Anti-nectin-4 antibody, conjugate including the same, and application thereof

Abstract

To provide an anti-nectin-4 antibody.SOLUTION: There are provided an anti-nectin-4 antibody or antigen-binding fragment thereof, an antibody conjugate comprising the same, and use thereof in manufacture of a medicament, particularly in the manufacture of a medicament for treating and / or preventing cancer.SELECTED DRAWING: Figure 10B

Description

[Technical field]

[0001] This application is a joint venture between the US Food and Drug Administration and the US Food and Drug Administration and the US Food and Drug Administration and the US Food and Drug Administration and the US Food and Drug Administration and the US Food and Drug Administration and the US tibody,conjugate including same,and appl Chinese Patent Application No. 20201097236 entitled "Compounding Method and Method for Manufacturing a Semiconductor Device Using a Combination of Polymers and Polymeric Materials" Priority of US Patent Application Publication No. 5.6 and "Anti-Nectin- 4 antibody,conjugate including same,and Chinese Patent Application No. 2020116 entitled "Chinese Patent Application No. 2020116" This application claims priority to US Pat. No. 24826.7, the entire contents of which are incorporated herein by reference. INCORPORATED INTO THE SPECIFICATION.

[0002] The present invention belongs to the field of biomedicine. In particular, the present invention relates to an anti-Nectin-4 antibody and a method for the treatment thereof. and a conjugate comprising the same, in particular in the treatment and / or prevention of nectin-4-related diseases Concerning its application. [Background technology]

[0003] Nectin-4, also known as PVRL, belongs to the immunoglobulin superfamily. It belongs to the nectin family of proteins and mediates calcium ion-independent cell adhesion junctions. The nectin family consists of nectin-1, nectin-2, and nectin- Nectin-3 and Nectin-4, which have a size of about 510 to 550 amino acids, The extracellular segment is formed by three Ig-like domains, a transmembrane region, and an intracellular tail region. It belongs to the type I transmembrane protein family, which is a structure containing three Ig-like domains. It has one main and two IgC domains, and the C-terminus of the intracellular domain is the afadin protein. It consists of Glu / Ala-X-Tyr-Val, which is used to bind to the PDZ domain of proteins. Nectin-4 does not have this protein sequence, but still Furthermore, it can bind to afadin protein within cells.

[0004] Nectin-4 inhibits anoikis and downregulates the PI3K-AKT signaling pathway By activating Nectin-4, it can promote the survival and proliferation of tumor cells. , hepatocellular carcinoma, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer and food Expressed in ductal cancer and strongly expressed in non-small cell lung cancer, bladder cancer, breast cancer, and pancreatic cancer Studies have shown that high expression of Nectin-4 in tumors is associated with poor prognosis. Therefore, Nectin-4 is an ideal candidate for the development of drugs against tumors. Can be used as a target.

[0005] Currently, enfortumab vedotin, an ADC drug targeting nectin-4 (e.g. (see, for example, WO2012 / 047724) are already being used for the treatment of urothelial carcinoma. Enfortumab vedotin is a conjugate of an anti-Nectin-4 fully human antibody. It is a gated VC-MMAE drug with a DAR value of 4. Four patients achieved complete response (CR) and 41 patients achieved partial response (PR). The objective response rate (ORR) was 41%, and the median OS was 13.6 months. The median duration of disease was 5.75 months, and the median progression-free survival (PFS) was 5.4 In the checkpoint inhibitor treatment group, 89 patients had a median survival time of 1 month. The ORR was 40%, and 23 patients in the checkpoint inhibitor-naïve group had an ORR of 4. In the liver metastasis group, the ORR was 39% in 33 patients. Dotin has excellent antitumor efficacy.

[0006] Nectin-4 is expressed in the placenta and also in several normal tissues, such as the skin and gingiva. It was weakly to moderately expressed in the uterine tract, breast, and bladder. The main clinical toxicities and side effects of the drug were fatigue (54%) and grade 3 or higher side effects. The most common causes of anaemia were anemia (8%), hyponatremia (7%), urinary tract infection (7%) and hyperglycemia (6%). Four patients developed respiratory failure, urinary obstruction, diabetic ketoacidosis, and multiple organ failure. These results suggest that the toxicity of enfortumab vedotin is The effects and side effects are shown to be too severe. Summary of the Invention

