Nectin-4 antibody and use thereof

Abstract

Provided are an anti-Nectin-4 antibody or an antigen-binding fragment thereof, an antibody conjugate and the use thereof, relating to the technical field of biology. Specifically, the obtained Nectin-4 antibody has higher affinity and good cell endocytosis rate. The antibody conjugate containing the antibody has a remarkable cell killing effect, and has a very strong tumor growth inhibition effect. Also provided is the use of the antibody or the antigen-binding fragment thereof and the antibody conjugate in preparing a drug used for treating cancers, which plays an important role in treating human tumors.

Description

Nectin-4 antibody and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antibody specifically binding to nectin-4 and a fragment thereof, as well as uses of the antibody. Background Art

[0002] The nectin cell adhesion molecule family includes four members: nectin-1, nectin-2, nectin-3, and nectin-4. Among them, nectin-1, nectin-2, and nectin-3 are widely expressed in normal adult tissues, while nectin-4 is widely expressed in cancer cell tissues.

[0003] In recent years, nectin cell adhesion molecule 4 (Nectin-4), a member of the nectin family of immunoglobulin-like adhesion molecules, has been recognized as a key factor in the development of malignant tumors, including urothelial carcinoma, gastric cancer, thyroid cancer, breast cancer, esophageal cancer, and ovarian cancer. Abnormal expression of nectin-4 is associated with tumor cell development through proliferation, angiogenesis, and decreased apoptosis, suggesting that nectin-4 is a potential therapeutic target.

[0004] Antibody-drug conjugates (ADCs) are a technology that uses the specific recognition ability of antibodies for specific antigens on the surface of tumor cells to accurately deliver anti-tumor drugs (such as cytotoxic agents, cytostatics, small molecule chemotherapeutics, etc.) to tumor target cells, causing them to accumulate and release intracellularly, thereby precisely killing tumors. Antibody-drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker (connector) that couples the antibody or antibody-like ligand to the drug. Due to their appropriate molecular weight, high stability, strong targeting, and minimal toxic side effects, antibody-drug conjugates have been considered the most promising anti-tumor drugs.

[0005] Currently, the leading drug targeting nectin-4 is enfortumab vedotin, an antibody-drug conjugate (ADC) consisting of an anti-nectin-4 monoclonal antibody conjugated to the cell-killing drug monomethyl auristatin E (MMAE). It is primarily used to treat bladder cancer, particularly urothelial carcinoma, and received FDA Breakthrough Therapy Designation in March 2018. Furthermore, other studies have shown that the adhesion factor Nnectin-4 is not only an effective prognostic factor for breast cancer, but also a therapeutic target for triple-negative breast cancer (TNBC). In vitro and in vivo studies have confirmed that anti-nectin-4 antibody-drug conjugates (ADCs) have a promising therapeutic effect in both localized and metastatic TNBC.

[0006] Seattle Genetics collaborated with Astellas to randomly conjugate the anti-nectin-4 antibody enfortumab using the company's proprietary linker, mc-vc-MMAE, to the anti-nectin-4 antibody enfortumab, resulting in the anti-nectin-4 antibody-drug conjugate enfortumab vedotin (Padcev). Clinical trial results showed that among patients receiving chemotherapy and PD-1 / PD-L1 inhibitors, those receiving enfortumab vedotin had a median overall survival of 12.9 months, 3.9 months longer than the chemotherapy control group, demonstrating a favorable tumor treatment effect. However, clinical studies also found that the use of enfortumab vedotin is often accompanied by fever, itchy skin, peripheral neuropathy, dry eyes, and neutropenia. These adverse reactions are directly related to excessive attachment of the small molecule to the antibody and unstable attachment method.

[0007] Therefore, there is an urgent need in the art to provide an efficient, simple, and practical chemical coupling method for the research and development of antibody-drug conjugates targeting nectin-4. Summary of the Invention

[0008] To address the above issues, the present invention provides an antibody molecule or functional fragment thereof that specifically binds to nectin-4, as well as an antibody conjugate comprising the antibody or functional fragment thereof. The antibody molecule or functional fragment thereof described herein has higher affinity and improved cellular endocytosis rate; the antibody conjugate comprising the antibody or functional fragment thereof exhibits significant cell-killing effects and a strong tumor inhibition rate.

[0009] The first aspect of the present invention is to provide an anti-nectin-4 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR selected from the amino acid sequence of SEQ ID NO: 1-17, 46 or a variant thereof; and the light chain variable region comprises a light chain CDR selected from the amino acid sequence of SEQ ID NO: 18-30 or a variant thereof.

[0010] In some embodiments, the present invention provides an anti-nectin-4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0011] (1) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively; or (2) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively; or (3) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:24, SEQ ID NO:19 and SEQ ID NO:25, respectively. NO:25; or (4) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:25, respectively; or (5) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:9, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:24, SEQ ID NO:19, SEQ ID NO:25, respectively; or (6) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: NO:30; or (7) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:17, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively;or (8) the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:46, and SEQ ID NO:17, respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.

[0012] In some preferred embodiments, for the antibody or antigen-binding fragment thereof, the amino acid sequences of HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from the following groups:

[0013] (1) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:17, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; or (2) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:46 and SEQ ID NO:17, respectively; and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively.

[0014] In a second aspect, the present invention provides an anti-nectin-4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL): the amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-37 and SEQ ID NOs: 47-52, and the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-43 and SEQ ID NOs: 53-57; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-37, SEQ ID NOs: 47-52, SEQ ID NOs: 38-43 and SEQ ID NOs: 53-57.

