Nectin-4 antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-Nectin-4 antibody or an antigen binding fragment thereof, an antibody conjugate and application thereof. Specifically, the Nectin-4 antibody obtained by the method disclosed by the invention has higher affinity and good endocytosis rate; an antibody conjugate containing the antibody has a remarkable cell killing effect and has a very strong tumor growth inhibition effect. The invention also relates to an application of the antibody or the antigen binding fragment thereof and the antibody conjugate in preparation of drugs for treating cancers, and the antibody or the antigen binding fragment thereof and the antibody conjugate play an important role in treatment of human tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to an antibody and its fragment that specifically binds to Nectin-4, and the use of such antibodies. Background Art

[0002] The Nectin cell adhesion molecule family contains 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) has been considered an important factor in the formation of malignant tumors including urothelial carcinoma, gastric cancer, thyroid cancer, breast cancer, esophageal cancer, ovarian cancer, etc. Nectin-4 is a member of the Nectin family of immunoglobulin-like adhesion molecules. The abnormal expression of Nectin-4 is related to the development of tumor cells caused by proliferation, angiogenesis, and reduced apoptosis, indicating that Nectin-4 is a potential therapeutic target.

[0004] Antibody-drug conjugate (ADC) is a technology that uses the specific recognition ability of antibodies to specific antigens on the surface of tumor cells to precisely deliver anti-tumor drugs (such as cytotoxic agents, cell inhibitors, small molecule chemotherapeutics, etc.) to tumor target cells, causing intracellular accumulation and release, and then 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 conjugates the antibody or antibody-like ligand to the drug. Due to its appropriate molecular weight, high stability, strong targeting, and low toxicity and side effects, antibody-drug conjugates have been considered the most promising anti-tumor drugs.

[0005] The main drug targeting Nectin-4 at present is Enfortumab vedotin, which is an antibody-drug conjugate composed of a monoclonal antibody against Nectin-4 and a cell-killing drug, monomethyl auristatin E (MMAE). It is mainly used for treating bladder cancer, especially urothelial carcinoma, and was granted Breakthrough Therapy Designation by the FDA in March 2018. In addition, other studies have shown that the adhesion factor Nnectin-4 can not only be used as an effective prognostic factor for breast cancer, but also as an effective therapeutic target for patients with triple-negative breast cancer (TNBC). In vitro and in vivo studies have confirmed that antibody-drug conjugates (ADCs) against Nectin-4 have good efficacy for local and metastatic TNBC.

[0006] Seattle Genetics and Astellas collaborated to randomly conjugate the company's unique linker mc-vc-MMAE with the Anti-Nectin-4 antibody enfortumab to obtain an Anti-Nectin-4 antibody-drug conjugate, Enfortumab Vedotin (Padcev). The clinical trial results showed that among patients who received chemotherapy and PD-1 / PD-L1 inhibitors, the median overall survival of patients who received Enfortumab Vedotin was 12.9 months, which was 3.9 months longer than that of the chemotherapy control group, showing good tumor treatment effects. However, clinical studies also found that the use of Enfortumab Vedotin was often accompanied by fever, skin itching, peripheral neuropathy, dry eye, and neutrophil decline. These adverse reactions are directly related to excessive conjugation of small molecules and antibodies and unstable conjugation methods.

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

[0008] In view of the above problems, the present invention provides an antibody molecule or its functional fragment that specifically binds to Nectin-4, and an antibody conjugate containing the antibody or its functional fragment. Among them, the antibody molecule or its functional fragment of the present invention has higher affinity and good endocytosis rate; the antibody conjugate containing the antibody or its functional fragment has significant cell killing effect and strong tumor inhibition rate.

[0009] A 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 sequences SEQ ID NO: 1-17, 46 or variants thereof; and the light-chain variable region comprises a light-chain CDR selected from the amino acid sequences SEQ ID NO: 18-30 or variants thereof.

[0010] In some embodiments, the present invention provides 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 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 shown as 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 shown as 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 shown as 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 shown as 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 shown as 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 shown as SEQ ID NO:24, SEQ ID NO:19, and SEQ ID NO:25 respectively; or (4) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:11 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:25 respectively; or (5) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:7, SEQ ID NO:12, and SEQ ID NO:9 respectively; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:24, SEQ ID NO:19, and SEQ ID NO:25 respectively; or (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; or (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;or the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of (8) are respectively as shown in SEQ ID NO:13, SEQ ID NO:46, and SEQ ID NO:17; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0012] In some preferred embodiments, for the antibody or its antigen-binding fragment, the amino acid sequences of the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the 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 respectively as shown in SEQ ID NO:13, SEQ ID NO:16, and SEQ ID NO:17; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; or (2) The amino acid sequences of the HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:13, SEQ ID NO:46, and SEQ ID NO:17; and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0014] In a second aspect of the present invention, there is provided an anti-Nectin-4 antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment 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 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 with the amino acid sequences 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.

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

[0016] (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 with the said amino acid sequence; or (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 with the said amino acid sequence; or (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; or (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; or (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; or (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; or (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; or (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; or (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;an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; or (10) 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: 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; or (11) 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 an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; or (12) 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: 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; or (13) 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 an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; or (14) 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: 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; or (15) 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: 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; or (16) 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 an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; or (17) 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; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence;or (18) 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: 57; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence; or (19) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 50 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (20) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 50 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (21) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 50 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (22) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 50 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (23) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 50 and a 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 with said amino acid sequence; or (24) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 51 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (25) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 51 and a light chain variable region amino acid sequence 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 said amino acid sequence; or (26) a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 51 and a light chain variable region amino acid sequence as shown in SEQ ID NO: 55;an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the said amino acid sequence; or (27) 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: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; or (28) 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: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; or (29) 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: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; or (30) 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: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; or (31) 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: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; or (32) 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: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; 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 with the said amino acid sequence.;

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

[0018] In the third aspect of the present invention, there is provided an anti-Nectin-4 antibody or an antigen-binding fragment thereof, which is murine, chimeric, or humanized; wherein the FR region sequences on the light and heavy chain variable regions of the humanized antibody are derived from the human germline light and heavy chains or their mutant sequences respectively; the antibodies of the present invention further include heavy chain constant regions and light chain constant regions.