[0007] In one general aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4. and a synthetic fragment thereof, the antibody or antigen-binding fragment thereof comprising: (1a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5 DR-H3 or variants thereof; and / or according to the Chothia numbering system The following three light chain CDRs are defined as follows: CDR-L1 comprising the sequence set forth in SEQ ID NO:6; or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof or a CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (1b) The following three heavy chain CDRs, as defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof; CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5; H3 or a variant thereof; and / or any of the following as defined according to the Abm numbering system: The three light chain CDRs below: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or its barrier a CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; or a variant thereof comprising the sequence set forth in (1c) the following three heavy chain CDRs as defined according to the Kabat numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 11, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5 DR-H3 or variants thereof; and / or as defined according to the Kabat numbering system The following three light chain CDRs are defined: CDR-L1 comprising the sequence set forth in SEQ ID NO:6; a CDR-L2 or variant thereof comprising the sequence set forth in SEQ ID NO: 7; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or The following three heavy chain CDRs are defined according to the Kabat numbering system: SEQ ID NO:1 CDR-H1 comprising the sequence as set forth in claim 1 or a variant thereof, the sequence as set forth in SEQ ID NO: 63 or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO:5 3 or variants thereof; and / or as defined according to the Kabat numbering system The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof ant, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, SEQ ID NO: 8. A CDR-L3 or a variant thereof comprising the sequence of or (1d) the following three heavy chain CDRs as defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, as set forth in SEQ ID NO: 14 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 15 DR-H3 or its variants; and / or as defined according to the IMGT numbering system The following three light chain CDRs are used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or A variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof , a CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (2a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, SEQ ID NO: 22 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 23 and / or the Chothia numbering system The following three light chain CDRs are defined according to the following: CDR- comprising the sequence set forth in SEQ ID NO: 24 L1 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO:25 a variant thereof, a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26; Or The following three heavy chain CDRs are defined according to the Chothia numbering system: No. 18, or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 R-H3 or a variant thereof; and / or according to the Chothia numbering system The following three light chain CDRs are defined: CDR-L1 comprising the sequence set forth in SEQ ID NO:24; or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a barrier thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof; or (2b) the following three heavy chain CDRs defined according to the Abm numbering system: No. 27, or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 R-H3 or variants thereof; and / or as defined according to the Abm numbering system The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or variants thereof, A CDR-L3 comprising the sequence set forth in column number 26 or a variant thereof; Or The following three heavy chain CDRs are defined according to the Abm numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 28 CDR-H2 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or variants thereof; and / or the following as defined according to the Abm numbering system: Three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO:24 or a variant thereof CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof, SEQ ID NO: 2 6. A CDR-L3 or a variant thereof comprising the sequence according to claim 6; or (2c) the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 30 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 CDR-H3 or a variant thereof; and / or according to the Kabat numbering system The following three light chain CDRs are defined: CDR-L1 comprising the sequence set forth in SEQ ID NO:24; or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a barrier thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof; or The following three heavy chain CDRs, defined according to the Kabat numbering system: SEQ ID NO:4 4 or a variant thereof, the sequence of SEQ ID NO: 30. or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 23 H3 or variants thereof; and / or as defined according to the Kabat numbering system The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or variants thereof, A CDR-L3 comprising the sequence set forth in column number 26 or a variant thereof; or (2d) The following three heavy chain CDRs as defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 31 or a variant thereof, or a variant thereof, comprising the sequence set forth in SEQ ID NO: 33 DR-H3 or its variants; and / or as defined according to the IMGT numbering system The following three light chain CDRs are used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or A variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof , a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof; or The following three heavy chain CDRs, as defined according to the IMGT numbering system: SEQ ID NO:54 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 32 CDR-H2 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 33 3 or variants thereof; and / or any of the following as defined according to the IMGT numbering system: The three light chain CDRs below: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a variant thereof ant, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof, SEQ ID NO: No. 26, or a variant thereof; or (3a) the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 39 or a variant thereof, SEQ ID NO: 40 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 41 and / or the Chothia numbering system The following three light chain CDRs are defined according to the following: CDR- comprising the sequence set forth in SEQ ID NO: 42 L1 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof, a CDR-L3 comprising the sequence set forth in SEQ ID NO: 8; or teeth (3b) the following three heavy chain CDRs defined according to the Abm numbering system: No. 45, or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 R-H3 or variants thereof; and / or as defined according to the Abm numbering system The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or variants thereof, A CDR-L3 or a variant thereof comprising the sequence set forth in column number 8; or (3c) the following three heavy chain CDRs as defined according to the Kabat numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 47, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 CDR-H3 or a variant thereof; and / or according to the Kabat numbering system The following three light chain CDRs are defined: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42; or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a barrier thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or The following three heavy chain CDRs, defined according to the Kabat numbering system: SEQ ID NO:4 7 or a variant thereof, the sequence of SEQ ID NO: 64 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 41 H3 or variants thereof; and / or as defined according to the Kabat numbering system The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or variants thereof, A CDR-L3 or a variant thereof comprising the sequence set forth in column number 8; or (3d) the following three heavy chain CDRs as defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 49 or a variant thereof, as set forth in SEQ ID NO: 50 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 51 DR-H3 or its variants; and / or as defined according to the IMGT numbering system The following three light chain CDRs are used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 52 or A variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:53 or a variant thereof , CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof Including, A variant may have one or more amino acid substitutions, deletions or deletions compared to the sequence from which it is derived. has an addition (e.g., a substitution, deletion or addition of one, two or three amino acids), , conservative substitutions are preferred.

[0008] In a preferred embodiment, the antibody or antigen-binding fragment thereof is a human or mouse immunoglobulin. It further comprises framework regions derived from the

[0009] In one aspect, the present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising administering to the patient an antibody or antigen-binding fragment thereof of the present invention and at least one therapeutic agent. The present invention provides an antibody conjugate comprising a therapeutic agent.

[0010] In yet another aspect, the present invention provides chimeric antigen receptors (CARs) and CAR-expressing immunoglobulins. Provide immune effector cells.

[0011] In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof of the present invention, and Fusion proteins comprising biologically active polypeptides are also contemplated.

[0012] The present invention relates to an antibody or an antigen-binding fragment thereof, an antibody conjugate, an immune effector, and a method for treating The subject invention relates to a method for the preparation of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ... The present invention also relates to pharmaceutical compositions comprising an excipient.

[0013] In yet another general aspect, the invention provides a method for treating cancer comprising the steps of: The antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, and multimeric The present invention relates to the use of a bispecific antibody, a fusion protein or a pharmaceutical composition.

[0014] The present invention relates to an antibody or an antigen-binding fragment thereof of the present invention in the treatment and / or prevention of cancer. , antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceuticals The invention also relates to the use of pharmaceutical compositions.

[0015] In another general aspect, the invention provides a method of treating cancer in a subject in need thereof. The method also provides a method, wherein the cancer is associated with Nectin-4, comprising: The subject is administered the antibody or antigen-binding fragment thereof, antibody conjugate, immune effector, or Administering a therapeutically effective amount of the cell, multispecific antibody, fusion protein or pharmaceutical composition. Includes.

[0016] Cancers include stomach cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial cancer, urethral cancer, and renal pelvis cancer. , ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, ductal carcinoma, triple-negative breast cancer, The cancer may be selected from pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, and esophageal cancer. preferable. [Brief description of the drawings]

