidin with PBS (2% BSA) at a ratio of 1:10000, add 100 μL / well to the corresponding wells, incubate at room temperature for 1 hour; wash five times with 300 μL PBST; add 100 μL In the wells corresponding to TMB, color was developed at room temperature for 10 minutes; 50 μL 2N H 2 SO 4 was added to terminate the reaction, and the plate reader was used to read the color at 450 nm.

[0393] The experimental results are shown in Figures 7A and 7B. 31 Hz, 56 Hz, and 74 Hz can all competitively inhibit the binding of human nectin-4 to human nectin-1. The competitive IC 50 The values ​​were 27.62 ng / mL, 26.90 ng / mL, and 27.22 ng / mL, respectively, which are comparable to Enfortumab (27.11 ng / mL).

[0394] Example 6 Specificity Detection of Anti-Human Nectin-4 Humanized Monoclonal Antibody

[0395] The nectin family includes nectin-1, nectin-2, nectin-3, and nectin-4. While the sequence homology among these members is approximately 25%, their structures and functions are relatively similar. To test whether the humanized anti-human nectin-4 monoclonal antibody described in this study recognizes only nectin-4 and not nectin-1, nectin-2, and nectin-3, an ELISA assay was used to determine the specificity of the antibody. The specific steps are as follows: dilute human nectin-1, nectin-2, nectin-3 and nectin-4 antigens to 1 μg / mL with CBS coating solution, and coat 100 μL per well on the ELISA plate and incubate at 4°C overnight; the next day, discard the liquid in the wells and wash once with 300 μL PBS; add 100 μL PBS (containing 2% BSA) and block at 37°C for 2 hours; dilute 31HZ, 56HZ, 74HZ and Enfortumab antibodies with PBS (containing 2% BSA), take 100 μL and add them to the corresponding wells, incubate at 37°C for 2 hours; wash 3 times with 300 μL PBST; dilute HRP-labeled goat anti-human secondary antibody at a ratio of 1:10,000 with PBS (containing 2% BSA), take 100 μL and add them to the corresponding wells, incubate at 37°C for 1 hour; wash 5 times with 300 μL PBST; add 100 μL TMB color development solution was added to the corresponding wells and color was developed at room temperature for 5-20 minutes; 50 μL 2N H2SO4 was added to terminate the reaction, and the OD450 nm reading was read on a microplate reader and imported into Graphpad Prism for curve fitting.

[0396] The experimental results are shown in Figures 8A, 8B, and 8C. 31HZ, 56HZ, and 74HZ all specifically recognize human nectin-4, but do not recognize nectin-1, nectin-2, and nectin-3.

[0397] Example 7 Preparation of Anti-Human Nectin-4 ADC

[0398] TL001 was coupled with an anti-human nectin-4 humanized monoclonal antibody to prepare an anti-human nectin-4 ADC. TL001 has the structure of formula (1):

[0399]

[0400] For the preparation method of TL001, please refer to the description in WO2019 / 114666, the relevant content of which is incorporated herein by reference in its entirety.

[0401] The preparation method of anti-human nectin-4 antibody conjugate (anti-human nectin-4 ADC) is as follows:

[0402] (1) Coupling: 30 mg of anti-human nectin-4 humanized monoclonal antibody was diluted with diluent (20 mM PB + 105 mM NaCl, pH 7.7), and sodium edetate solution with a final concentration of 5 mM was added and mixed. TCEP solution (molar ratio of 4.4:1 to antibody) was added and mixed, and the mixture was allowed to stand at room temperature for 30 minutes. TL001 dissolved in dimethyl sulfoxide (molar ratio of 9:1 to antibody) was added to the above solution system, mixed, and allowed to stand at room temperature for 2 hours to obtain coupled samples, which were named 31HZ-TL001, 56HZ-TL001, 74HZ-TL001, and Enfortumab-TL001. After buffer exchange, the anti-human nectin-4 ADC was aliquoted and stored at 4°C.