[0019] Preferably, the antibody comprises the heavy chain constant domains of human IgG1, IgG2, IgG3, IgG4 and the light chain constant domains of human κ or λ type or variants thereof, wherein the variants include substitutions of one or several amino acids.

[0020] Preferably, the antibody comprises the human IgG1 constant domain or a variant thereof, and the κ constant domain or a variant thereof, wherein the variants include substitutions of one or several amino acids.

[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 with 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 of the present invention, there is provided 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] In a fifth aspect of the present invention, there is provided an expression vector comprising the isolated polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0027] In a sixth aspect of the present invention, there is provided a host cell comprising the above expression vector. The host cell can be a prokaryotic cell or a eukaryotic cell. In a preferred example, the host cell is Expi 293F cell.

[0028] A seventh aspect of the present invention is to provide an antibody conjugate comprising the above-mentioned anti-Nectin 4 antibody or its antigen-binding fragment 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 radioisotopes, anti-tumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0030] In some embodiments, the antibody conjugate of the present invention has the following structure:

[0031]

[0032] Where: n is 1-8;

[0033] H7-2-3 is an anti-Nectin-4 antibody, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 of the antibody are shown in 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 of the antibody are shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 respectively.

[0034] Furthermore, the antibody conjugate of the present invention has the following structure:

[0035]

[0036] Where: n is 1-8;

[0037] H7-2-3 is an anti-Nectin-4 antibody, and the antibody has the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:49 and the amino acid sequence of the light chain variable region shown in SEQ ID NO:55.

[0038] In the eighth aspect of the present invention, there is provided a method for preparing the isolated monoclonal antibody, the antibody specifically binding to Nectin-4, or an antigen-binding fragment thereof as described in the first aspect. The antibodies of the present invention can be generated 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 generating monoclonal antibodies, such as transforming B lymphocytes with viruses or oncogenes or using phage display techniques with 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. Patent Nos. 4,816,567, 5,225,539, 5,530,101, 5,585,089, 5,693,762, or 6,180,370, etc.

[0039] A preferred animal system for preparing hybridomas secreting monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established method. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0040] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0041] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from antibody-producing lymphocytes of a defined strategy, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering, also see Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0042] The ninth aspect of the present invention provides the use of the antibody or its antigen-binding fragment and the antibody conjugate in the manufacture of a medicament for treating cancer. The cancer includes, but is not limited to, bladder 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.

[0043] The technical solution of the present invention has achieved beneficial technical effects: the Nectin-4 antibody obtained in this application has higher affinity and good endocytosis rate; the antibody conjugate containing this antibody has significant cell killing effect and strong tumor inhibition rate. Description of the Drawings

[0044] Figure 1 Detection results of the binding ability of the chimeric antibody to Nectin-4 protein;

[0045] Figure 2 Detection results of the ability of the chimeric antibody to competitively bind Nectin-4 with Nectin-1;

[0046] Figure 3 Determination results of the binding ability of the humanized antibody to Nectin-4;

[0047] Figure 4 Detection results of the ability of the humanized antibody to competitively bind Nectin-4 with Nectin-1;

[0048] Figure 5 Determination results of the FACS binding ability of the humanized antibody;

[0049] Figure 6 Results of the killing activity of the antibody-drug conjugate against tumor cells in vitro;

[0050] Figure 7 Tumor volume changes of the subcutaneous xenograft model of human pancreatic cancer BxPC3 in female NOG mice with the conjugated antibody;

[0051] Figure 8 Changes in the body weight of female NOG mice with the subcutaneous xenograft model of human pancreatic cancer BxPC3 with the conjugated antibody. Detailed Embodiments

[0052] The present invention will be further explained and illustrated below in conjunction with embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the protection scope of the present invention.

[0053] In the following examples, the materials used in the experiments are all commercially available or can be prepared with reference to the existing publicly disclosed technologies; those without specified sources and specifications are commercially available; the various processes and methods not described in detail are conventional methods well known in the art.

[0054] The positive control antibody used in the examples is Enfortumab, which is prepared according to the amino acid sequences in Patent CN103402538A (the heavy chain amino acid sequence is shown as SEQ ID NO:74, and the light chain amino acid sequence is shown as SEQ ID NO:75); the sequence of human Nectin-4 protein (Q96NY8) is searched from Uniprot, and the amino acid sequence is shown as SEQ ID NO:76. The extracellular region sequence is from amino acids 32 to 349. A signal peptide and Kozak sequence are added to the N-terminus of the protein sequence, and a 6×His tag is added to the C-terminus. After sequence optimization by Nanjing Genscript Biotech Co., Ltd., it is cloned into the PCDNA3.4 vector, transiently transfected into 293F cells, and the Nectin-4-His protein is purified, with the amino acid sequence shown as SEQ ID NO:77.

[0055] Screening and Identification of Hybridoma Cells in Example 1

[0056] Five 6-8-week-old female BALB / c mice were selected for immunization. At the first immunization, the antigen protein Nectin-4-His was mixed with complete Freund's adjuvant at 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 each mouse was injected with 100 μL; 14 days after the first immunization, a second immunization was carried out. The Nectin-4-His antigen protein was mixed with incomplete Freund's adjuvant at 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; 7 days after the three immunizations, tail blood was collected, and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA).