[0017] [Figure 1A] 1 is a graph showing the results of flow cytometry of A549-Nectin-4 stable cell line. [Figure 1B] 1 is a graph showing the results of flow cytometry of T24-Nectin-4 stable cell line. [Diagram 2] 1 is a graph showing the results of affinity detection of anti-human Nectin-4 mouse antibody. [Figure 3A] 1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 31HZ and human Nectin-4 protein. [Figure 3B] 1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 74HZ and human Nectin-4 protein. [Figure 3C] 1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 56HZ and human Nectin-4 protein. [Figure 3D] 1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 31HZ and cynomolgus monkey Nectin-4 protein. [Figure 3E] 1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 74HZ and cynomolgus monkey Nectin-4 protein. [Figure 3F]1 is a graph showing the results of ELISA detection of the affinity between the anti-human Nectin-4 humanized monoclonal antibody 56HZ and cynomolgus monkey Nectin-4 protein. [Figure 4A] 1 is a graph showing the results of flow cytometry for the affinity of anti-human Nectin-4 humanized monoclonal antibodies 31HZ and 74HZ to T47D cells. [Figure 4B] 1 is a graph showing the results of flow cytometry regarding the affinity between the anti-human Nectin-4 humanized monoclonal antibody 56HZ and NCI-N87 cells. [Figure 5A] 1 is a graph showing the results of detecting the endocytosis activity of anti-human Nectin-4 humanized monoclonal antibodies 31HZ and 74HZ in T47D cells. [Figure 5B] 1 is a graph showing the results of detecting the endocytosis activity of anti-human Nectin-4 humanized monoclonal antibodies 31HZ and 74HZ in MDA-MB-468 cells. [Figure 5C] 1 is a graph showing the results of detecting the endocytosis activity of anti-human Nectin-4 humanized monoclonal antibodies 31HZ and 74HZ in A549-Nectin-4 cells. [Figure 5D] 1 is a graph showing the results of detecting the endocytosis activity of 56HZ, an anti-human Nectin-4 humanized monoclonal antibody, in T24-Nectin-4 cells. [Figure 6A] 1 is a graph showing the detection results of 31HZ and 74HZ, which are anti-human Nectin-4 humanized monoclonal antibodies that recognize the human Nectin-4 domain. [Figure 6B] 1 is a graph showing the detection results of 56HZ, an anti-human Nectin-4 humanized monoclonal antibody that recognizes the human Nectin-4 domain. [Figure 7A] 1 is a graph showing the results of detecting the competitive activity of anti-human Nectin-4 humanized monoclonal antibodies 31HZ and 74HZ. [Figure 7B]1 is a graph showing the results of detecting the competitive activity of 56HZ, an anti-human Nectin-4 humanized monoclonal antibody. [Figure 8A] 1 is a graph showing the results of detecting the specificity of 31HZ, an anti-human Nectin-4 humanized monoclonal antibody. [Figure 8B] 1 is a graph showing the results of detecting the specificity of 74HZ, an anti-human Nectin-4 humanized monoclonal antibody. [Figure 8C] 1 is a graph showing the results of detecting the specificity of 56HZ, an anti-human Nectin-4 humanized monoclonal antibody. [Figure 9A] 1 is a graph showing the results of killing of A549-Nectin-4 cells by anti-human Nectin-4 ADCs 31HZ-TL001 and 74HZ-TL001. [Figure 9B] 1 is a graph showing the results of A549 cell killing by anti-human nectin-4 ADCs 31HZ-TL001 and 74HZ-TL001. [Figure 9C] 1 is a graph showing the results of killing of T24-nectin-4 cells by anti-human nectin-4 ADCs 31HZ-TL001 and 56HZ-TL001. [Figure 9D] 1 is a graph showing the results of T24 cell killing by anti-human nectin-4 ADCs 31HZ-TL001 and 56HZ-TL001. [Figure 10A] 1 is a graph showing the results of killing of T47D cells by anti-human nectin-4 ADCs 31HZ-TL001 and 74HZ-TL001. [Figure 10B] 1 is a graph showing the results of MDA-MB-468 cell killing by anti-human nectin-4 ADCs 31HZ-TL001 and 74HZ-TL001. [Figure 10C] 1 is a graph showing the results of killing of NCI-N87 cells by anti-human nectin-4 ADCs 31HZ-TL001 and 56HZ-TL001. [Figure 11]1 is a graph showing the change in tumor volume over time for each group of mice in a subcutaneous NCI-H322M cell xenograft tumor model in NOD / SCID mice. [Figure 12] 1 is a graph showing the change in body weight over time for each group of mice in a subcutaneous NCI-H322M cell xenograft tumor model in NOD / SCID mice. [Figure 13] Graph showing the change in tumor volume over time for each group of mice in the subcutaneous HuPrime® gastric cancer BL9200 PDX model in NOD / SCID mice. [Figure 14] 1 shows the change in body weight over time for each group of mice in the subcutaneous HuPrime® gastric cancer BL9200 PDX model in NOD / SCID mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition In the present invention, unless otherwise specified, the technical and scientific terms used herein are understood to be within the skill of the art. The terms "protein" and "nucleic acid" have their commonly understood meanings. Terminology and experimental procedures related to chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are included. These are terms and conventional procedures widely used in the corresponding fields. For better understanding, definitions and explanations of relevant terms are provided below.

[0019] As used herein, "at least one" or "one or more" means Species) are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 (species) or more.

[0020] The word "comprising" or its equivalent "including," "containing," and "having" ng) are open-ended and may include additional unrecited elements, steps, or Constituents are not excluded. The expression "consisting of" means Any unspecified element, step or component is excluded. The phrase "consisting essentially of" means Any elements, steps or components specifically recited herein, as well as any fundamental aspects of the claimed subject matter. Optional elements, steps or components that do not materially affect the fundamental and novel characteristics of the invention. The word "comprising" means that the scope is limited to: "consisting essentially of" and " It should be understood that this expression includes the expression "consisting of" It is.

[0021] The words "optionally" or "optionally" refer to the event or condition that is subsequently described. Certain events or circumstances may or may not occur and this statement does not include the occurrence of such events or circumstances. "Conditions" are intended to cover situations where an event or circumstance occurs, as well as situations where the event or circumstance does not occur.

[0022] As used herein, a "polypeptide" refers to two polypeptides linked by a peptide bond. A "protein" refers to a polymer formed from one or more amino acids. Unless otherwise specified, the term "polypeptide" refers to a peptide that is formed by covalent or non-covalent bonding. The terms "peptide" and "protein" are used interchangeably herein.

[0023] As used herein, an "antibody" refers to an antibody that binds to an antigen through at least one antigen-binding site. It refers to an immunoglobulin or a fragment thereof that specifically binds to an original epitope. The definition of an antibody includes antigen-binding fragments. Thus, the term "antibody" refers to a multispecific antibody. Antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, Antibodies include single domain antibodies, and antigen-binding fragments. Antibodies include synthetic antibodies (e.g., chemically or Antibodies produced by biological coupling, antibodies treated with enzymes The antibodies provided herein may be any Immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY); Any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and Ig A2), or subclasses (e.g., IgG2a and IgG2b). In yet another embodiment, the antibody of the present invention is a murine monoclonal antibody. The antibody is a humanized monoclonal antibody.