[0403] (2) Detection:

[0404] LC-MS molecular weight analysis of anti-human Nectin-4 ADC was performed using the following conditions:

[0405] Chromatographic determination conditions:

[0406] Liquid chromatography column: Thermo MAbPac RP 3.0 × 100 mm;

[0407] Mobile phase A: 0.1% FA / 98% H2O / 2% ACN;

[0408] Mobile phase B: 0.1% FA / 2% H2O / 98% ACN;

[0409] Flow rate: 0.25 mL / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 1 μL;

[0410] Time (min) 220 2225 2630 Mobile phase A (%) 7560 557575 Mobile phase B (%) 2540 9595 2525

[0411] Switching valve: 0-3min to waste, 3-22min to MS, 22-30min to waste

[0412] Mass spectrometry conditions:

[0413] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0414] Parameters: GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; m / z 600-4000; Time bins to sum 40.

[0415] The theoretical and measured molecular weights of the light and heavy chains of the conjugated anti-human 31HZ-TL001 (the heavy chain is calculated based on the major glycoform G0F) are shown in the table below:

[0416]

[0417] The light chain of 31HZ-TL001 was conjugated to 0-1 toxins (the ratios of LC and DAR1 were 0.1% and 99.9%, respectively), and the heavy chain was conjugated to 0-4 toxins (the ratios of HC, DAR1, DAR2, DAR3, and DAR4 were 0.0%, 2.0%, 10.2%, 86.1%, and 1.8%, respectively). The coupling ratio (DAR) of the antibody to the toxin was calculated to be 7.75, and the calculation formula was: DAR = light chain DAR1×2+heavy chain (DAR1×1+DAR2×2+DAR3×3+DAR4×4)×2.

[0418] Using the same method to detect the DAR values ​​of 56HZ-TL001, 74HZ-TL001, and Enfortumab-TL001, they were 7.71, 7.98, and 8.01, respectively:

[0419] According to the above mass spectrometry analysis results, the structure of the anti-human nectin-4 ADC can be determined as follows:

[0420]

[0421] wherein A is a humanized monoclonal antibody against human nectin-4; and γ refers to the number of TL001s connected to A via forming a thiol bond with the sulfhydryl group of A, which is an integer of 1-10.

[0422] Enfortumab-VC-MMAE was prepared by conjugating VC-MMAE to enfortumab using the same preparation and detection methods as described above, with a molar ratio of antibody to VC-MMAE of 1:5. The resulting DAR value for this batch of enfortumab-VC-MMAE was 5.09.

[0423] Example 8 Detection of the killing activity of anti-human nectin-4 ADC against tumor cell lines that highly express human nectin-4

[0424] The A549-Nectin-4 stable cell line was selected to detect the killing activity of 31HZ-TL001, 74HZ-TL001, and Enfortumab-TL001 against the human Nectin-4 high-expressing cell line. The specific experimental steps are as follows: digest A549-Nectin-4 cells, collect cells by centrifugation, resuspend cells to 100,000 / mL in DMEM+4% FBS medium, and plate cells in a 96-well plate at 100 μL / well; gradiently dilute the ADC molecule with DMEM basal medium, starting at a final concentration of 150 μg / mL, dilute 4-fold, 11 concentration points, add 100 μL to each well, and the final serum concentration is 2%; culture in a 37°C, 5% CO2 incubator for 72 hours; add CCK8 (Rhinogen), 20 μL / well, incubate in a 37°C, 5% CO2 incubator for 0.5-2.5 hours, read at 450 nm using a microplate reader (MD) every half hour and import into Graphpad Prism for curve fitting.

[0425] The experimental results are shown in Figures 9A and 9B. 31HZ-TL001, 74HZ-TL001 and Enfortumab-TL001 can effectively kill A549-Nectin-4 cells. 50 The values ​​were 1390ng / mL, 1364ng / mL and 2049ng / mL, respectively, indicating that 31HZ-TL001 and 74HZ-TL001 had better killing activity against tumor cell lines that overexpressed human nectin-4 than Enfortumab-TL001; at the same time, the killing activity of 31HZ-TL001, 74HZ-TL001 and Enfortumab-TL001 against A549 cells that did not overexpress human nectin-4 was 50 The values ​​were 5982ng / mL, 6251ng / mL, and 5604ng / mL, respectively. Overall, judging from the cytotoxicity against A549-Nectin-4 and A549 cells, 31HZ-TL001, 74HZ-TL001, and Enfortumab-TL001 showed nectin-4-specific cytotoxicity.