[0057] Three days before cell fusion, 150 μg of the Nectin-4-His antigen protein was directly injected into the abdominal cavity of the mice for booster immunization. Three days later, the mice were sacrificed, and their spleens were taken out and fused with the mouse myeloma SP2 / 0 cell line. 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 1-2×10 7 cells / ml using BTX Buffer, and fusion was carried out using an electrofusion instrument. After fusion, HAT medium was added and plated at 3×10 4 cells per well, and cultured in an incubator at 37°C and 5% CO2.

[0058] When culturing until 14 days, positive hybridoma cells were detected by ELISA. The Nectin-4-His protein was used to coat a 96-well ELISA plate. The 96-well ELISA plate was coated at a concentration of 5 μg / mL in an amount of 100 μL / well and coated overnight at 4°C; the next day, the coating solution was discarded and 200 μL / well of 5% skim milk powder was added; blocked at 37°C for 2 h, washed 3 times with PBST, added 50 μL of hybridoma cell culture supernatant, and incubated at 37°C for 1 h; after washing 3 times with PBST, added secondary antibody goat anti-mouse IgG-HRP and incubated at 37°C for 1 h. Washed 3 times with PBST, added chromogenic solution for color development for 10 min, and terminated the color development.

[0059] Flow cytometry was used to detect the binding ability of the hybridoma cell culture supernatant to Nectin-4 expressed by 293F cells. 293F-Nectin-4 cells were collected, and the cells were plated at 10 5 cells / well in a 96-well plate and washed once with PBS. 100 μL of the hybridoma cell culture supernatant was taken and incubated with the cells at 37°C for 1 h. After incubation, centrifuged once at 1000 r / min and the supernatant was gently aspirated. Washed 2 times with PBS at the same rotation speed. 100 μL of DPBS with fluorescent secondary antibody was added to each well and incubated at 37°C for 1 h. After the reaction, centrifuged and washed 2 times with PBS. Finally, 200 μL of PBS was added to each well to resuspend the cells, and detected by flow cytometry. The obtained positive clones were monoclonalized, and finally 7 hybridoma cell lines with the best binding activity were screened: 8A6-A9, 20H6-G5, 38A3-E7, 48F6-C3, 50D3-C10, 56H10-B1, 75B10-A11.

[0060] Example 2 Obtaining Monoclonal Antibody Sequences

[0061] The 7 screened hybridoma cells were cultured, and RNA was extracted respectively, and cDNA was amplified by RT-PCR method; the hybridoma cell culture supernatant was taken, and the antibody subtype was identified by IsoStripTM Mouse Monoclonal Antibody Subtype Identification Kit. According to the subtype identification results, specific nested PCR primers were designed, and the variable regions of the heavy and light chains of these 7 antibodies were amplified by RACE PCR (GenScript), and the PCR products were subcloned into the pMD18-T vector system (TaKaRa). Vector-specific primers were used to verify and sequence the inserted fragments. The amino acid / DNA sequences encoding the variable regions of the heavy and light chains of the generated antibodies were obtained. The CDR sequences of the heavy chain of the antibody, the CDRs of the light chain, and the amino acid sequences of the variable regions of the heavy and light chains of the antibody are shown in SEQ ID NO: 1-43 and listed in Table 1, and the CDRs were determined according to Kabat numbering.

[0062] Table 1 Heavy and light chain variable region sequence lists of hybridoma antibodies against Nectin-4

[0063] Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL 8A6 - A9 1 2 3 18 19 20 31 38 20H6 - G5 4 5 6 21 22 23 32 39 38A3 - E7 7 8 9 24 19 25 33 40 48F6 - C3 7 10 11 26 27 25 34 41 50D3 - C10 7 12 9 24 19 25 35 40 56H10 - B1 13 14 15 28 29 30 36 42 75B10 - A11 13 16 17 28 29 30 37 43

[0064] Example 3 Preparation of chimeric antibodies

[0065] The obtained murine antibody heavy chain variable region sequence was linked to the heavy chain constant region of human IgG1 antibody, and the light chain variable region sequence was added with the κ light chain constant region (the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 45). A signal peptide sequence and a Kozak sequence were added before the antibody amino acid sequence, and DNA sequence optimization and synthesis were carried out by GenScript Biotech Corporation. After sequence synthesis, it was cloned into the pCDNA3.4 vector (Invitrogen), and the plasmid was extracted and transfected into 293F cells. The 293F cells were cultured to the logarithmic growth phase, and the density was adjusted to 3×10 6 cells / mL with Expi 293F expression medium. 6 mL of OPTI MEM medium was added with 40 μg each of the light chain plasmid and the heavy chain plasmid, and 6 mL of OPTI MEM medium was added with 320 μL of Expi Fectamine TM 293 transfection reagent. The transfection reagent was added to the DNA mixture, mixed well and left standing for 15 min, and then added to the cells. Cultured at 37 °C, 8% CO2, 100 r / min for 20 h, 600 μL of Enhancer I and 6 mL of Enhancer II were added. After culturing for 5 days, samples were collected and purified, and the obtained chimeric antibodies were named chi-1, chi-2, chi-3, chi-4, chi-5, chi-6, and chi-7 respectively.

[0066] Example 4 Purification and identification of chimeric antibodies

[0067] First, a Protein A affinity column was prepared and equilibrated with PBS (pH 7.4); the cell culture supernatant centrifuged (1500 r / min, 10 min) and filtered through a 0.45 μm filter membrane was passed through the column, and then washed with PBS (pH 7.4) until the OD450 value was close to zero; eluted with a 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), and the eluate in the peak region was collected and adjusted to pH 6.0 to obtain the purified antibody, which was stored at -20 °C for later use.