[0024] As used herein, a "conventional antibody" or "full length antibody" generally comprises four polypeptides. Polypeptide: Contains two heavy chains (HC) and two light chains (LC). Each light chain has Each heavy chain contains a "light chain variable region (VL)" and a "light chain constant region (CL)" up to the C-terminus. The heavy chain variable region (VH) and the heavy chain constant region (CH) are included from the N-terminus to the C-terminus. The heavy chain constant region can comprise, from N-terminus to C-terminus, a CH1, a CH2, and a CH3. In certain immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region is The "Fc" fragment comprises CH2 and CH3 and is capable of binding to an Fc receptor. The "hinge region" refers to the region that provides the site of synthesis between the Fab fragment and the Fc fragment of an immunoglobulin. Refers to the part of an antibody that connects the constant region and the transmembrane fragment. In some cases, the hinge region is the region that connects the constant region and the transmembrane fragment. It refers to the part of the T cell receptor that connects the domains. When used herein, hinge region may refer to any functional equivalent. The positions of VH, VL, CL, CH1, CH2, CH3 and hinge regions of the antibody were determined based on known amino acid sequences. The applicable algorithms and software may be determined in accordance with the For information on software, see, for example, William R. Strohl, Lila M .Strohl, (2012), Antibody structure-functi on relationships, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibo dy Engineering, Woodhead Publishing, 37~56 can be found on page 1.

[0025] Generally, VL and VH each contain three highly variable "complementarity determining regions (CDRs)" ) and four relatively conserved "framework regions (FR)", which are From the end to the C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 In the present specification, the light chain variable region CDR (CDRL) is referred to as CDR-L1, CDR-L2, CDR-L3, CDR-L4, CDR-L5, CDR-L6, CDR-L7, CDR-L8, CDR-L9, CDR-L10, CDR-L11, CDR-L12, CDR-L13, CDR-L14, CDR-L15, CDR-L16, CDR-L The heavy chain variable region CDR (CDRH) can be referred to as CDR-L2 and CDR-L3. These can be referred to as CDR-H1, CDR-H2, and CDR-H3. The binding specificity of an antigen-binding site can be determined by the six CDRs formed. In some cases, other fragments (e.g., Single domain antibodies, also known as nanobodies, are generally considered to have antigen-binding capacity. Those skilled in the art can use methods well known in the art, for example, Kabat, Abm, or Ch. The Othia numbering system can be used to identify CDRs. Numerical CDR numbering systems, e.g., Chothia, Abm, Kabat and IMG T can be used for the same variable region. Although the CDRs identified by the same numbering system may differ, CDRs identified by the same numbering system are It will be understood by those skilled in the art that each of these sites represents an efficient antigen-binding site capable of binding to a given antigen-binding site. The description of the CDR numbering system can be found, for example, in the Kabat numbering system. For more information, see Kabat, EA et al. (1991) Sequences of Prot eins of Immunological Interest, Fifth Edi tion, USDepartment of Health and Human Services vices, NIH Publication No.91-3242;Chothia For the numbering system see: Chothia, C. et al. (1987) J. Mol. Biol. l.196:901-917; or for the IMGT numbering system: Lefran c,M.-P., 2011(6), IMGT, the International I mmunoGeneTics Information System Cold Sp ring Harb Protoc.;Abm numbering system:Marti n, ACR and J. Allen (2007) “Bioinformatics t tools for antibody engineering”, S. Dubel (ed. ), Handbook of Therapeutic Antibodies.Wei Nheim: Wiley-VCH Verlag, pp. 95-118. do.

[0026] As used herein, the terms "framework region" and "framework" are used interchangeably. As used herein, "framework region," "framework" Alternatively, the term "FR" residues refers to residues other than the CDR residues defined above in the variable region of an antibody. Refers to amino acid residues.

[0027] The "Fv" fragment, consisting of one VH and one VL, binds to the antibody via non-covalent interactions. It is generally believed to be the smallest antigen-binding fragment that contains the original binding site. Also known as single domain antibodies (VHH, sdAb or nanobodies) containing only the variable regions of A "single-chain Fv (scFv)" is a peptide linker consisting of VH and VL. The disulfide-stabilized Fv(d "sFv" or "disulfide-stabilized single-chain Fv (scdsFv or dsscFv)" These can be obtained by introducing disulfide bonds into Fv or scFv, respectively. In some embodiments, for example, antibody conjugates in the CARs of the invention, multispecific It is preferred that the specific antibody or antigen-binding fragment used is an scFv or scdsFv.

[0028] As used herein, "Fab" refers to one complete antibody light chain (VL-CL) and It comprises an antibody heavy chain variable region and one heavy chain constant region (VH-CH1, also known as Fd). Single-chain Fab (scFab) is a peptide linker that connects the CL and CH1 in Fab. "F(ab')2" is basically a fusion protein consisting of a hinge region and a fusion protein consisting of a fusion protein consisting of a hinge region and a fusion protein consisting of a hinge region. It contains two Fab fragments linked by disulfide bonds at F It is one half of F(ab')2 and reduces the disulfide bond in the hinge region of F(ab')2. It can be obtained by subtracting

[0029] The antibodies or antigen-binding fragments may be "monovalent," "bivalent," "trivalent," or "tetravalent," or more. The antibody may also be multivalent, i.e., it may contain multiple (e.g., one, two, three, four or more) antigens. It has a binding site.

[0030] Two or more (e.g., two, three, four, five, or six) antibodies or antigen-binding fragments When binding to an antigen, a "multispecific antibody" is used, e.g., a bispecific, trispecific, or tetraspecific antibody. They can also be called polyspecific antibodies, which bind to two, three, or four antigens, respectively. The present invention represents a multispecific antibody capable of

[0031] As used herein, a "diabody" refers to a molecule that is composed of two scFvs. and each scFv has a VH and VL. are linked by a short peptide linker (approximately 5-10 amino acid residues), Thus, the VH chain and the VL chain are paired (i.e., the VH of the first scFv and the VL of the second scFv The VL of the first scFv pairs with the VH of the second scFv, and the VL of the first scFv pairs with the VH of the second scFv. Diabodies are bispecific antibodies.