[0426] The same experimental method as described above was used to detect the killing activity of 31HZ-TL001 and 56HZ-TL001 against the human nectin-4 high-expressing cell line T24-nectin-4. The experimental results are shown in Figures 9C and 9D. Both 31HZ-TL001 and 56HZ-TL001 can effectively kill T24-nectin-4 cells. 50The values ​​were 1277ng / mL and 1568ng / mL, respectively, indicating that the killing activities of the two were basically equivalent; the killing activity of 31HZ-TL001 and 56HZ-TL001 on T24 cells that did not overexpress human nectin-4 was EC 50 The values ​​were 2884ng / mL and 3665ng / mL respectively, and the killing activity was significantly weaker than that of T24-Nectin-4 cells, indicating that the killing effect of both was specific to Nectin-4.

[0427] Example 9 Detection of the killing activity of anti-human nectin-4 ADC against tumor cell lines endogenously expressing human nectin-4

[0428] The T47D tumor cell line was selected to detect the killing activity of 31HZ-TL001, 74HZ-TL001, and Enfortumab-TL001 against tumor cell lines endogenously expressing human nectin-4. The specific experimental steps are as follows: digest T47D cells, collect cells by centrifugation, resuspend cells to 100,000 / mL in 1640+4% FBS medium, and plate cells in a 96-well plate at 100 μL / well; gradiently dilute the ADC molecule with 1640 basal medium, starting at a final concentration of 150 μg / mL, dilute 4-fold, and add 11 concentration points to the corresponding wells, 100 μL per well, for a final serum concentration of 2%; culture in a 37°C, 5% CO2 incubator for 72 hours; add CCK8 (Rhinogen) at 20 μL / well, incubate in a 37°C, 5% CO2 incubator for 0.5-2.5 hours, read at 450 nm using a microplate reader (MD) every half hour, and import the results into Graphpad Prism for curve fitting.

[0429] The experimental results are shown in Figure 10A. 31HZ-TL001, 74HZ-TL001 and Enfortumab-TL001 can effectively kill T47D cells. 50 The values ​​were 206.1 ng / mL, 611.3 ng / mL and 229.2 ng / mL, respectively, indicating that the killing activity of 31HZ-TL001 against T47D cells was comparable to that of Enfortumab-TL001.

[0430] The same experimental method as described above was used to test the killing activity of 31HZ-TL001, 74HZ-TL001 and Enfortumab-TL001 against the tumor cell line MDA-MB-468 that endogenously expresses human nectin-4. The experimental results are shown in Figure 10B. 31HZ-TL001, 74HZ-TL001 and Enfortumab-TL001 can effectively kill MDA-MB-468 cells. 50The values ​​were 1030 ng / mL, 2277 ng / mL and 1190 ng / mL, respectively, indicating that the killing activity of 31HZ-TL001 against MDA-MB-468 cells was comparable to that of Enfortumab-TL001.

[0431] The same experimental method as described above was used to detect the killing activity of 31HZ-TL001 and 56HZ-TL001 against the tumor cell line NCI-N87 endogenously expressing human nectin-4. The experimental results are shown in Figure 10C. Both 31HZ-TL001 and 56HZ-TL001 can effectively kill NCI-N87 cells. 50 The values ​​were 9376ng / mL and 10935ng / mL, respectively, indicating that the killing activities of the two were basically equivalent.

[0432] In summary, the cytotoxic activity of 31HZ-TL001 and 56HZ-TL001 against tumor cell lines endogenously expressing human nectin-4 is basically equivalent to that of Enfortumab-TL001.

[0433] Example 10 Anti-human Nectin-4 ADC in vivo CDX model efficacy test

[0434] The CDX model was constructed using the human non-small cell lung cancer cell line NCI-H322M to evaluate the anti-tumor effect of ADC molecules. The specific steps are as follows: NCI-H322M cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells in the exponential growth phase were collected, resuspended in PBS, and plated at 1×10 7 The cells (suspended in 0.1 mL PBS) were subcutaneously inoculated into female NOD / SCID mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) to establish a subcutaneous xenograft tumor model. 3 At 4 hr, animals were randomly divided into groups based on tumor volume, with 5 mice per group. The day of grouping was designated Day 0, and the groups included vehicle (normal saline) (negative control), 31HZ-TL001 (3 mg / kg and 10 mg / kg), 56HZ-TL001 (3 mg / kg and 10 mg / kg), Enfortumab-TL001 (10 mg / kg), and Enfortumab-VC-MMAE (10 mg / kg). All samples were injected into the tail vein twice weekly for a total of 6 doses.