[0068] Dilute the Nectin-4-His protein to 0.5 μg / mL and plate it in a 96-well plate, 100 μL per well, and coat it overnight at 4°C. The next day, after discarding the supernatant, block it with 300 μL of 2% skim milk at 37°C for 2 h. After discarding the blocking solution, wash it 3 times with PBST. Dilute the chimeric antibody to 40 μg / mL and perform 12-fold serial dilutions; plate it in a 96-well plate, 100 μL per well, and react it at 37°C for 1 h. After the reaction, wash it 3 times with PBST and add 100 μL of goat anti-human IgG-Fc secondary antibody, react it at 37°C for 1 h, wash it 3 times with PBST again, then add 100 μL of TMB chromogenic solution to develop color for 10 min. After the reaction, add 100 μL of 1 M hydrochloric acid solution to each well to terminate the reaction, and read and record the data at 450 nm. The results are as Figure 1 shown in Table 2, the binding ability of the chimeric antibody and the control antibody to the Nectin-4 protein is comparable.

[0069] Table 2 EC50 values of the chimeric antibody binding to Nectin-4

[0070] Antibody EC50 value (ng / ml) chi - 1 38.85 chi - 2 30.47 chi - 3 31.38 chi - 4 26.89 chi - 5 26.17 chi - 6 30.82 chi - 7 22.27 Enfortumab 26.97

[0071] Example 5 Identification of the competitive ability of the chimeric antibody with Nectin-4 ligand Nectin1 by competitive ELISA

[0072] Dilute the Nectin-4-his protein to 2 μg / mL and plate it in a 96-well plate, 100 μL per well, and coat it overnight at 4°C. The next day, after discarding the supernatant, block it with 300 μL of 2% skim milk at 37°C for 2 h. After discarding the blocking solution, wash it 3 times with PBST. Dilute the chimeric antibody to 40 μg / mL and perform 12-fold serial dilutions; take 50 μL each of the diluted antibody and the solution of Ncetin1 (2 μg / mL) in a 1:1 ratio, plate it in a 96-well plate, and react it at 37°C for 1 h. After the reaction, wash it 3 times with PBST and add 100 μL of goat anti-human IgG-Fc secondary antibody, react it at 37°C for 1 h, wash it 3 times with PBST again, then add 100 μL of TMB chromogenic solution to develop color for 10 min. After the reaction, add 100 μL of 1 M hydrochloric acid solution to each well to terminate the reaction, and read and record the data at 450 nm. The results are as Figure 2 shown in Table 3, the ability of the chimeric antibody chi-7 to competitively bind Nectin-4 is better than that of the control antibody.

[0073] Table 3 EC50 values of the chimeric antibody competitively binding Nectin-4 with Nectin1

[0074] Antibody EC50 value (ng / ml) chi - 1 759.7 chi - 2 550.4 chi - 3 622.6 chi - 4 537.2 chi - 5 500.4 chi - 6 532.1 chi - 7 352.1 Enfortumab 696.1

[0075] Example 6 Affinity determination of the chimeric antibody

[0076] Using Biacore (T200), the affinity between the monoclonal antibody and the antigen Nectin-4 was determined. Ultra-pure water was filtered through a 0.22 μm filter membrane to prepare HBS-EP buffer; the stock solution of the NECTIN-4 monoclonal antibody was diluted to 6 μg / mL with HBS-EP buffer as the ligand; the NECTIN-4 protein was serially diluted 8-fold starting from 100 nM with HBS-EP buffer, and then an additional zero point was added as the analyte; an appropriate amount of Glycine 1.5 was aspirated as the regeneration solution; the ligand, analyte, regeneration solution, and HBS-EP buffer were placed on the sample tray; the program was set as follows: the ligand flow rate was 10 μL / min for 20 s; the analyte flow rate was 30 μL / min, the binding time was 100 s, and the dissociation time was 600 s; the regeneration solution flow rate was 30 μL / min for 30 s. The program was started. The SPR signal was collected and saved by the Biacore T200 Control Software, and then the data was processed using the Biacore T200 Evaluation analysis 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 superior to that of the control.

[0077] Table 4 Determination of the affinity of the chimeric antibody

[0078] Chimeric antibody KD (M) chi - 1 <![CDATA[5.88×10 -9 > chi - 2 <![CDATA[7.27×10 -10 > chi - 3 <![CDATA[1.87×10 -9 > chi - 4 <![CDATA[7.93×10 -10 <!-- 10 -->]]> chi - 5 <![CDATA[5.70×10 -9 > chi - 6 <![CDATA[1.44×10 -9 > chi - 7 <![CDATA[1.59×10 -9 > Enfortumab <![CDATA[6.87×10 -9 >

[0079] Example 7 Determination of the endocytosis effect of the chimeric antibody by flow cytometry

[0080] MCF-7 cells were collected and the cell density was adjusted to 2×10 6 cells / mL with 2% FBS + PBS. 100 μL of the cell suspension was added to each well of a U-shaped 96-well plate. The protein density was adjusted to 40 μg / mL and 8 μg / mL, and 100 μL of each was added to the U-shaped 96-well plate and mixed well. The reaction was carried out at 4°C for 1 h. After the reaction, the cells were washed twice with PBST, then resuspended in 200 μL of 2% FBS + PBS and evenly distributed into two U-shaped 96-well plates. One plate was placed at 4°C and the other at 37°C. After standing for 20 h, the cells were centrifuged at 2000 r / min for 3 min and washed twice with PBST. 100 μL of APC secondary antibody diluted 1:400 was added to each well, and the plate was incubated at 4°C for 1 h and then washed twice with PBS. The data was analyzed using the instrument. The results are shown in Table 5, indicating that the endocytosis rate of chi-7 among the 7 chimeric antibodies was the highest.