[0015] In some embodiments, the antibodies of the invention include antibodies comprising a combination of heavy chain variable regions (VH) and light chain variable regions (VL) selected from the following possibilities:

[0016] (1) the heavy chain variable region amino acid sequence of SEQ ID NO:31, and the light chain variable region amino acid sequence of SEQ ID NO:38; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (2) the heavy chain variable region amino acid sequence of SEQ ID NO:32, and the light chain variable region amino acid sequence of SEQ ID NO:39; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (3) the heavy chain variable region amino acid sequence of SEQ ID NO:33, and the light chain variable region amino acid sequence of SEQ ID NO:40; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (4) the heavy chain variable region amino acid sequence of SEQ ID NO: NO:34, and the light chain variable region amino acid sequence as shown in SEQ ID NO:41; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (5) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:35, and the light chain variable region amino acid sequence as shown in SEQ ID NO:40; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (6) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:36, and the light chain variable region amino acid sequence as shown in SEQ ID NO:42; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (7) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:37 NO:37, and the light chain variable region amino acid sequence as shown in SEQ ID NO:43; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (8) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:47, and the light chain variable region amino acid sequence as shown in SEQ ID NO:43; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (9) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:48, and the light chain variable region amino acid sequence as shown in SEQ ID NO:53;or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (10) the heavy chain variable region amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region amino acid sequence set forth in SEQ ID NO:54; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (11) the heavy chain variable region amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region amino acid sequence set forth in SEQ ID NO:55; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (12) the heavy chain variable region amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region amino acid sequence set forth in SEQ ID NO: NO:56; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (13) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:48, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:57; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (14) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:49, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:53; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (15) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:49, and the light chain variable region amino acid sequence as set forth in SEQ ID NO: NO:54; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (16) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:49, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:55; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (17) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:49, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:56; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;or (18) the heavy chain variable region amino acid sequence of SEQ ID NO:49, and the light chain variable region amino acid sequence of SEQ ID NO:57; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (19) the heavy chain variable region amino acid sequence of SEQ ID NO:50, and the light chain variable region amino acid sequence of SEQ ID NO:53; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (20) the heavy chain variable region amino acid sequence of SEQ ID NO:50, and the light chain variable region amino acid sequence of SEQ ID NO:54; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (21) the heavy chain variable region amino acid sequence of SEQ ID NO:51 NO:50, and the light chain variable region amino acid sequence as shown in SEQ ID NO:55; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (22) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:50, and the light chain variable region amino acid sequence as shown in SEQ ID NO:56; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (23) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:50, and the light chain variable region amino acid sequence as shown in SEQ ID NO:57; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (24) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:50, and the light chain variable region amino acid sequence as shown in SEQ ID NO:58; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; NO:51, and the light chain variable region amino acid sequence as shown in SEQ ID NO:53; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (25) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:51, and the light chain variable region amino acid sequence as shown in SEQ ID NO:54; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (26) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:51, and the light chain variable region amino acid sequence as shown in SEQ ID NO:55;or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (27) the heavy chain variable region amino acid sequence of SEQ ID NO:51, and the light chain variable region amino acid sequence of SEQ ID NO:56; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (28) the heavy chain variable region amino acid sequence of SEQ ID NO:51, and the light chain variable region amino acid sequence of SEQ ID NO:57; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (29) the heavy chain variable region amino acid sequence of SEQ ID NO:52, and the light chain variable region amino acid sequence of SEQ ID NO:53. NO:53; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (30) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:52, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:54; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (31) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:52, and the light chain variable region amino acid sequence as set forth in SEQ ID NO:55; or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; or (32) the heavy chain variable region amino acid sequence as set forth in SEQ ID NO:52, and the light chain variable region amino acid sequence as set forth in SEQ ID NO: NO:56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto; or (33) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:52, and the light chain variable region amino acid sequence as shown in SEQ ID NO:57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0017] In some preferred embodiments, the antibody of the present invention is selected from the following groups: (1) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:37, and the light chain variable region amino acid sequence as shown in SEQ ID NO:43; or (2) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:47, and the light chain variable region amino acid sequence as shown in SEQ ID NO:43; or (3) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:48, and the light chain variable region amino acid sequence as shown in SEQ ID NO:55; or (4) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:48, and the light chain variable region amino acid sequence as shown in SEQ ID NO:57; or (5) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:49, and the light chain variable region amino acid sequence as shown in SEQ ID NO:55; or (6) the heavy chain variable region amino acid sequence as shown in SEQ ID NO:49, and the light chain variable region amino acid sequence as shown in SEQ ID NO:56.

[0018] In a third aspect, the present invention provides an anti-nectin-4 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is murine, chimeric, or humanized; wherein the FR region sequences on the light chain and heavy chain variable regions of the humanized antibody are respectively derived from human germline light chain and heavy chain or mutant sequences thereof; the antibody of the present invention also includes a heavy chain constant region and a light chain constant region.

[0019] Preferably, the antibody comprises a heavy chain constant domain of human IgG1, IgG2, IgG3, IgG4 and a light chain constant domain of human kappa or lambda type or a variant thereof, wherein the variant comprises one or several amino acid substitutions.

[0020] Preferably, the antibody comprises a human IgG1 constant domain or a variant thereof, and a kappa constant domain or a variant thereof, wherein the variant comprises one or several amino acid substitutions.

[0021] More preferably, the antibody comprises the heavy chain constant region shown in SEQ ID NO:44 and the light chain constant region shown in SEQ ID NO:45.

[0022] More preferably, the antibody of the present invention comprises a complete structure having two light chains and two heavy chains, and the antibody is selected from the following groups: (1) a heavy chain amino acid sequence as shown in SEQ ID NO:58, and a light chain amino acid sequence as shown in SEQ ID NO:62; or (2) a heavy chain amino acid sequence as shown in SEQ ID NO:59, and a light chain amino acid sequence as shown in SEQ ID NO:63; or (3) a heavy chain amino acid sequence as shown in SEQ ID NO:60, and a light chain amino acid sequence as shown in SEQ ID NO:64; or (4) a heavy chain amino acid sequence as shown in SEQ ID NO:61, and a light chain amino acid sequence as shown in SEQ ID NO:65.

[0023] In a fourth aspect, the present invention provides an isolated polynucleotide molecule encoding the antibody or antigen-binding fragment thereof of the present invention.

[0024] In some embodiments, the polynucleotide molecule has a heavy chain nucleotide sequence as shown in SEQ ID NOs: 66-69, and a light chain nucleotide sequence as shown in SEQ ID NOs: 70-73.