[0435] After administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was recorded daily.

[0436] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy:

[0437] TGI (%) = [1-(V T末 -V T始 ) / (V C末 -V C始 )]*100%

[0438] Where V T末 : Mean tumor volume of the treatment group at the end of the experiment;

[0439] V T始 : Mean tumor volume of the treatment group at the beginning of drug administration;

[0440] V C末 : Mean tumor volume of negative control group at the end of the experiment;

[0441] V C始 : Mean tumor volume of the negative control group at the beginning of drug administration.

[0442] As shown in Table 9 and Figures 11 and 12, 31HZ-TL001, 56HZ-TL001, Enfortumab-TL001, and Enfortumab-VC-MMAE significantly inhibited tumor growth in the NCI-H322M xenograft model in a dose-dependent manner. After six doses (on day 21), compared to the negative control group, at a 10 mg / kg dose level, the TGIs for 31HZ-TL001, 56HZ-TL001, Enfortumab-TL001, and Enfortumab-VC-MMAE were 155.79%, 163.56%, 156.31%, and 173.79%, respectively. All treated groups showed no significant weight loss during the observation period, indicating that the ADC was well tolerated by the animals.

[0443] Table 9 NCI-H322M+NOD / SCID mouse CDX model

[0444]

[0445] Note: TGI: tumor growth inhibition rate; PR: partial tumor regression; CR: complete tumor regression. P values ​​are compared with the vehicle group.

[0446] These results demonstrate that both 31HZ-TL001 and 56HZ-TL001 effectively inhibited tumor growth in a dose-dependent manner, as measured by their TGIs at 3 and 10 mg / kg doses. At a 10 mg / kg dose, both 31HZ-TL001 and 56HZ-TL001 caused partial tumor regression in all five mice, demonstrating comparable efficacy to enfortumab-TL001 and enfortumab-VC-MMAE. Furthermore, 31HZ-TL001 and 56HZ-TL001 were well tolerated by tumor-bearing mice.

[0447] Example 11 Anti-human Nectin-4 ADC in vivo PDX model efficacy testing

[0448] from Tumor tissue was harvested from mice bearing BL9200 bladder cancer xenografts (Crown Biotech (Taicang) Co., Ltd., a bladder cancer tumor with high Nectin-4 expression, derived from a 63-year-old male patient). Tumors were cut into 3×3×3mm diameter tumor masses and inoculated subcutaneously at the right anterior scapula of NOD / SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). When the average tumor volume of the tumor-bearing mice reached approximately 150-250mm, the tumor was inoculated subcutaneously at the right anterior scapula of the NOD / SCID mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). 3 At 4 hr, mice were randomly divided into groups according to tumor size, with 7 mice per group. The day of grouping was defined as Day 0. There were 4 groups: Human IgG1 isotype control antibody (negative control) group (IgG1 3 mg / kg), Human IgG1-TL001 (abbreviated as IgG1-TL001, 3 mg / kg; see Example 7 for preparation method), 56HZ-TL001 1 mg / kg group and 3 mg / kg group. All samples were injected into the tail vein twice a week for a total of 6 doses. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The measurement and calculation methods are as described in Example 10.

[0449] The experimental results are shown in Table 10 and Figures 13 and 14. After six doses (on day 20), compared to the negative control group (Human IgG1 (hIgG1) 3 mg / kg), 56HZ-TL001 3 mg / kg inhibited tumor growth by 105.55% (p < 0.05), and four mice in this group experienced partial tumor regression. The tumor growth inhibitory effect of 56HZ-TL001 3 mg / kg was significantly superior to that of the isotype control ADC (hIgG1-TL001 3 mg / kg) at the same dose (p < 0.001). One mouse in the hIgG1 group died, while no animals in the other groups died and were active normally. During the dosing period, mice in all groups experienced a slight decrease in weight, with the IgG1 group experiencing the greatest weight loss due to ineffective treatment, indicating that the tumor-bearing mice tolerated the ADC well.