[0081] Table 5 Determination of the endocytosis rate of the chimeric antibody by flow cytometry

[0082]

[0083] Example 8: Removal of PTM Sites from Chimeric Antibody

[0084] Since the CDR2 sequence of the chi-7 heavy chain (amino acid sequence shown in SEQ ID NO: 16) has NG deamidation sites and NTT glycosylation sites, after mutation, it becomes SG and NPT to remove the deamidation and glycosylation sites (amino acid sequence shown in SEQ ID NO: 46). The mutated chi-7 is 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. It is linked to the constant region of human IgG1 antibody and co-transfected with the plasmid linked to cVL7 and the κ light chain constant region to obtain the protein chi-7-PTM, and the affinity is measured. The results are shown in Table 6 below.

[0085] Table 6: Affinity Measurement of chi-7-PTM Antibody

[0086] Antibody name ka (1 / Ms) kd (1 / s) KD (M) chi - 7 <![CDATA[6.13×10 5 > <![CDATA[1.08×10 -3 > <![CDATA[1.76×10 -9 > chi - 7 - PTM <![CDATA[7.01×10 5 > <![CDATA[1.47×10 -3 > <![CDATA[2.09×10 -9 >

[0087] Example 9: Humanization of Chimeric Antibody

[0088] To design a humanized antibody, the chimeric antibody chi-7-PTM (amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 47, and amino acid sequence of the light chain variable region shown in SEQ ID NO: 43) was selected as the template for further humanization design. The humanization of the murine antibody was carried out by the complementary determining region (CDR) transplantation method, referring to US Patents 4816567; 5225539; 5530101; 5585089; 5693762 and 6180370. The specific humanization method is as follows.

[0089] The light chain and heavy chain variable region sequences of the antibody chi-7-PTM were aligned with the human immunoglobulin gene database on the NCBI website (http: / / www.ncbi.nlm.nih.gov / igblast / ). The human germline IGVH and IGVK with the highest homology to the antibody chi-7-PTM, high expression level, and also used in other marketed drugs were selected as the frameworks for humanization. The selected light chain germline receptor sequence of the antibody chi-7-PTM is human IGKV7-3*01, and the selected heavy chain germline receptor sequence is human IGHV1-2*06. Homology modeling of the variable region of the antibody chi-7-PTM was performed, and amino acids within variable region 3A were selected for back mutation after alignment with the murine antibody to obtain 5 heavy chain variable region sequences, named VH1, VH2, VH3, VH4, VH5 respectively, and 5 light chain variable region sequences, named VL1, VL2, VL3, VL4, VL5 respectively. The sequences are shown in Table 7 below.

[0090] Amino acid sequences of the variable regions of the heavy and light chains after chi-7-PTM reverse mutation in Table 7

[0091] Heavy chain variable region Amino acid sequence Light chain variable region Amino acid sequence VH1 SEQ ID NO:48 VL1 SEQ ID NO:53 VH2 SEQ ID NO:49 VL2 SEQ ID NO:54 VH3 SEQ ID NO:50 VL3 SEQ ID NO:55 VH4 SEQ ID NO:51 VL4 SEQ ID NO:56 VH5 SEQ ID NO:52 VL5 SEQ ID NO:57

[0092] Example 10 Transient expression of humanized antibody

[0093] The humanized heavy chain variable region was linked to the heavy chain constant region of human IgG1 antibody, and the light chain variable region sequence was linked to the κ light chain constant region (the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO: 44, and the amino acid sequence of the light chain constant region is shown as 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 Biotech Corporation. After sequence synthesis, it was cloned into the pCDNA3.4 vector (Invitrogen) and the plasmid was extracted. 5 humanized heavy chain plasmids and 5 humanized light chain plasmids were obtained. The heavy chain plasmids and light chain plasmids were orthogonally combined, transfected into 293F cells for transient expression, and 25 humanized antibodies were obtained. The constructed humanized antibodies are shown in Table 8.

[0094] Table 8 Humanized antibodies

[0095]

[0096]

[0097] Example 11 Affinity determination of humanized antibody

[0098] Using Biacore (T200), the affinity between the antibody and the antigen Nectin-4-His was determined. Ultra-pure water was filtered through a 0.22 μm filter membrane to prepare HBS-EP buffer; the Nectin-4 humanized antibody was diluted to 6 μg / mL with HBS-EP buffer as the ligand; the Nectin-4-His protein was serially diluted 8-fold starting from 100 nM with HBS-EP buffer, and then an additional zero point was added as the analyte; an appropriate amount of Glycine 1.5 was aspirated as the regeneration solution; the ligand, analyte, regeneration solution, and HBS-EP buffer were placed on the sample tray; the program was set as follows: ligand flow rate 10 μL / min for 20 s; analyte 30 μL / min, binding time 100 s, dissociation time 600 s; regeneration solution 30 μL / min for 30 s. The program was started. The SPR signal was collected and saved by the Biacore T200 Control Software, and then the data was processed using the Biacore T200 Evaluation analysis software. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and the KD value was calculated. The results are shown in Table 9. Based on the affinity of the antibody and the number of amino acid mutations in the antibody, H7-1-3, H7-1-5, H7-2-3, and H7-2-4 were selected for subsequent experiments.