[0032] As used herein, an "antigen-binding fragment" of an antibody is less than full-length, but contains the same antigen-binding domain as the full-length antibody. and / or a portion of a variable region of the polypeptide (e.g., one or more CDRs and / or The full-length antibody specifically binds to the antigen. An antigen-binding fragment refers to a portion of a full-length antibody that retains at least a portion of its ability to bind to an antigen. Examples of such antibodies include antibody derivatives prepared by enzymatically treating full-length antibodies, and synthetic antibodies. These may be synthetic or recombinantly produced derivatives. Examples of synthetic fragments include, but are not limited to, Fv, scFv, dsFv, scDv, sFv, Fab, scFab, Fab', F(ab')2, diabody, Fd and Fd Antigen-binding fragments include, for example, fragments of the nucleotide sequence ... For example, disulfide bonds and / or peptide linkers and / or non-covalent interactions. Thus, it may comprise multiple peptide chains formed by linking them together.

[0033] As used herein, the term "monoclonal antibody" refers to a highly homogeneous antibody. This refers to a population of antibodies that may be present in trace amounts. Monoclonal antibodies are generally made of Monoclonal antibodies useful in the present invention are For example, transgenic animals using recombinant DNA techniques (e.g., production from immortalized B cells (e.g., B cell hybridomas); or production from bacterial cells, eukaryotic cells or plant cells, or phage antibody libraries. The present invention can be prepared by any method known in the art, such as by isolation from a soluble form of the compound. The antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody. The disclosed antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

[0034] A "chimeric antibody" is a chimeric antibody that is a mixture of a portion (e.g., CDR, FR, variable region, constant region, or any of these) of a (combination of) is identical or homologous to the corresponding sequence of an antibody derived from a particular species, and the remaining The term "antibody" refers to an antibody in which the sequence is identical or homologous to the corresponding sequence of an antibody from another species. As used herein, a "chimeric antibody" includes portions that belong to different antibody classes or subclasses. In certain embodiments, chimeric antibodies include antibodies comprising a murine antibody variable region and a human antibody variable region. and a human antibody constant region.

[0035] As used herein, a "humanized antibody" refers to an antibody that contains non-human and human antibody sequences. A humanized antibody therefore refers to an antibody that contains minimal sequences derived from non-human immunoglobulin. The non-human antibody may be an antibody derived from any non-human species. The antibody may be an antibody that contains a portion derived from a non-human species (e.g., a chimeric antibody). Species may include, for example, mouse, rat, rabbit, alpaca, or non-human primate. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. A fused antibody can be derived from a non-human antibody (e.g., a murine antibody or a chimeric antibody), e.g., a non-human antibody. by grafting the CDR sequences of a human antibody (e.g., a murine antibody) into the framework regions of a human antibody. In some cases, the antigen-binding ability and / or stability of the humanized antibody may be improved. In order to maintain the integrity of the non-human antibody (e.g., mouse antibody) framework sequences, The amino acid residues can be retained in the framework regions of the human antibody, i.e., Perform "reverse mutation" (see, for example, Morrison et al. (1984) Proc. Natl. A cad.Sci.81(21):6851-6855;Neuberger et al. (1984 )Nature 312:604-608).

[0036] As used herein, the "percent (%) sequence identity" of an amino acid sequence, the "sequence "Identity" has a definition that is widely accepted in the art and is used in sequence alignments. Two points determined by a test (e.g., manual inspection or by a known algorithm) The percent identity refers to the percent identity between the polypeptide sequences. Percent identity can be determined using methods known to those skilled in the art. Use e.g. BLAST, BLAST-2, Clustal Omega, FAS Determined using publicly available computer software such as TA software It is possible.

[0037] "Affinity" or "binding affinity" refers to the binding between an antibody and an antigen through non-covalent interactions. The strength of the binding between a molecule and a substance is measured using the equilibrium dissociation constant K D or EC 50 It can be reported as K D are the equilibrium association constant (ka) and equilibrium dissociation constant The constant (kd) can be calculated by measuring: K D =kd / ka. affinity For example, biofilm interferometry (e.g., Octet Fortebio detection system) (e.g., ELISA, radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay, etc.) using conventional techniques known in the art, such as immunoassays, or flow cytometry. It can be determined.

[0038] As used herein, "specific binding" of an antibody to an antigen refers to a high affinity binding between the antibody and the antigen. Generally, the K between a specifically bound antibody and an antigen is D The value must be at least Also about 10 -6 ~ At least about 10 -9 Less than M, e.g., at least about 10 -6 M, less About 10 -7 M, at least about 10 -8 M, at least about 10 -9 It's M.

[0039] As used herein, the term "derived from" or "derived from" a reference amino acid sequence. An amino acid sequence "derived from" may be a partial or complete match to a reference amino acid sequence. For example, the amino acid sequence of the heavy chain constant region derived from a human immunoglobulin is The amino acid sequence has a similar structure to that of the heavy chain constant region of the wild-type human immunoglobulin from which it is derived. At least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% sequence identity. has.

[0040] As used herein, a "variant" of a polypeptide or amino acid sequence refers to a or one or more amino acid mutations compared to the polypeptide or amino acid sequence from which it is derived. The amino acid mutations include substitutions, deletions, or additions of amino acids. Amino acids within a non-essential region of a polypeptide or protein are generally It will be appreciated by those skilled in the art that suitable conservative amino acid substitutions can be made without altering the biological activity of the compound. As will be appreciated (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / C (See, e.g., Ummings Pub. Co., p. 224). Suitable conservative substitutions are readily determined by those skilled in the art. Some non-limiting examples of common conservative substitutions of amino acid residues are given below: In certain cases, the amino acid substitutions are non-conservative substitutions. To alter the properties of the antibody fragment, e.g., to alter the type of antibody glycosylation modification, interchain To alter the ability to form disulfide bonds or activity for antibody conjugates Amino acid mutations or modifications can be made to the antibody or antibody fragment to provide suitable groups. It will be understood by those skilled in the art that antibodies or antibodies therefor containing such amino acid mutations or modifications can be used. Antigen-binding fragments of the above are also included within the scope of the antibody or antigen-binding fragment thereof of the present invention. [Table 1]

[0041] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" The term generally refers to a small number of nucleic acids, which may consist of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). An oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives. Refers to...