[0450] These results demonstrate that 56HZ-TL001 significantly inhibits tumor growth in the BL9200 bladder cancer PDX xenograft model. Furthermore, 56HZ-TL001 was well tolerated by tumor-bearing mice.

[0451] Table 10 Bladder cancer + NOD / SCID mouse PDX model

[0452]

[0453] Note: TGI: tumor growth inhibition rate; PR: partial tumor regression; CR: complete tumor regression; / : not applicable.

[0454] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published so far, and these changes are all within the scope of protection of the present invention. The scope of protection of the present invention is given by the appended claims and any equivalents thereof.

[0455] Sequence Listing

[0456]

[0457]

[0458]

[0459]

[0460]

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Nectin-4, comprising: (1a) The following three heavy-chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:3 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:4 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:5 or a variant thereof; and / or The following three light-chain CDRs defined according to the Chothia numbering system: CDR-L1 comprising the sequence shown in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; Or (1b) The following three heavy-chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:9 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:10 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:5 or a variant thereof; and / or The following three light-chain CDRs defined according to the Abm numbering system: CDR-L1 comprising the sequence shown in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; Or (1c) The following three heavy-chain CDRs defined according to the Kabat numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:11 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:12 or 63 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:5 or a variant thereof; and / or The following three light-chain CDRs defined according to the Kabat numbering system: CDR-L1 comprising the sequence shown in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; Or (1d) The following three heavy-chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:13 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:14 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:15 or a variant thereof; and / or The following three light-chain CDRs defined according to the IMGT numbering system: CDR-L1 comprising the sequence shown in SEQ ID NO:16 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:17 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; Or (2a) The following three heavy-chain CDRs defined according to the Chothia numbering system: A CDR-H1 comprising the sequence shown in SEQ ID NO:21 or 18 or a variant thereof, A CDR-H2 comprising the sequence shown in SEQ ID NO:22 or a variant thereof, A CDR-H3 comprising the sequence shown in SEQ ID NO:23 or a variant thereof; and / or The following three light chain CDRs defined according to the Chothia numbering system: A CDR-L1 comprising the sequence shown in SEQ ID NO:24 or a variant thereof, A CDR-L2 comprising the sequence shown in SEQ ID NO:25 or a variant thereof, A CDR-L3 comprising the sequence shown in SEQ ID NO:26 or a variant thereof; Or (2b) The following three heavy chain CDRs defined according to the Abm numbering system: A CDR-H1 comprising the sequence shown in SEQ ID NO:27 or 36 or a variant thereof, A CDR-H2 comprising the sequence shown in SEQ ID NO:28 or a variant thereof, A CDR-H3 comprising the sequence shown in SEQ ID NO:23 or a variant thereof; and / or The following three light chain CDRs defined according to the Abm numbering system: A CDR-L1 comprising the sequence shown in SEQ ID NO:24 or a variant thereof, A CDR-L2 comprising the sequence shown in SEQ ID NO:25 or a variant thereof, A CDR-L3 comprising the sequence shown in SEQ ID NO:26 or a variant thereof; Or (2c) The following three heavy chain CDRs defined according to the Kabat numbering system: A CDR-H1 comprising the sequence shown in SEQ ID NO:29 or 44 or a variant thereof, A CDR-H2 comprising the sequence shown in SEQ ID NO:30 or a variant thereof, A CDR-H3 comprising the sequence shown in SEQ ID NO:23 or a variant thereof; and / or The following three light chain CDRs defined according to the Kabat numbering system: A CDR-L1 comprising the sequence shown in SEQ ID NO:24 or a variant thereof, A CDR-L2 comprising the sequence shown in SEQ ID NO:25 or a variant thereof, A CDR-L3 comprising the sequence shown in SEQ ID NO:26 or a variant thereof; Or (2d) The following three heavy chain CDRs defined according to the IMGT numbering system: A CDR-H1 comprising the sequence shown in SEQ ID NO:31 or 54 or a variant thereof, A CDR-H2 comprising the sequence shown in SEQ ID NO:32 or a variant thereof, A CDR-H3 comprising the sequence shown in SEQ ID NO:33 or a variant thereof; and / or The following three light chain CDRs defined according to the IMGT numbering system: A CDR-L1 comprising the sequence shown in SEQ ID NO:34 or a variant thereof, A CDR-L2 comprising the sequence shown in SEQ ID NO:35 or a variant thereof, A CDR-L3 comprising the sequence shown in SEQ ID NO:26 or a variant thereof; Or (3a) The following three heavy chain CDRs defined according to the Chothia numbering system: A CDR-H1 comprising the sequence shown in SEQ ID NO:39 or a variant thereof, A CDR-H2 comprising the sequence shown in SEQ ID NO:40 or a variant thereof, a CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Chothia numbering system: a CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, a CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, a CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (3b) the following three heavy chain CDRs defined according to the Abm numbering system: a CDR-H1 comprising the sequence shown in SEQ ID NO:45 or a variant thereof, a CDR-H2 comprising the sequence shown in SEQ ID NO:46 or a variant thereof, a CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Abm numbering system: a CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, a CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, a CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (3c) the following three heavy chain CDRs defined according to the Kabat numbering system: a CDR-H1 comprising the sequence shown in SEQ ID NO:47 or a variant thereof, a CDR-H2 comprising the sequence shown in SEQ ID NO:48 or 64 or a variant thereof, a CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof; and / or the following three light chain CDRs defined according to the Kabat numbering system: a CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, a CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, a CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (3d) the following three heavy chain CDRs defined according to the IMGT numbering system: a CDR-H1 comprising the sequence shown in SEQ ID NO:49 or a variant thereof, a CDR-H2 comprising the sequence shown in SEQ ID NO:50 or a variant thereof, a CDR-H3 comprising the sequence shown in SEQ ID NO:51 or a variant thereof; and / or the following three light chain CDRs defined according to the IMGT numbering system: a CDR-L1 comprising the sequence shown in SEQ ID NO:52 or a variant thereof, a CDR-L2 comprising the sequence shown in SEQ ID NO:53 or a variant thereof, a CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; wherein, the variant has one or more amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitution is a conservative substitution; preferably, the antibody or antigen-binding fragment further comprises a framework region derived from a human or murine immunoglobulin; Preferably, the antibody or antigen-binding fragment binds to human Nectin-4.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) The following six CDRs of the heavy and light chains defined according to the Chothia numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 3 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 4 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; or (b) The following six CDRs of the heavy and light chains defined according to the Abm numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 9 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 10 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; or (c) The following six CDRs of the heavy and light chains defined according to the Kabat numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 11 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 12 or 63 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 5 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 6 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 7 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; or (d) The following six CDRs of the heavy and light chains defined according to the IMGT numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 13 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 14 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 15 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 16 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 17 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; wherein, the variant has one or more amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) The following six CDRs of heavy and light chains defined according to the Chothia numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 21 or 18, or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 22, or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 23, or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 24, or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 25, or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 26, or a variant thereof; or (b) The following six CDRs of heavy and light chains defined according to the Abm numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 27 or 36, or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 28, or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 23, or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 24, or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 25, or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 26, or a variant thereof; or (c) The following six CDRs of heavy and light chains defined according to the Kabat numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 29 or 44, or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 30, or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 23, or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 24, or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 25, or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 26, or a variant thereof; or (d) The following six CDRs of heavy and light chains defined according to the IMGT numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO: 31 or 54, or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO: 32, or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO: 33, or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO: 34, or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO: 35, or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO: 26, or a variant thereof; wherein, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