[0099] Table 9 Determination of the Affinity of Humanized Antibodies

[0100] Antibody KD (M) kon (1 / Ms) kdis (1 / s) Enfotumab <![CDATA[9.3117×10 -9 > <![CDATA[9.88×10 4 > <![CDATA[9.20×10 -4 > cHi - 7 - PTM <![CDATA[2.9911×10 -9 > <![CDATA[2.483×10 5 > <![CDATA[7.427×10 -4 > H7-1-1 <![CDATA[7.2382×10 -9 > <![CDATA[4.736×10 5 > <![CDATA[3.428×10 -3 > H7-1-2 <![CDATA[7.0759×10 -9 > <![CDATA[4.928×10 5 > <![CDATA[3.487×10 -3 > H7-1-3 <![CDATA[3.4106×10 -9 > <![CDATA[2.779×10 5 > <![CDATA[9.45×10 -4 > H7-1-4 <![CDATA[3.5254×10 -8 > <![CDATA[2.830×10 5 > <![CDATA[9.977×10 -3 > H7-1-5 <![CDATA[3.3683×10 -9 > <![CDATA[2.745×10 5 > <![CDATA[9.246×10 -4 > H7-2-1 <![CDATA[7.2843×10 -9 > <![CDATA[5.505×10 5 > <![CDATA[4.010×10 -3 > H7-2-2 <![CDATA[5.3222×10 -9 > <![CDATA[6.146×10 5 > <![CDATA[3.271×10 -3 > H7-2-3 <![CDATA[1.4059×10 -9 > <![CDATA[6.942×10 5 > <![CDATA[0.976×10 -3 > H7-2-4 <![CDATA[3.8485×10 -9 > <![CDATA[2.918×10 5 > <![CDATA[1.123×10 -3 > H7-2-5 <![CDATA[5.3344×10 -9 > <![CDATA[2.542×10 5 > <![CDATA[1.356×10 -3 > H7-3-1 <![CDATA[9.1983×10 -9 > <![CDATA[4.827×10 5 > <![CDATA[4.440×10 -3 > H7-3-2 <![CDATA[9.1692×10 -9 > <![CDATA[4.851×10 5 > <![CDATA[4.448×10 -3 > H7-3-3 <![CDATA[3.8392×10 -9 > <![CDATA[2.873×10 5 > <![CDATA[1.103×10 -3 > H7-3-4 <![CDATA[3.9986×10 -9 > <![CDATA[2.821×10 5 > <![CDATA[1.128×10 -3 > H7-3-5 <![CDATA[4.0431×10 -9 > <![CDATA[2.644×10 5 > <![CDATA[1.069×10 -3 > H7-4-1 <![CDATA[8.3674×10 -9 > <![CDATA[5.084×10 5 > <![CDATA[4.254×10 -3 > H7-4-2 <![CDATA[9.5775×10 -9 > <![CDATA[4.971×10 5 > <![CDATA[4.761×10 -3 > H7-4-3 <![CDATA[3.5227×10 -9 > <![CDATA[3.151×10 5 > <![CDATA[1.110×10 -3 > H7-4-4 <![CDATA[3.4184×10 -9 > <![CDATA[3.136×10 5 > <![CDATA[1.072×10 -3 > H7-4-5 <![CDATA[2.8468×10 -8 > <![CDATA[3.297×10 5 > <![CDATA[9.386×10 -3 > H7-5-1 <![CDATA[5.3383×10 -9 > <![CDATA[7.139×10 5 > <![CDATA[3.811×10 -3 > H7-5-2 <![CDATA[1.3082×10 -8 > <![CDATA[6.146×10 5 > <![CDATA[8.040×10 -3 > H7-5-3 <![CDATA[4.7492×10 -9 > <![CDATA[3.649×10 5 > <![CDATA[1.733×10 -3 > H7-5-4 <![CDATA[4.7887×10 -9 > <![CDATA[3.738×10 5 > <![CDATA[1.790×10 -3 > H7-5-5 <![CDATA[4.7079×10 -9 > <![CDATA[3.543×10 5 > <![CDATA[1.668×10 -3 >

[0101] The amino acid sequences of the heavy and light chains of H7-1-3 are shown in SEQ ID NO:58 and SEQ ID NO:62 respectively, and the nucleotide sequences of the heavy and light chains of H7-1-3 are shown in SEQ ID NO:66 and SEQ ID NO:70 respectively; the amino acid sequences of the heavy and light chains of H7-1-5 are shown in SEQ ID NO:59 and SEQ ID NO:63 respectively, and the nucleotide sequences of the heavy and light chains of H7-1-5 are shown in SEQ ID NO:67 and SEQ ID NO:71 respectively; the amino acid sequences of the heavy and light chains of H7-2-3 are shown in SEQ ID NO60: and SEQ ID NO:64 respectively, and the nucleotide sequences of the heavy and light chains of H7-2-3 are shown in SEQ ID NO:68 and SEQ ID NO:72 respectively; the amino acid sequences of the heavy and light chains of H7-2-4 are shown in SEQ ID NO:61 and SEQ ID NO:65 respectively, and the nucleotide sequences of the heavy and light chains of H7-2-4 are shown in SEQ ID NO:69 and SEQ ID NO:73 respectively.

[0102] Binding of the Humanized Antibody at the Protein Level in Example 12

[0103] Dilute the Nectin-4-his protein to 0.5 μg / mL and coat a 96-well plate with 100 μL per well overnight at 4°C. The next day, discard the supernatant, then block with 300 μL of 2% non-fat milk at 37°C for 2 h. After discarding the blocking solution, wash 3 times with PBST. Dilute the humanized antibody to 40 μg / mL and perform 12-fold serial dilutions; coat a 96-well plate with 100 μL per well and react at 37°C for 1 h. After the reaction, wash 3 times with PBST and add 100 μL of goat anti-human IgG-FC secondary antibody, react at 37°C for 1 h, wash 3 times with PBST again, then add 100 μL of TMB chromogenic solution for 10 min of chromogenesis. After the reaction, add 100 μL of 1 M hydrochloric acid solution to each well to terminate the reaction, and read and record the data at 450 nm. The results are as Figure 3 shown in Table 10, the protein binding ability of the humanized antibody was not reduced compared to the PTM-modified antibody.