[0025] In some preferred embodiments, the nucleotide molecule encoding the antibody or antigen-binding fragment thereof of the present invention is selected from the following groups: (1) the nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 66, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 70; or (2) the nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 67, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 71; or (3) the nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 68, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 72; or (4) the nucleic acid molecule has a heavy chain variable region nucleotide sequence as shown in SEQ ID NO: 69, and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 73.

[0026] The fifth aspect of the present invention is to provide an expression vector comprising an isolated polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0027] The sixth aspect of the present invention is to provide a host cell comprising the above-mentioned expression vector. The host cell can be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is an Expi 293F cell.

[0028] The seventh aspect of the present invention provides an antibody conjugate comprising the above-mentioned anti-nectin 4 antibody or antigen-binding fragment thereof and an effector molecule, wherein the effector molecule is conjugated to the anti-nectin 4 antibody.

[0029] In some embodiments, the effector molecule is selected from a radioisotope, an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0030] In some embodiments, the antibody conjugates of the present invention have the following structure:

[0031] Where: n is 1-8;

[0032] H7-2-3 is an anti-nectin-4 antibody, the HCDR1, HCDR2, and HCDR3 amino acid sequences of the antibody are shown in SEQ ID NO: 13, SEQ ID NO: 46, and SEQ ID NO: 17, respectively, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the antibody are shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0033] Furthermore, the antibody conjugate of the present invention has the following structure:

[0034] Where: n is 1-8;

[0035] H7-2-3 is an anti-nectin-4 antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO:49 and a light chain variable region amino acid sequence as shown in SEQ ID NO:55.

[0036] In an eighth aspect, the present invention provides a method for preparing the isolated monoclonal antibody, the antibody that specifically binds to nectin-4, or an antigen-binding fragment thereof described in the first aspect. The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). The somatic cell hybridization method is preferred. In principle, other techniques for producing monoclonal antibodies, such as viral or oncogene-transformed B lymphocytes or phage display technology using antibody gene libraries, can be used to prepare the antibodies of the present invention. Chimeric or humanized antibodies are also well known in the art, such as U.S. Patents 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, or 6,180,370.

[0037] The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0038] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., as described in Spieker-Polet et al, Proc. Natl. Acad. Sci. USA 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0039] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from a defined strategy of antibody-producing lymphocytes, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details of recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K. Clo Antibody Engineering ISBN 1-58829-092-1.

[0040] A ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof and antibody conjugate in the manufacture of a medicament for treating cancer. The cancers include, but are not limited to, bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, prostate cancer, colorectal cancer, glioma, and mesothelioma.

[0041] The technical solution of the present invention has achieved beneficial technical effects: the Nectin-4 antibody obtained in the present application has higher affinity and good cell endocytosis rate; the antibody conjugate containing the antibody has a significant cell killing effect and a strong tumor inhibition rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the results of the detection of the binding ability of the chimeric antibody to the Nectin-4 protein;

[0043] Figure 2 Detection results of the ability of chimeric antibodies to compete with nectin-1 for binding to nectin-4;

[0044] Figure 3 shows the results of the humanized antibody binding assay to Nectin-4;

[0045] Figure 4 shows the results of the humanized antibody's ability to compete with nectin-1 for binding to nectin-4;

[0046] Figure 5: FACS binding ability assay results of humanized antibodies;

[0047] Figure 6 shows the results of the killing activity of antibody-drug conjugates on tumor cells in vitro;

[0048] Figure 7 Effects of conjugated antibodies on the changes in tumor volume in the subcutaneous transplanted tumor model of human pancreatic cancer cells BxPC3 in female NOG mice;

[0049] Figure 8 Effects of conjugated antibodies on the body weight changes of female NOG mice in a subcutaneous transplanted tumor model of human pancreatic cancer cells BxPC3. DETAILED DESCRIPTION

[0050] The present invention will be further explained and illustrated below in conjunction with the examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0051] In the following examples, the materials used in the experiments can be purchased or prepared according to existing disclosed technologies; those without indicating the source and specifications are commercially available; various processes and methods not described in detail are conventional methods known in the art.

[0052] The positive control antibody used in the examples was Enfortumab, which was prepared according to the amino acid sequence in patent CN103402538A (the heavy chain amino acid sequence is shown in SEQ ID NO: 74, and the light chain amino acid sequence is shown in SEQ ID NO: 75). The sequence of the human nectin-4 protein (Q96NY8) was found from Uniprot, and the amino acid sequence is shown in SEQ ID NO: 76. Amino acids 32 to 349 are the extracellular region sequence. A signal peptide and Kozak sequence were added to the N-terminus of the protein sequence, and a 6×His tag was added to the C-terminus. After sequence optimization by Nanjing GenScript Biotechnology Co., Ltd., it was cloned into the PCDNA3.4 vector, transiently transfected into 293F cells, and purified to obtain the nectin-4-His protein, the amino acid sequence of which is shown in SEQ ID NO: 77.

[0053] Example 1 Screening and identification of hybridoma cells

[0054] Five female BALB / c mice aged 6-8 weeks were selected for immunization. During the first immunization, the antigen protein Nectin-4-His was mixed with complete Freund's adjuvant in a volume ratio of 1:1 and fully emulsified using a single-channel emulsifier, and then the mice were immunized for the first time by heel and subcutaneous injection. The immunization dose was 50 μg / mouse, and 100 μL was injected into each mouse. The second immunization was performed 14 days after the first immunization. The Nectin-4-His antigen protein was mixed with incomplete Freund's adjuvant in a volume ratio of 1:1 and fully emulsified using a single-channel emulsifier, and then the mice were immunized by subcutaneous injection. The immunization dose was 50 μg / mouse. The third immunization was the same as the second operation. Seven days after the third immunization, the tail was cut off and blood was collected, and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA).