4. The antibody or antigen-binding fragment thereof of claim 1, which comprises: (a) The following six CDRs of heavy and light chains defined according to the Chothia numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:39 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:40 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (b) The following six CDRs of the heavy and light chains defined according to the Abm numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:45 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:46 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (c) The following six CDRs of the heavy and light chains defined according to the Kabat numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:47 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:48 or 64 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:41 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO:42 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:43 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; or (d) The following six CDRs of the heavy and light chains defined according to the IMGT numbering system: CDR-H1 comprising the sequence shown in SEQ ID NO:49 or a variant thereof, CDR-H2 comprising the sequence shown in SEQ ID NO:50 or a variant thereof, CDR-H3 comprising the sequence shown in SEQ ID NO:51 or a variant thereof, CDR-L1 comprising the sequence shown in SEQ ID NO:52 or a variant thereof, CDR-L2 comprising the sequence shown in SEQ ID NO:53 or a variant thereof, CDR-L3 comprising the sequence shown in SEQ ID NO:8 or a variant thereof; Wherein, said variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions); preferably, said substitution is a conservative substitution.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, which comprises: VH having the sequence shown in SEQ ID NO:1 or SEQ ID NO:55 or a variant thereof and / or VL having the sequence shown in SEQ ID NO:2 or SEQ ID NO:56 or a variant thereof; or a VH having a sequence as set forth in SEQ ID NO:19 or SEQ ID NO:57 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:20 or SEQ ID NO:58 or a variant thereof; or a VH having a sequence as set forth in SEQ ID NO:37 or SEQ ID NO:59 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:38 or SEQ ID NO:60 or a variant thereof; wherein, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, comprising: (1) a VH having a sequence as set forth in SEQ ID NO:1 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:2 or a variant thereof; or (2) a VH having a sequence as set forth in SEQ ID NO:19 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:20 or a variant thereof; or (3) a VH having a sequence as set forth in SEQ ID NO:37 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:38 or a variant thereof; or (4) a VH having a sequence as set forth in SEQ ID NO:55 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:56 or a variant thereof; or (5) a VH having a sequence as set forth in SEQ ID NO:57 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:58 or a variant thereof; or (6) a VH having a sequence as set forth in SEQ ID NO:59 or a variant thereof and / or a VL having a sequence as set forth in SEQ ID NO:60 or a variant thereof; wherein, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

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

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, which further comprises: The heavy chain constant region of human immunoglobulin or a variant thereof, and the light chain constant region of human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; the light chain constant region is selected from the κ or λ light chain constant regions. More preferably, the heavy chain constant region is an IgG1 heavy chain constant region. Most preferably, the heavy chain constant region has the sequence shown in SEQ ID NO: 61; the light chain constant region is a κ light chain constant region; and / or the light chain constant region has the sequence shown in SEQ ID NO:

62.

9. An antibody or an antigen-binding fragment thereof according to any one of claims 1-8, which comprises a heavy chain and a light chain, wherein: (a) the heavy chain comprises: VH having the sequence shown in SEQ ID NO: 1 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 2 and a light chain constant region having the sequence shown in SEQ ID NO: 62; or (b) the heavy chain comprises: VH having the sequence shown in SEQ ID NO: 19 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 20 and a light chain constant region having the sequence shown in SEQ ID NO: 62; or (c) the heavy chain comprises: VH having the sequence shown in SEQ ID NO: 37 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 38 and a light chain constant region having the sequence shown in SEQ ID NO: 62; or (d) the heavy chain comprises: VH having the sequence shown in SEQ ID NO: 55 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 56 and a light chain constant region having the sequence shown in SEQ ID NO: 62; or (e) the heavy chain comprises: VH having the sequence shown in SEQ ID NO: 57 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 58 and a light chain constant region having the sequence shown in SEQ ID NO: 62; or (f) The heavy chain comprises: VH having the sequence shown in SEQ ID NO: 59 and a heavy chain constant region having the sequence shown in SEQ ID NO: 61, and the light chain comprises: VL having the sequence shown in SEQ ID NO: 60 and a light chain constant region having the sequence shown in SEQ ID NO:

62.

10. An antibody or antigen-binding fragment thereof according to any one of claims 1-9, selected from scFv, Fab, Fab', F(ab') 2 , Fv fragment, disulfide-stabilized Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-10, which binds to the IgV domain of Nectin-4.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, which is conjugated to at least one label; preferably, the label is selected from enzymes, fluorescent dyes, radioisotopes, biotin, and colloidal gold.