[0104] Table 10 Determination of the Binding Ability of the Humanized Antibody to Nectin-4

[0105] Antibody EC50 value (ng / ml) chi - 7 - PTM 22.05 H7-1-3 23.18 H7-1-5 34.11 H7-2-3 20.72 H7-2-4 21.43 Enfortumab 28.45

[0106] Example 13 Competitive ELISA to Identify the Competitive Ability of the Humanized Antibody with the Nectin-4 Ligand Nectin1

[0107] Dilute the Nectin-4-his protein to 2 μg / mL and coat a 96-well plate with 100 μL per well overnight at 4°C. The next day, discard the supernatant, then block with 300 μL of 2% non-fat milk at 37°C for 2 h. After discarding the blocking solution, wash 3 times with PBST. Dilute the humanized antibody to 40 μg / mL and perform 12-fold serial dilutions; take 50 μL each of the diluted antibody and the ligand Ncetin1 solution (2 μg / mL) in a 1:1 ratio, coat a 96-well plate, and react at 37°C for 1 h. After the reaction, wash 3 times with PBST and add 100 μL of goat anti-human IgG-FC secondary antibody, react at 37°C for 1 h, wash 3 times with PBST again, then add 100 μL of TMB chromogenic solution for 10 min of chromogenesis. After the reaction, add 100 μL of 1 M hydrochloric acid solution to each well to terminate the reaction, and read and record the data at 450 nm. The results are as Figure 4 shown in Table 11, the results show that the humanized antibody can compete for the Nectin-4 binding ligand Nectin1, and the effect is better than the control.

[0108] Table 11 EC50 Values of the Humanized Antibody and Nectin1 in Competing for Binding to Nectin-4

[0109]

[0110]

[0111] Example 14 Determination of the cell-binding ability of humanized antibodies by flow cytometry

[0112] Take 293F-Nectin-4 cells in the logarithmic growth phase, centrifuge at 1200 r / min for 5 min, discard the supernatant, resuspend the cells with 5 mL of DPBS, and adjust the cell density to 2×10 6 cells / mL, and inoculate the cells into a U-bottom 96-well plate at a volume of 50 μL / well. Take the antibody, adjust the antibody concentration to 20 μg / mL, and dilute it in 12 gradients at a 3-fold dilution. Add it to the U-bottom 96-well plate at 50 μL / well, react at 37 °C for 1 h, centrifuge at 1200 r / min for 5 min, discard the supernatant, wash twice with 200 μL of DBS, add 100 μL of DPBS with fluorescent secondary antibody to each well, react at 37 °C for 1 h. After the reaction, centrifuge and wash twice with DPBS. Finally, resuspend the cells with 200 μL of DPBS in each well, load onto the instrument and analyze the data. The results are as Figure 5 shown in Table 12, the EC50 values of H7-1-3, H7-2-3, and H7-2-4 antibodies are all less than that of the control antibody Enfortumab.

[0113] Table 12 Cell-binding ability of humanized antibodies

[0114] Antibody EC50 value (μg / ml) chi - 7 - PTM 0.1937 H7-1-3 0.1568 H7-1-5 0.2001 H7-2-3 0.1752 H7-2-4 0.1580 Enfortumab 0.1836

[0115] Example 15 Determination of the endocytosis effect of humanized antibodies on tumor cells by flow cytometry

[0116] Collect MCF-7 cells, adjust the cell density to 2×10 with 2% FBS + PBS 6cells / mL, add 100 μL per well into a U-shaped 96-well plate. Adjust the density of the humanized antibody to 40 μg / mL and 8 μg / mL, add 100 μL per well into the U-shaped 96-well plate, mix well, and react at 4°C for 1 h. After the reaction, wash twice with PBST, then resuspend the cells with 200 μL of 2% FBS + PBS, and evenly distribute them into two U-shaped 96-well plates. One plate is placed at 4°C, and the other plate is placed at 37°C. After standing for 20 h, centrifuge at 2000 r / min for 3 min and wash twice with PBST. Add 100 μL of APC secondary antibody diluted 1:400 per well, incubate at 4°C for 1 h, then wash twice with PBS. Detect the antibody endocytosis amount by flow cytometry, and calculate the endocytosis efficiency as (fluorescence value at 4°C - fluorescence value at 37°C) / fluorescence value at 4°C. The endocytosis efficiency of each antibody is shown in Table 13 below. The endocytosis efficiency of H7-2-3 is 74% at a concentration of 8 μg / mL and 68% at 40 μg / mL, both higher than the endocytosis efficiency of the control antibody Enfortumab.

[0117] Table 13 Endocytosis rate of humanized antibodies determined by flow cytometry

[0118]

[0119] Example 16 Preparation of anti-Nectin-4 antibody conjugate

[0120]

[0121] Reduction of antibody: For the antibody prepared in the example, dilute the antibody H7-2-3 to a concentration of 18.31 mg / mL. Take 5.0 mL of this solution and add it to a 250 mL glass reaction flask container. Under magnetic stirring, add 1.5 μM TCEP aqueous solution (5.83 mL; equivalent to 2.5 equivalents per molecule of antibody), stir at room temperature for 2 h to reduce the disulfide bond in the inner hinge region of the antibody.