[0055] Three days before cell fusion, mice were intraperitoneally injected with 150 μg of Nectin-4-His antigen protein for booster immunization. Three days later, the mice were sacrificed, their spleens removed, and fused with mouse myeloma SP2 / 0 cells. The prepared SP2 / 0 cells were mixed with mouse spleen cells at a ratio of 1:5 to 1:10, and the cell density was adjusted to 1 to 2 × 10 cells / mL using BTX Buffer. 7 After fusion, HAT medium was added and 3×10 4 The cells were plated and cultured in a 37°C, 5% CO2 incubator.

[0056] After 14 days of culture, positive hybridoma cells were detected by ELISA. Nectin-4-His protein was coated onto a 96-well ELISA plate at a concentration of 5 μg / mL (100 μL / well) and incubated overnight at 4°C. The next day, the coating solution was discarded and 200 μL / well of 5% skim milk powder was added. The plates were blocked at 37°C for 2 hours, washed three times with PBST, and 50 μL of hybridoma cell culture supernatant was added, incubated at 37°C for 1 hour. After washing three times with PBST, the secondary antibody goat anti-mouse IgG-HRP was added and incubated at 37°C for 1 hour. After washing three times with PBST, the plates were incubated with a colorimetric solution for 10 minutes, and then color development was terminated.

[0057] The binding ability of hybridoma cell culture supernatant to Nectin-4 expressed by 293F cells was detected by flow cytometry. 293F-Nectin-4 cells were collected and the cells were cultured at 10 5The cells were plated in 96-well plates and washed once with PBS. 100 μL of hybridoma cell culture supernatant was incubated with the cells at 37°C for 1 hour. After incubation, the cells were centrifuged once at 1000 r / min and the supernatant was gently aspirated. The cells were washed twice with PBS at the same speed. 100 μL of DPBS with fluorescent secondary antibody was added to each well and the cells were reacted at 37°C for 1 hour. After the reaction, the cells were centrifuged and washed twice with PBS. Finally, 200 μL of PBS was added to each well to resuspend the cells. The cells were detected by flow cytometry and the positive clones were monocloned. Finally, 7 hybridoma cell lines with the best binding activity were screened: 8A6-A9, 20H6-G5, 38A3-E7, 48F6-C3, 50D3-C10, 56H10-B1, and 75B10-A11.

[0058] Example 2 Monoclonal Antibody Sequence Acquisition

[0059] The seven hybridoma cells screened were cultured, RNA was extracted, and cDNA was amplified by RT-PCR. The hybridoma cell culture supernatants were used to identify the antibody subtypes using the IsoStrip™ Mouse Monoclonal Antibody Subtype Identification Kit. Based on the subtype identification results, specific nested PCR primers were designed and RACE PCR (GenScript) was used to amplify the variable regions of the heavy and light chains of the seven antibodies. The PCR products were subcloned into the pMD18-T vector system (TaKaRa). The inserts were verified and sequenced using vector-specific primers. The amino acid / DNA sequences encoding the heavy and light chain variable regions of the generated antibodies were obtained. The antibody heavy chain CDR sequences, light chain CDR sequences, and amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 1-43 and are listed in Table 1. The CDRs are numbered according to Kabat numbering.

[0060] Table 1 Heavy chain and light chain variable region sequences of anti-Nectin-4 hybridoma antibodies

[0061] Example 3 Preparation of chimeric antibodies

[0062] The obtained murine antibody heavy chain variable region sequence was linked to the heavy chain constant region of the human IgG1 antibody, and the light chain variable region sequence was added with the kappa light chain constant region (the heavy chain constant region amino acid sequence is shown in SEQ ID NO:44, and the light chain constant region amino acid sequence is shown in SEQ ID NO:45). A signal peptide sequence and a Kozak sequence were added before the antibody amino acid sequence, and the DNA sequence was optimized and synthesized by GenScript Biotechnology Co., Ltd. After sequence synthesis, it was cloned into the pCDNA3.4 vector (Invitrogen), the plasmid was extracted, and 293F cells were transfected. 293F cells were cultured to the logarithmic growth phase and the density was adjusted to 3×10 6 / mL, OPTI MEM medium 6mL plus light chain plasmid and heavy chain plasmid 40μg each, OPTIMEM medium 6mL plus Expi Fectamine TM 293 transfection reagent (320 μL). Add the transfection reagent to the DNA mixture, mix thoroughly, let stand for 15 minutes, and then add it to the cells. Incubate at 37°C, 8% CO2, and 100 rpm for 20 hours. Add 600 μL of Enhancer I and 6 mL of Enhancer II. After 5 days of culture, collect samples and purify them. The resulting chimeric antibodies were named chi-1, chi-2, chi-3, chi-4, chi-5, chi-6, and chi-7.

[0063] Example 4 Purification and identification of chimeric antibodies

[0064] First, a Protein A affinity column was prepared and equilibrated with PBS (pH 7.4). The cell culture supernatant, which was centrifuged (1500 r / min, 10 min) and filtered through a 0.45 μm filter, was passed through the column and then washed with PBS (pH 7.4) until the OD450 value was close to zero. The antibody was eluted with 50 mmol / L, pH 3.5 glycine-hydrochloric acid buffer (50 mmol / L glycine solution adjusted to pH 3.5 with 1 mol / L hydrochloric acid). The eluate in the peak region was collected and adjusted to pH 6.0 to obtain the purified antibody, which was then stored at -20°C for future use.

[0065] Nectin-4-His protein was diluted to 0.5 μg / mL and plated onto a 96-well plate, 100 μL per well, for overnight coating at 4°C. The next day, the supernatant was discarded and the plates were blocked with 300 μL of 2% skim milk at 37°C for 2 hours. The blocking solution was discarded and the plates were washed three times with PBST. The chimeric antibody was diluted to 40 μg / mL and diluted four-fold in 12 steps. 100 μL per well of the 96-well plate were plated onto the plates, and the plates were incubated at 37°C for 1 hour. After the reaction, the plates were washed three times with PBST and 100 μL of goat anti-human IgG-FC secondary antibody was added. The plates were incubated at 37°C for 1 hour, washed three times with PBST, and developed with 100 μL of TMB colorimetric solution for 10 minutes. The reaction was terminated by adding 100 μL of 1 M hydrochloric acid solution to each well. The data were read at 450 nm. As shown in Figure 1 and Table 2, the chimeric antibody and the control antibody showed comparable binding to nectin-4 protein.