13. An antibody conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-11 and at least one therapeutic agent.

14. The antibody conjugate of claim 13, wherein the therapeutic agent is a cytotoxic agent.

15. The antibody conjugate of claim 13 or 14, which has the structure shown in formula (Ⅰ), A-(L-D) γ (Ⅰ)； wherein, A is the antibody or antigen-binding fragment thereof according to any one of claims 1-11; L comprises an amino acid or a peptide composed of 2 to 10 amino acids; more preferably, L is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, One of 1 and 2 represents the position where L is connected to A, and the other represents the position where L is connected to D; D is selected from cytotoxic agents, therapeutic antibodies, radioisotopes, oligonucleotides and their analogs, bioactive peptides, protein toxins, and enzymes; preferably, D is a molecule having anti-tumor biological activity; Further preferably, D is γ is an integer from 1 to 10.

16. The antibody conjugate of claim 15, wherein the (L-D) moiety has the structure of formula (1) and is connected by forming a thioether bond with the thiol group on A, The antibody conjugate has the structure of formula (2): where γ is an integer from 1 to 10, and A is the antibody or antigen-binding fragment thereof according to any one of claims 1-11.

17. The antibody conjugate of claim 13, wherein the therapeutic agent is a radioisotope; Preferably, the radioactive isotope is selected from 212 Bi,[[]] 213 Bi,[[]] 131 I,[[]] 125 I,[[]] 111 In,[[]] 177 Lu,[[]] 186 Re,[[]] 188 Re,[[]] 153 Sm,[[]] 90 Y.[[]] 18. A composition comprising the antibody conjugate according to any one of claims 13-17, wherein the molar ratio (DAR value) of the therapeutic agent to the antibody or antigen-binding fragment thereof in the composition is a decimal or integer between 1 and 10 (for example, a decimal or integer between 1 and 8, for example, 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.79, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0, 7.03, 7.1, 7.12, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

19. A chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11, a transmembrane domain, and one or more intracellular T cell signaling domains.

20. An immune effector cell expressing the CAR according to claim 19 on its surface; preferably, the immune effector cell is selected from cytotoxic T cells, natural killer cells, and natural killer T cells.

21. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-11.

22. An expression vector comprising the nucleic acid molecule of claim 21.

23. A host cell comprising the nucleic acid molecule of claim 21 or the expression vector of claim 24; preferably, the host cell is a eukaryotic cell; more preferably, a mammalian cell; most preferably, a Chinese hamster ovary cell; or preferably, the host cell is a prokaryotic cell; more preferably, the host cell is Escherichia coli.

24. A multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11 and at least one other antibody or antigen-binding fragment thereof or antibody analogue such that the multispecific antibody is capable of binding Nectin-4 and at least one other antigen; preferably, the multispecific antibody is a bispecific antibody, trispecific antibody, or tetra-specific antibody.

25. A fusion protein comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11, and an additional bioactive polypeptide; preferably, the additional bioactive polypeptide is a polypeptide or protein having therapeutic activity, binding activity, or enzymatic activity.

26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-11, an antibody conjugate according to any one of claims 13-17, the composition of claim 18, the immune effector cell of claim 20, the multispecific antibody of claim 24, or the fusion protein of claim 25, and a pharmaceutically acceptable carrier and / or excipient.

27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-11, an antibody conjugate according to any one of claims 13-17, the composition of claim 18, the immune effector cell of claim 20, the multispecific antibody of claim 24, the fusion protein of claim 25, or the pharmaceutical composition of claim 26 in the manufacture of a medicament for the treatment of cancer; preferably, the cancer is selected from gastric cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, urethral cancer, renal pelvic cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, breast ductal carcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, and esophageal cancer.

28. The use of claim 27, wherein the medicament is used in combination with one or more therapeutic agents selected from the following: anti-tumor drugs, chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors.

29. Use according to claim 27, wherein the drug is used in combination with one or more treatment regimens selected from the following: surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.

30. A method for detecting the presence of Nectin-4 in a sample or determining its expression level, which comprises: (a) contacting the sample with an antibody according to any one of claims 1-11 or an antigen-binding fragment thereof, and (b) detecting the formation of an immune complex between the antibody or antigen-binding fragment and Nectin-4 in the sample or determining the amount of the immune complex to detect the presence of Nectin-4 in the sample or determine the expression level of Nectin-4 in the sample.

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