[0042] As used herein, "isolated" refers to a material (e.g., a nucleic acid molecule or polypeptide) that is that the food product (peptide) is separated from the source or environment in which it occurs, i.e., It means that it is substantially free of other components.

[0043] As used herein, a "vector" refers to a vector used to introduce exogenous nucleic acid into a host cell. A vehicle used to introduce a vector into a suitable host cell to transform the host cell. Once transformed, the exogenous nucleic acid is amplified or expressed. Vectors are generally expressed as episomes. However, genes or parts of them can also be designed to be integrated into chromosomes of the genome. As used herein, the definition of vector includes plasmids, linearized plasmids, and the like. Plasmid, viral vector, cosmid, phage vector, phagemid, artificial chromosome (eg, yeast artificial chromosomes and mammalian artificial chromosomes).

[0044] As used herein, an "expression vector" is a vector capable of expressing DNA. refers to a vector in which the DNA is linked to regulatory sequences (e.g. Regulatory sequences include sequences that are operably linked to a promoter, a ribosome binding site, or the like. It may contain promoter and terminator sequences, and may optionally contain an origin of replication, a selection It may also include a selection marker, an enhancer, a polyadenylation signal, etc. The vector, when introduced into a suitable host cell, results in expression of the cloned DNA. The vector may be a plasmid, a phage vector, a recombinant virus or other vector. Expression vectors are well known to those skilled in the art and are capable of replicating in eukaryotic and / or prokaryotic cells. Expression vectors as well as expression vectors that remain episomal or that integrate into the genome of the host cell. The present invention includes an expression vector comprising the

[0045] As used herein, a "host cell" refers to a cell that can accept, maintain, and replicate a vector. A host cell is a cell used to produce or amplify a nucleic acid or vector. The host cell may also be used to express the polypeptide encoded thereby. It may be a eukaryotic cell or a prokaryotic cell.

[0046] As used herein, the term "treatment" refers to a disease / symptom. Amelioration of a condition, e.g., reducing or eliminating a disease / symptom, preventing the onset, exacerbation and / or progression of a disease / symptom. Treatment refers to preventing or delaying the progression or deterioration of a condition. and / or healing.

[0047] An "effective amount" refers to a compound that reduces the severity of the symptoms of a disease, the frequency and duration of asymptomatic periods of a disease, To increase time or prevent injury or disability due to contracting a disease "Effective amount" refers to a sufficient amount to prevent, cure, ameliorate, or prevent a disease or condition. For example, in the treatment of tumors, The antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, or An "effective amount" of a pharmaceutical composition is an amount that reduces tumor cell growth or tumor cell proliferation compared to an untreated subject. Growth is increased by at least about 10%, preferably by at least about 20%, more preferably by at least About 30%, more preferably at least about 40%, more preferably at least about 50%, More preferably, the concentration is at least about 60%, more preferably at least about 70%, and even more preferably Preferably, the inhibition is at least about 80%. Conventional animal models of tumors in the art, such as spontaneous tumors, induced tumors, and The efficacy of the antibody can be evaluated using animal models involving cell culture and transplanted tumors. The ability of the compound to inhibit the growth of the IL-1 protein is tested using in vitro assays well known in the art. The antibody or antigen-binding fragment thereof, antibody conjugate, and immune effector of the present invention can also be used. An effective amount of the vector cell or pharmaceutical composition may be used to reduce tumor size or otherwise improve the condition of the subject. It is possible to alleviate symptoms in (e.g., prevent and / or treat metastasis or recurrence of) Those skilled in the art will be able to determine the appropriate dosage based on, for example, the subject's age, health condition, sex, severity of symptoms, and the particular The effective amount can be determined based on factors such as the composition or route of administration. One or more doses may be administered.

[0048] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" The active ingredient is preferably a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. These refer to excipients and are well known in the art (e.g., Remington's Pharmaceuticals, In: Mechanical Sciences., edited by Gennaro AR, 19th ed., P Ennsylvania: Mack Publishing Company, 1995 (see, for example, US Pat. No. 6,333,623), which include, but are not limited to, pH adjusters, surfactants, Adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for enhancing absorption For example, pH adjusters are not the only Examples of surfactants include, but are not limited to, phosphate buffer. However, cationic, anionic, or nonionic surfactants, such as Tween- 80. Examples of ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, parabens. Examples of antibacterial and antifungal agents include chlorobutanol, phenol, and sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugar, Na Examples of agents that delay absorption include, but are not limited to, Diluents include, but are not limited to, monostearate and gelatin. Although not included, water, aqueous buffers (e.g., buffered saline), alcohols and polyols ( Examples of preservatives include, but are not limited to, glycerol. For example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, Stabilizers include various antibacterial and antifungal agents such as phenol, sorbic acid, etc. It has the meaning commonly understood by those skilled in the art and is intended to stabilize the desired activity of the active ingredient in the drug. Examples of suitable additives include, but are not limited to, monosodium glutamate, gelatin, S PGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose , glucan, or glucose), amino acids (e.g., glutamic acid, glycine), proteins Proteins (e.g., dried whey, albumin or casein) or their degradation products (e.g., lamin, These include progesterone, acetylcholine, and acetylcholine hydrolysates.

[0049] As used herein, examples of mammals include, but are not limited to, humans. , non-human primates, rats, mice, cows, horses, pigs, sheep, dogs, and cats. As used herein, the term "object" refers to a subject, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject ( A subject is a cancer patient, a human or an animal. The terms "target" and "subject" are used interchangeably.

[0050] Anti-Nectin-4 antibody or antigen-binding fragment thereof In one general aspect, the present invention provides an anti-Nectin-4 antibody that specifically recognizes and binds to Nectin-4. A cutin-4 antibody or an antigen-binding fragment thereof is provided.