[0066] Table 2 EC50 values ​​of chimeric antibodies binding to nectin-4

[0067] Example 5 Competitive ELISA to identify the competitive ability of chimeric antibodies with nectin-4 ligand Nectin1

[0068] Nectin-4-his protein was diluted to 2 μg / mL and plated onto a 96-well plate, 100 μL per well, for overnight coating at 4°C. The next day, the supernatant was discarded and the plates were blocked with 300 μL of 2% skim milk at 37°C for 2 h. The blocking solution was discarded and the plates were washed three times with PBST. The chimeric antibody was diluted to 40 μg / mL and diluted fourfold in 12 steps. 50 μL of each of the diluted antibody and Ncetin1 solution (2 μg / mL) were plated onto a 96-well plate at a 1:1 ratio and incubated at 37°C for 1 h. After the reaction, the plates were washed three times with PBST and 100 μL of goat anti-human IgG-FC secondary antibody was added. The plates were incubated at 37°C for 1 h, washed again three times with PBST, and then developed with 100 μL of TMB colorimetric solution for 10 min. The reaction was terminated by adding 100 μL of 1 M hydrochloric acid solution to each well. The data were read at 450 nm and recorded. The results are shown in FIG2 and Table 3 , and the chimeric antibody chi-7 has a better ability to compete with nectin-4 than the control antibody.

[0069] Table 3 EC50 values ​​of chimeric antibodies competing with Nectin1 for binding to Nectin-4

[0070] Example 6 Affinity determination of chimeric antibodies

[0071] The affinity of a monoclonal antibody for the antigen nectin-4 was determined using Biacore (T200). Ultrapure water was filtered through a 0.22 μm filter to prepare HBS-EP buffer. The nectin-4 monoclonal antibody stock solution was diluted to 6 μg / mL using HBS-EP buffer to serve as the ligand. The nectin-4 protein was diluted in HBS-EP buffer in eight two-fold dilutions starting at 100 nM, followed by a zero dilution, to serve as the analyte. An appropriate amount of glycoside 1.5 was pipetted as the regeneration buffer. The ligand, analyte, regeneration buffer, and HBS-EP buffer were placed on the sample tray. The program was set as follows: ligand flow rate 10 μL / min, 20 s; analyte flow rate 30 μL / min, association time 100 s, dissociation time 600 s; regeneration buffer 30 μL / min, 30 s. The program was started. SPR signals were acquired and saved using Biacore T200 Control Software, and data were processed using Biacore T200 Evaluation Software. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and the KD value was calculated as shown in Table 4 below. The affinity of the chimeric antibody was better than that of the control.

[0072] Table 4 Affinity determination of chimeric antibodies

[0073] Example 7 Flow cytometry determination of the endocytic effect of chimeric antibodies

[0074] MCF-7 cells were collected and the cell density was adjusted to 2×10 6 The protein concentration was adjusted to 40 μg / mL and 8 μg / mL, and 100 μL was added to each well of a U-shaped 96-well plate. The protein concentration was adjusted to 40 μg / mL and 8 μg / mL, and 100 μL was added to each well of the U-shaped 96-well plate, mixed, and incubated at 4°C for 1 hour. After the reaction, the cells were washed twice with PBST, resuspended in 200 μL of 2% FBS + PBS, and evenly divided into two U-shaped 96-well plates. One plate was incubated at 4°C and the other at 37°C. After incubation for 20 hours, the cells were centrifuged at 2000 rpm for 3 minutes and washed twice with PBST. 100 μL of a 1:400 dilution of APC secondary antibody was added to each well. The cells were incubated at 4°C for 1 hour, washed twice with PBS, and analyzed. The results are shown in Table 5. The results show that chi-7 had the highest cellular internalization rate among the seven chimeric antibodies.

[0075] Table 5 Internalization rate of chimeric antibodies determined by flow cytometry

[0076] Example 8 Chimeric Antibody Removal of PTM Sites

[0077] Because the CDR2 sequence of the chi-7 heavy chain (amino acid sequence shown in SEQ ID NO:16) contains an NG deamidation site and an NTT glycosylation site, it was mutated to SG and NPT to remove the deamidation and glycosylation sites (amino acid sequence shown in SEQ ID NO:46). The mutated chi-7 was named chi-7-PTM. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:47, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:43. These proteins were linked to the constant region of a human IgG1 antibody and co-transfected with a plasmid containing cVL7 linked to the kappa light chain constant region to obtain the chi-7-PTM protein. Affinity assays were performed, and the results are shown in Table 6.

[0078] Table 6 Chi-7-PTM antibody affinity determination

[0079] Example 9 Humanization of Chimeric Antibodies

[0080] To design humanized antibodies, the chimeric antibody chi-7-PTM (heavy chain variable region amino acid sequence as shown in SEQ ID NO: 47, light chain variable region amino acid sequence as shown in 43) was selected as a template for further humanization design. Humanization of mouse-derived antibodies was performed by complementarity-determining region (CDR) grafting, as described in U.S. Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370. Humanization was performed specifically according to the following method.

[0081] The light and heavy chain variable region sequences of the chi-7-PTM antibody were compared with the human immunoglobulin gene database at the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). Human germline IGVH and IGVK, which share the highest homology with the chi-7-PTM antibody and are also highly expressed and used in other drug-developed drugs, were selected as the framework for humanization. The light chain germline receptor sequence selected for the antibody chi-7-PTM is human IGKV7-3*01, and the heavy chain germline receptor sequence selected is human IGHV1-2*06. Homology modeling was performed on the variable region of the antibody chi-7-PTM, and amino acids within the variable region 3A were selected and compared with mouse antibodies and back mutations were performed to obtain five heavy chain variable region sequences, named VH1, VH2, VH3, VH4, and VH5, respectively. Five light chain variable region sequences were obtained, named VL1, VL2, VL3, VL4, and VL5, respectively. The sequences are shown in Table 7 below.