[0122] Conjugation of Antibody with Linker-Drug Conjugate Compound: Slowly add DMSO (82.88 μL; equivalent to 5.0 equivalents per molecule of antibody) containing 3.0 mM (Reference: Lyon RP, Meyer DL, Setter JR, et al. Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues. Methods Enzymol. 2012;502:123-38. doi:10.1016 / B978-0-12-416039-2.00006-9) of the linker-drug conjugate compound (Vc-MMAE) to the above solution system, stir at room temperature for 1 hour to conjugate the antibody with the linker-drug conjugate compound, and finally obtain the antibody-drug conjugate H7-2-3-MMAE with a DAR value of 4.0.

[0123] Example 17 Killing Activity of Anti-Nectin-4 Antibody-Drug Conjugate against Tumor Cells in Vitro

[0124] The anti-Nectin-4 antibody-drug conjugate can effectively kill tumor cells expressing the Nectin-4 target. We selected the breast cancer cell line MCF-7 from ATCC expressing Nectin-4 as the target cells to evaluate the killing activity of the anti-Nectin-4 antibody-drug conjugate H7-2-3-MMAE against tumor cells in vitro.

[0125] Collect MCF-7 cells by trypsin digestion, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells with 10% FBS + EMEM medium and count to adjust the cell density to 1×10 4 / ml, and seed 100 μl of cells per well in a 96-well plate; Prepare samples: Dilute the test article with 10% FBS + EMEM medium to 180 μg / ml, serially dilute it 9-fold in 6-fold dilutions, and set another 0 concentration point, for a total of 10 concentration points. Mix 50 μl of each concentration with the cells, and set three replicates for each concentration point; Incubate in a 37°C / CO2 incubator for 5 days. After incubation, add 50 μl of CTG to each well and detect the chemiluminescence with a multifunctional microplate reader to evaluate cell viability, and fit a four-parameter curve to calculate the killing EC50 value. The results are as Figure 6 shown in and Table 14. H7-2-3-MMAE has a significant killing effect on tumor cells in vitro and is more effective than the control molecule Enfortumab vedotin.

[0126] Table 14 Cell Killing Activity of Antibody-Drug Conjugates

[0127] Antibody - drug conjugate EC50 value (μg / ml) H7 - 2 - 3 - MMAE 0.8843 Enfortumab vedotin 4.750

[0128] Example 18 In Vivo Efficacy Experiment of Anti-Nectin-4 Antibody-Drug Conjugate

[0129] Study on the Effect of ADC Molecule on Subcutaneous Xenograft Model of Human Pancreatic Cancer Cell BxPC3 in Female NOG Mice

[0130] A subcutaneous xenograft model of human pancreatic cancer cell BxPC-3 was established using female NOG mice to investigate the therapeutic effect of the ADC molecule on this model. The human pancreatic cancer cell BxPC-3 used in this experiment was cultured in RPMI 1640 medium supplemented with 10% FBS in a 37 °C incubator containing 5% CO2. Mice were inoculated before the cells were continuously cultured for ten generations. BxPC-3 cells (cell concentration adjusted to 1×10 8 cells / mL) were mixed with Matrigel at a volume ratio of 1:1, and 0.1 mL / animal was inoculated subcutaneously into the right flank of female NOG mice. The inoculation dose of BxPC-3 cells per mouse was 5×10 6 cells. When the average tumor volume of the mice reached about 145 mm 3 , the mice were randomly divided into 3 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. Administration started on the day of grouping (defined as D0 day), and the drug was administered by tail vein injection at a dose of 6 mg / kg. The drug was administered only once throughout the experimental period. Tumor volume and body weight were measured weekly, and the results are shown in Figure 7 and Figure 8 .

[0131] The maximum diameter (D) and minimum diameter (d) of the tumor were measured weekly using an electronic vernier caliper, the tumor volume was calculated, and the tumor growth inhibition rate of each treatment group was calculated according to the formula.

[0132] Tumor volume V (mm 3 ) = [D × d 2 / 2;

[0133] Relative tumor proliferation rate T / C (%) = (T n - T0) / (C n - C0) × 100%, where T n , C n are the tumor volumes of the treatment group and the control group on day Dn during the experiment; T0 and C0 are the tumor volumes of the treatment group and the control group on day D0 at the start of the experiment;

[0134] Tumor growth inhibition rate TGI (%) = 1 - T / C (%).

[0135] The results are shown in Table 15. The results indicate that the ADC molecule has significant anti-tumor effects. The tumor volume reaches the minimum on the 17th day, and the tumor inhibition rate reaches the maximum. After that, the tumor volume increases. At the same dose, H7-2-3-MMAE has a better effect on inhibiting tumor growth than the control molecule Enfortumab vedotin.

[0136] Table 15 Tumor inhibition effects of antibody-drug conjugates in animals

[0137]

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, 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, 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, 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, SEQ ID NO:23; (3) The amino acid sequences of HCDR1, HCDR, and HCDR3 are respectively as shown in SEQ ID NO:7, SEQ ID NO:8, 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, 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, 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, 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, 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, SEQ ID NO:25; (6) The amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30; (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; (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 NOs: 31-37, 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, 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, SEQ ID NOs: 53-57.

3. The anti-nectin-4 antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequences of the antibody heavy chain variable region and light chain variable region are selected from the following groups: (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; (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; (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; (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 sequences; (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 sequences; (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 sequences; (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 sequences; (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 sequences; (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 sequences; (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 sequences; (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 said amino acid sequences; (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 said 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 said 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 said 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 said 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 said 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 said 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 said 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 said amino acid sequence; (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; (21) 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: 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; (22) 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: 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; (23) 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: 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; (24) 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: 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; (25) 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: 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; (26) 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: 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; (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; (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 heavy chain variable region and the light chain variable region of the antibody 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 to 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 contains the polynucleotide according to claim 6.

8. An antibody conjugate, characterized in that, The antibody conjugate has 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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