[0051] Nectin-4 is a full or partial amino acid sequence of human nectin-4 or non-human nectin-4. The nectin-4 may be a protein comprising the amino acid sequence. The nectin-4 is preferably human nectin-4. Human nectin-4 is preferably, for example, Uniprot identification number (ID): Q96NY8 The non-human Nectin-4 may have an amino acid sequence represented by the ... It may be a mammalian nectin-4, such as a primate cynomolgus monkey nectin-4, e.g., U It may have the amino acid sequence represented by niprot identification number (ID): L0N6D9. The non-human nectin-4 may also be, for example, rat nectin-4, e.g., Un The peptide may have the amino acid sequence represented by the iprot identification number (ID): D3ZET1. The non-human nectin-4 may also be, for example, mouse nectin-4, e.g., Uni The antibody may have the amino acid sequence represented by the prot identification number (ID): Q8R007.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof binds to the IgV domain of Nectin-4. In some embodiments, the antibody or antigen-binding fragment thereof is capable of specifically binding to The fragment is capable of specifically binding to the IgV domain of human nectin-4.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to Nectin-4. , it is possible to block its interaction with nectin-1.

[0054] In some embodiments, the anti-Nectin-4 antibody comprises three heavy chain CDRs (CDRH): - A heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3. 1, CDR-H2 and CDR-H3 are selected from the CDRH group in Table 1. [Table 2]

[0055] In some embodiments, the anti-Nectin-4 antibody comprises three light chain CDRs (CDRLs): - comprising a light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3. 1, CDR-L2 and CDR-L3 are selected from the CDRL group in Table 2. [Table 3]

[0056] Complementarity Determining Regions (CDRs) In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs, defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5; CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof. In another particular embodiment, the antibody specifically binds to Nectin-4. Antibodies or antigen-binding fragments thereof are defined according to the Chothia numbering system as follows: The three light chain CDRs below: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof , CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof, The CDR-L3 comprises the sequence, or a variant thereof.

[0057] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs defined according to the Abm numbering system: SEQ ID NO: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 9, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5; CDR-H3 or a variant thereof comprising the sequence set forth in SEQ ID NO:5; In other particular embodiments, an antibody or a variant thereof that specifically binds to Nectin-4 is The antigen-binding fragment is composed of three light chains, defined according to the Abm numbering system: CDR: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence described in SEQ ID NO: 8 R-L3 or a variant thereof.

[0058] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 11 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence described in SEQ ID NO:5. In another particular embodiment, the IL-11 polypeptide is specifically targeted to Nectin-4. Binding antibodies or antigen-binding fragments thereof are defined according to the Kabat numbering system. The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or its barrier a CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; The present invention also includes a CDR-L3 comprising the sequence set forth above or a variant thereof.

[0059] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the IMGT numbering system: No. 13 or a variant thereof, the sequence of SEQ ID NO: 14 or a variant thereof; and CDR-H3 comprising the sequence set forth in SEQ ID NO: 15. or a variant thereof. In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be any of the following three types defined according to the IMGT numbering system: light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof; CDR-L2 or a variant thereof comprising the sequence set forth in sequence number 17, and the sequence set forth in SEQ ID NO:8 or a variant thereof.

[0060] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , (a) Six heavy and light chains defined according to the Chothia numbering system: CDR: CDR-H1 comprising the sequence set forth in SEQ ID NO: 3 or a variant thereof, SEQ ID NO: 4 or a variant thereof, the sequence of SEQ ID NO: 5 CDR-H3 or a variant thereof, and CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (b) the six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO:5 DR-H3 or a variant thereof, CDR-L1 or or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO:7 or a variant thereof , a CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 11 or a variant thereof, SEQ ID NO: 1 2 or 63, or a variant thereof, or a variant thereof, comprising the sequence set forth in SEQ ID NO:6. CDR-L1 or a variant thereof, CDR-L2 or or a variant thereof, a CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof T; or (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, SEQ ID NO: 14 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 15 CDR-H3 or a variant thereof, CDR-L comprising the sequence set forth in SEQ ID NO: 16 1 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof Variant, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof Includes.

[0061] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs, defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 23 In another particular embodiment, the antibody specifically binds to Nectin-4. Antibodies or antigen-binding fragments thereof that bind to the antibody are defined according to the Chothia numbering system. The following three light chain CDRs are included: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof 25 or a variant thereof, and the sequence It comprises a CDR-L3 comprising the sequence set forth in No. 26 or a variant thereof.

[0062] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs defined according to the Abm numbering system: SEQ ID NO: 27 or a variant thereof, the sequence of SEQ ID NO: 28 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23, In another particular embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three classes of antibodies, which are defined according to the Abm numbering system: Light chain CDR: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, SEQ ID NO: No. 25 or a variant thereof, and The CDR-L3 comprises the sequence, or a variant thereof.

[0063] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 29 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be any of the following, as defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof a CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof; The present invention also includes a CDR-L3 comprising the sequence set forth above or a variant thereof.

[0064] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the IMGT numbering system: No. 31 or a variant thereof, the sequence of which is set forth in SEQ ID NO: 32. or a variant thereof; and CDR-H3 comprising the sequence set forth in SEQ ID NO: 33. or a variant thereof. In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be any of the following three types defined according to the IMGT numbering system: light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a variant thereof; CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 35, The CDR-L3 comprises the sequence, or a variant thereof.

[0065] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , (a) Six heavy and light chains defined according to the Chothia numbering system: CDR: CDR-H1 comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, SEQ ID NO: No. 22 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof. CDR-L1 or a variant thereof, CDR-L2 or a CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof T; or (b) the six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 27 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 CDR-H3 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 29 or a variant thereof, SEQ ID NO: 3 0, or a variant thereof, the sequence set forth in SEQ ID NO: 23 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 24 L1 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO:25 a variant thereof, a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26; or (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 31 or a variant thereof, SEQ ID NO: 32 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 33 CDR-H3 or a variant thereof, CDR-L comprising the sequence set forth in SEQ ID NO: 34 1 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof Variant, CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof Includes.

[0066] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs, defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 18 or a variant thereof, or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 23 In another particular embodiment, the antibody specifically binds to Nectin-4. Antibodies or antigen-binding fragments thereof that bind to the antibody are defined according to the Chothia numbering system. The following three light chain CDRs are included: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof 25 or a variant thereof, and the sequence It comprises a CDR-L3 comprising the sequence set forth in No. 26 or a variant thereof.