[0082] Table 7 Amino acid sequences of heavy and light chain variable regions after chi-7-PTM reverse mutation

[0083] Example 10 Transient expression of humanized antibodies

[0084] The humanized heavy chain variable region is connected to the heavy chain constant region of human IgG1 antibody, and the light chain variable region sequence is connected to the kappa light chain constant region (the heavy chain constant region amino acid sequence is shown in SEQ ID NO:44, and the light chain constant region amino acid sequence is shown in SEQ ID NO:45). A signal peptide sequence and a Kozak sequence are added before the antibody amino acid sequence, and the DNA sequence is optimized and synthesized by GenScript Biotechnology Co., Ltd. After sequence synthesis, it is cloned into the pCDNA3.4 vector (Invitrogen) and the plasmid is extracted. Five humanized heavy chain plasmids and five humanized light chain plasmids are obtained. The heavy chain plasmid and the light chain plasmid are orthogonally combined, transfected into 293F cells, and transiently expressed to obtain 25 humanized antibodies. The humanized antibodies constructed are shown in Table 8.

[0085] Table 8 Humanized antibodies

[0086] Example 11 Humanized Antibody Affinity Determination

[0087] The affinity of the antibody for the antigen nectin-4-His was determined using a Biacore (T200). Ultrapure water was filtered through a 0.22 μm filter to prepare HBS-EP buffer. Humanized nectin-4 antibody was diluted to 6 μg / mL in HBS-EP buffer as the ligand. Nectin-4-His protein was diluted in HBS-EP buffer in eight 2-fold steps starting at 100 nM, followed by a zero dilution, as the analyte. An appropriate amount of Glycine 1.5 was pipetted as the regeneration buffer. The ligand, analyte, regeneration buffer, and HBS-EP buffer were placed on the sample tray. The program was set as follows: ligand flow rate 10 μL / min, 20 s; analyte flow rate 30 μL / min, association time 100 s, dissociation time 600 s; regeneration buffer 30 μL / min, 30 s. The program was started. SPR signals were acquired and saved using Biacore T200 Control Software, and data were processed using Biacore T200 Evaluation Software. Affinity kinetic curves were fitted according to a 1:1 Langmuir binding model, and KD values ​​were calculated. The results are shown in Table 9. Based on the affinity of the antibodies and the number of amino acid mutations in the antibodies, H7-1-3, H7-1-5, H7-2-3, and H7-2-4 were selected for subsequent experiments.

[0088] Table 9 Humanized antibody affinity determination

[0089] The heavy chain and light chain amino acid sequences of H7-1-3 are shown in SEQ ID NO:58 and SEQ ID NO:62, respectively, and the heavy chain and light chain nucleotide sequences of H7-1-3 are shown in SEQ ID NO:66 and SEQ ID NO:70, respectively; the heavy chain and light chain amino acid sequences of H7-1-5 are shown in SEQ ID NO:59 and SEQ ID NO:63, respectively, and the heavy chain and light chain nucleotide sequences of H7-1-5 are shown in SEQ ID NO:67 and SEQ ID NO:71, respectively; the heavy chain and light chain amino acid sequences of H7-2-3 are shown in SEQ ID NO:60 and SEQ ID NO:64, respectively, and the heavy chain and light chain nucleotide sequences of H7-2-3 are shown in SEQ ID NO:68 and SEQ ID NO:72, respectively; the heavy chain and light chain amino acid sequences of H7-2-4 are shown in SEQ ID NO:61 and SEQ ID NO:65, respectively, and the heavy chain and light chain nucleotide sequences of H7-2-4 are shown in SEQ ID NO:69 and SEQ ID NO:61, respectively. Shown in NO:73.

[0090] Example 12 Binding of humanized antibody protein level

[0091] Nectin-4-his protein was diluted to 0.5 μg / mL and plated on a 96-well plate, with 100 μL per well. The plate was coated overnight at 4°C. The next day, the supernatant was discarded and the plate was blocked with 300 μL of 2% skim milk at 37°C for 2 h. The blocking solution was discarded and the plate was washed three times with PBST. The humanized antibody was diluted to 40 μg / mL and diluted 4-fold in 12 steps. The plate was plated on a 96-well plate, with 100 μL per well. The plate was incubated at 37°C for 1 h. After the reaction, the plate was washed three times with PBST and 100 μL of goat anti-human IgG-FC secondary antibody was added. The plate was incubated at 37°C for 1 h and washed again three times with PBST. 100 μL of TMB colorimetric solution was added for 10 min. The reaction was terminated by adding 100 μL of 1 M hydrochloric acid solution to each well. The data were read at 450 nm and recorded. The results are shown in Figure 3 and Table 10. The protein binding ability of the humanized antibody was not reduced compared to the PTM-modified antibody.

[0092] Table 10 Determination of the binding ability of humanized antibodies to Nectin-4

[0093] Example 13 Competitive ELISA to identify the competitive ability of humanized antibodies with Nectin-4 ligand Nectin1

[0094] Nectin-4-his protein was diluted to 2 μg / mL and plated on a 96-well plate with 100 μL per well. The plate was coated at 4°C overnight. The next day, the supernatant was discarded and the plate was blocked with 300 μL of 2% skim milk at 37°C for 2 h. The blocking solution was discarded and the plate was washed three times with PBST. The humanized antibody was diluted to 40 μg / mL and diluted 4-fold in 12 gradients; 50 μL of the diluted antibody and ligand Ncetin1 solution (2 μg / mL) were taken at a 1:1 ratio and spread on a 96-well plate. The plates were reacted at 37°C for 1 hour. After the reaction, the plates were washed 3 times with PBST and 100 μL of goat anti-human IgG-FC secondary antibody was added. The plates were reacted at 37°C for 1 hour, washed again with PBST 3 times, and then 100 μL of TMB colorimetric solution was added for 10 minutes. After the reaction, 100 μL of 1M hydrochloric acid solution was added to each well to terminate the reaction. The data were read and recorded at 450 nm. The results are shown in Figure 4 and Table 11. The results show that the humanized antibodies can compete for the binding of Nectin-4 to the ligand Nectin1, and the effect is better than the control.

[0095] Table 11 EC50 values ​​of humanized antibodies competing with Nectin1 for binding to Nectin-4

[0096] Example 14 Flow cytometry determination of cell binding ability of humanized antibodies

[0097] Take 293F-Nectin-4 cells in the logarithmic growth phase, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend the cells in 5 mL of DPBS, and adjust the cell density to 2 × 10 6 / mL, and cells were seeded into U-bottom 96-well plates at a volume of 50μL / well. The antibody was taken and adjusted to a concentration of 20μg / mL. It was diluted in 12 steps of a 3-fold gradient and added to the U-shaped 96-well plate at 50μL / well. The plate was incubated at 37°C for 1 hour. After centrifugation at 1200r / min for 5 minutes, the supernatant was discarded and the cells were washed twice with 200μL of DBS. 100μL of DPBS with fluorescent secondary antibody was added to each well and incubated at 37°C for 1 hour. After the reaction, the cells were centrifuged and washed twice with DPBS. Finally, 200μL of DPBS was added to each well to resuspend the cells. The cells were loaded onto the instrument and the data were analyzed. The results are shown in Figure 5 and Table 12. The EC50 values ​​of H7-1-3, H7-2-3, and H7-2-4 antibodies were all lower than those of the control antibody Enfortumab.

[0098] Table 12 Cell binding ability of humanized antibodies

[0099] Example 15 Flow cytometry determination of the endocytic effect of humanized antibodies on tumor cells

[0100] MCF-7 cells were collected and the cell density was adjusted to 2×10 6 / mL, 100μL per well was added to a U-shaped 96-well plate. The humanized antibody density was adjusted to 40μg / mL and 8μg / mL, and 100μL per well was added to a U-shaped 96-well plate, mixed, and incubated at 4°C for 1 hour. After the reaction, the cells were washed twice with PBST, resuspended in 200μL of 2% FBS + PBS, and evenly divided into two U-shaped 96-well plates. One plate was placed at 4°C and the other at 37°C. After standing for 20 hours, the cells were centrifuged at 2000r / min for 3 minutes and washed twice with PBST. 100μL of a 1:400 dilution of APC secondary antibody was added to each well, incubated at 4°C for 1 hour, and then washed twice with PBS. The amount of antibody internalization was detected by flow cytometry. The internalization efficiency was calculated as (4°C fluorescence value - 37°C fluorescence value) / 4°C fluorescence value. The internalization efficiency of each antibody is shown in Table 13 below. The endocytic efficiency of H7-2-3 was 74% at a concentration of 8 μg / mL and 68% at a concentration of 40 μg / ml, both of which were higher than the endocytic efficiency of the control antibody Enfortumab.

[0101] Table 13 Flow cytometry determination of humanized antibody endocytosis rate

[0102] Example 16 Preparation of Anti-Nectin-4 Antibody-Drug Conjugates

[0103] Antibody reduction: For the antibody prepared in the example, antibody H7-2-3 was diluted to a concentration of 18.31 mg / mL. 5.0 mL of this solution was added to a 250 mL glass reaction flask. A 1.5 μM TCEP aqueous solution (5.83 mL; equivalent to 2.5 equivalents per antibody molecule) was added while stirring with a magnetic stirrer. The mixture was stirred at room temperature for 2 hours to reduce the disulfide bonds in the hinge region of the antibody.

[0104] Conjugation of the antibody to the linker-drug linker compound: To the above solution was slowly added DMSO (82.88 μL; equivalent to 5.0 equivalents per antibody molecule) containing a 3.0 mM linker-drug linker compound (Vc-MMAE) (reference: Lyon RP, Meyer DL, Setter JR, at al. Conjugation of anticancer drugs through endogenous monoclonal antibody cysteineresidues. Methods Enzymol. 2012; 502: 123-38. doi: 10.1016 / B978-0-12-416039-2.00006-9). The solution was stirred at room temperature for 1 hour to conjugate the antibody to the linker-drug linker compound, ultimately obtaining the antibody-drug conjugate H7-2-3-MMAE with a DAR value of 4.0.

[0105] Example 17 Cytotoxicity of anti-nectin-4 antibody-drug conjugates against tumor cells in vitro

[0106] Anti-Nectin-4 antibody-drug conjugates can effectively kill tumor cells expressing the Nectin-4 target. We selected ATCC-derived breast cancer cells MCF-7 expressing Nectin-4 as target cells to evaluate the killing activity of anti-Nectin-4 antibody-drug conjugate H7-2-3-MMAE on tumor cells in vitro.

[0107] MCF-7 cells were collected by trypsin digestion and centrifuged at 1000 rpm for 3 min. The supernatant was discarded and the cells were resuspended in 10% FBS + EMEM medium and counted to adjust the cell density to 1 × 10 4 / ml, 100μl cells per well were plated in a 96-well plate; sample preparation: the test sample was diluted to 180ug / ml with 10% FBS + EMEM medium, and 9 six-fold dilution gradients were set, with a 0 concentration point set, for a total of 10 concentration points. 50μl was mixed with cells in each well, and triplicate wells were set for each concentration point; incubated in a 37°C / CO2 incubator for 5 days. After the incubation period, 50μl CTG was added to each well and chemiluminescence was measured on a multifunctional microplate reader to evaluate cell viability. The killing EC50 value was calculated by fitting a four-parameter curve. The results are shown in Figure 6 and Table 14. H7-2-3-MMAE has a significant killing effect on tumor cells in vitro, and the effect is superior to the control molecule Enfortumab vedotin.

[0108] Table 14 Cell killing activity of antibody-drug conjugates

[0109] Example 18 In vivo efficacy experiment of anti-nectin-4 antibody-drug conjugates

[0110] Study on the effect of ADC molecules on the subcutaneous transplanted tumor model of human pancreatic cancer cells BxPC3 in female NOG mice

[0111] A subcutaneous transplant model of human pancreatic cancer cell line BxPC-3 was established in female NOG mice to investigate the therapeutic effects of ADC molecules in this model. Human pancreatic cancer cell line BxPC-3 cells used in this experiment were cultured in RPMI 1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the tenth generation of cell culture, mice were inoculated. BxPC-3 cells were harvested (cell concentration adjusted to 1×10 8 The cells were mixed with Matrigel at a volume ratio of 1:1 and 0.1 mL / mouse was inoculated subcutaneously in the right flank of female NOG mice. The inoculation volume of BxPC-3 cells per mouse was 5 × 10 6 When the average tumor volume of mice reached 145mm 3 At around 3 d, the mice were randomly divided into three groups according to tumor volume and body weight, namely: vehicle control PBS group, Enfortumab vedotin group, and H7-2-3-MMAE group, with 6 mice in each group. Drug administration began on the day of grouping (the day of grouping was defined as D0) by tail vein injection at a dose of 6 mg / kg. The drug was administered only once during the entire experimental period, and the tumor volume and body weight were measured every week. The results are shown in Figures 7 and 8.

[0112] The maximum diameter (D) and minimum diameter (d) of the tumor were measured with an electronic vernier caliper every week, and the tumor volume was calculated. The tumor growth inhibition rate of each drug-treated group was calculated according to the formula.

[0113] Tumor volume V (mm 3 )=[D×d 2 ] / 2;

[0114] Relative tumor growth rate T / C (%) = (T n -T0) / (C n -C0)×100%, where T n 、C n T0 and C0 are the tumor volumes of the treatment group and the control group on Dn day during the experiment; T0 and C0 are the tumor volumes of the treatment group and the control group on D0 day at the beginning of the experiment;

[0115] Tumor growth inhibition rate TGI (%) = 1-T / C (%).

[0116] The results, shown in Table 15, demonstrate that the ADC exhibited significant anti-tumor effects, with tumor volume reaching a minimum and tumor inhibition rate reaching a maximum on day 17, after which tumor volume increased. At the same dose, H7-2-3-MMAE demonstrated superior tumor growth inhibition compared to the control molecule, enfortumab vedotin.

[0117] Table 15 Antitumor effects of antibody-drug conjugates in animals

Claims

1. An anti-Nectin-4 antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region contains heavy-chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light-chain variable region contains light-chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: The amino acid sequences of HCDR1, HCDR2, HCDR3 of the heavy-chain variable region and LCDR1, LCDR2, and LCDR3 of the light-chain variable region are selected from the following groups: (1) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; (2) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; (3) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:24, SEQ ID NO:19, and SEQ ID NO:25; (4) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:11; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:25; (5) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:9; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:24, SEQ ID NO:19, and SEQ ID NO:25; (6) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 respectively; (7) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:17 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 respectively; (8) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:13, SEQ ID NO:46, and SEQ ID NO:17 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 respectively.

2. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31 - 37, SEQ ID NO:47 - 52, and the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38 - 43, SEQ ID NO:53 - 57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:31 - 37, SEQ ID NO:47 - 52, SEQ ID NO:38 - 43, SEQ ID NO:53 - 57.

3. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to claim 2, wherein The amino acid sequences of the antibody heavy chain variable region and light chain variable region are selected from the following groups: (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:31 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:38; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence; (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:32 and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:39; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence; (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 40; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 41; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (5) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 40; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (6) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 42; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (7) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (8) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 47, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 43; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (9) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 53; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (10) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 54; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (11) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (12) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (13) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (14) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 53; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (15) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 54; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (16) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (17) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (18) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (19) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 53; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (20) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 54; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (21) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (22) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (23) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 50, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (24) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 53; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (25) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 54; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (26) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 51, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 55; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence; (27) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:51, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (28) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:51, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (29) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:53; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (30) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:54; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (31) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:55; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (32) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:56; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; (33) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:52, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence.

4. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to claim 3, wherein The amino acid sequences of the antibody heavy chain variable region and light chain variable region are selected from the following groups: (1) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:43; (2) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:47, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:43; (3) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:55; (4) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:48, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:57; (5) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:55; (6) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:

56.

5. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that The antibody or its antigen-binding fragment further comprises a complete structure having two light chains and two heavy chains, and the antibody is selected from the following group: (1) The amino acid sequence of the heavy chain as shown in SEQ ID NO:58, and the amino acid sequence of the light chain as shown in SEQ ID NO:62; (2) The amino acid sequence of the heavy chain as shown in SEQ ID NO:59, and the amino acid sequence of the light chain as shown in SEQ ID NO:63; (3) The amino acid sequence of the heavy chain as shown in SEQ ID NO:60, and the amino acid sequence of the light chain as shown in SEQ ID NO:64; (4) The amino acid sequence of the heavy chain as shown in SEQ ID NO:61, and the amino acid sequence of the light chain as shown in SEQ ID NO:

65.

6. A polynucleotide, characterized in that, The polynucleotide encodes the anti-human Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-5.

7. An expression vector, characterized in that, The expression vector comprises the polynucleotide according to claim 6.

8. An antibody conjugate, characterized in that, The antibody conjugate has the following structure as shown in the following formula: Wherein, n is 1-8; H7-2-3 is an anti-Nectin-4 antibody or its antigen-binding fragment, and the anti-Nectin-4 antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO:13, SEQ ID NO:46, and SEQ ID NO:17, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region are respectively as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:

30.

9. The antibody conjugate according to claim 8, wherein The antibody has a heavy chain variable region sequence as shown in SEQ ID NO:49, and a light chain variable region sequence as shown in SEQ ID NO:

55.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-5 and / or the antibody conjugate according to any one of claims 8-9, and a pharmaceutically acceptable carrier.

11. Use of the anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-5, the antibody conjugate according to any one of claims 8-9, the polynucleotide according to claim 6, the expression vector according to claim 7, or the pharmaceutical composition according to claim 10 in the preparation of a drug for treating or preventing cancer.

12. The cancer according to claim 11, wherein The cancer is selected from hepatobladder cancer, breast cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, prostate cancer, colorectal cancer, glioma, mesothelioma.

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