[0067] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs defined according to the Abm numbering system: SEQ ID NO: 36 or a variant thereof, the sequence of SEQ ID NO: 28 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23, In another particular embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three classes of antibodies, which are defined according to the Abm numbering system: Light chain CDR: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, SEQ ID NO: No. 25 or a variant thereof, and The CDR-L3 comprises the sequence, or a variant thereof.

[0068] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 44 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be any of the following, as defined according to the Kabat numbering system: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof 25, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 26, or a variant thereof.

[0069] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the IMGT numbering system: No. 54, or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 33 In another particular embodiment, the IL-11 polypeptide is specific for Nectin-4. Antibodies or antigen-binding fragments thereof that specifically bind are defined according to the IMGT numbering system. The following three light chain CDRs are included: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof; and further comprising a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof. .

[0070] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , (a) Six heavy and light chains defined according to the Chothia numbering system: CDR: CDR-H1 comprising the sequence set forth in SEQ ID NO: 18 or a variant thereof, SEQ ID NO: No. 22 or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof. CDR-L1 or a variant thereof, CDR-L2 or a CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof T; or (b) the six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 36 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 CDR-H3 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 44 or a variant thereof, SEQ ID NO: 3 0, or a variant thereof, the sequence set forth in SEQ ID NO: 23 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 24 L1 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO:25 a variant thereof, a CDR-L3 comprising the sequence set forth in SEQ ID NO: 26; or (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 54 or a variant thereof, SEQ ID NO: 32 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 33 CDR-H3 or a variant thereof, CDR-L comprising the sequence set forth in SEQ ID NO: 34 1 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof Variant, CDR-L3 comprising the sequence set forth in SEQ ID NO: 26 or a variant thereof Includes.

[0071] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs, defined according to the Chothia numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 39 or a variant thereof, or a variant thereof, CDR-H2 comprising the sequence set forth in SEQ ID NO: 41 In another particular embodiment, the antibody specifically binds to Nectin-4. Antibodies or antigen-binding fragments thereof that bind to the antibody are defined according to the Chothia numbering system. The following three light chain CDRs are included: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof 43 or a variant thereof, and the sequence It comprises a CDR-L3 comprising the sequence set forth in No. 8 or a variant thereof.

[0072] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragment comprises the following three heavy chain CDRs defined according to the Abm numbering system: SEQ ID NO: 45 or a variant thereof, the sequence of SEQ ID NO: 46 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41, In another particular embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three classes of antibodies, which are defined according to the Abm numbering system: Light chain CDR: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof, SEQ ID NO: CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 43, and the sequence set forth in SEQ ID NO: 8. The present invention further comprises a CDR-L3 or a variant thereof comprising the sequence.

[0073] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 In other specific embodiments, including DR-H3 or a variant thereof, Binding antibodies or antigen-binding fragments thereof are defined according to the Kabat numbering system. The following three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof ant, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof, and SEQ ID NO: No. 8 or a variant thereof.

[0074] In certain embodiments, an antibody or its antigen-binding fragment that specifically binds to Nectin-4 The fragments consist of the following three heavy chain CDRs, defined according to the IMGT numbering system: No. 49 or a variant thereof, the sequence of which is set forth in SEQ ID NO: 50. or a variant thereof; and CDR-H3 comprising the sequence set forth in SEQ ID NO:51. or a variant thereof. In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be any of the following three types defined according to the IMGT numbering system: light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO:52 or a variant thereof; CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 53, and or a variant thereof.

[0075] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , (a) Six heavy and light chains defined according to the Chothia numbering system: CDR: CDR-H1 comprising the sequence set forth in SEQ ID NO: 39 or a variant thereof, SEQ ID NO: No. 40, or a variant thereof, CDR-H3 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof. CDR-L1 or a variant thereof, CDR-L2 or or variants thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or variants thereof or (b) the six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 45 or a variant thereof, CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 CDR-H3 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof a CDR-L3 or variant thereof comprising the sequence set forth in SEQ ID NO:8; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof, SEQ ID NO: 4 8 or 64, or a variant thereof, SEQ ID NO: 41 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 42 CDR-L1 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 43 2 or a variant thereof, CDR-L3 or a variant thereof comprising the sequence set forth in SEQ ID NO:8 riant; or (d) the following six heavy and light chain CDRs defined according to the IMGT numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 49 or a variant thereof, SEQ ID NO: 50 or a variant thereof, comprising the sequence set forth in SEQ ID NO: 51 CDR-H3 or a variant thereof, CDR-L comprising the sequence set forth in SEQ ID NO:52 1 or a variant thereof, CDR-L2 comprising the sequence set forth in SEQ ID NO:53 or a variant thereof Variant, CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof Includes.

[0076] Some embodiments of the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 In this study, a variant is at least 80% and at least 85% similar to the sequence from which it was derived. %, at least 90%, at least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 9 8%, at least 99%, or 100% sequence identity with, or derived from Substitution, deletion or addition of one or more amino acids compared to the sequence (e.g., one The substitutions are conservative substitutions. It is preferable that there is.

[0077] Framework Region (FR) In some embodiments, the VH further comprises a heavy chain framework region (FR). In an embodiment, the VL further comprises a light chain framework region (FR). The heavy and / or light chain framework regions may each independently be selected from any species of immunoglobulin. The heavy chain framework regions and the light chain framework regions may be derived from The regions each independently comprise a heavy chain framework region of a human or mouse immunoglobulin and Preferably, it is derived from a light chain framework region. In one embodiment, it is derived from a heavy chain framework region. The heavy and light chain framework regions are each independently derived from a mouse immunoglobulin. heavy and light chain framework regions of human immunoglobulins; Amino acid sequences derived from the light chain framework region and the light chain framework region, or a combination thereof In one embodiment, the heavy chain framework region and the light chain framework region comprise Independently, the heavy and light chain framework regions of a human germline antibody It includes an amino acid sequence derived from the region.

[0078] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention is and / or an antibody or antigen thereof. The VL of the binding fragment contains a light chain framework region derived from a human immunoglobulin. Thus, in certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise: In such an embodiment, the antibody or antigen-binding fragment of the present invention is a humanized antibody or antigen-binding fragment thereof. The heavy chain framework region and / or the light chain framework region of the antigen-binding fragment may be The heavy chain may contain one or more non-human (e.g., murine) amino acid residues, e.g., The framework region and / or the light chain framework region may contain one or more amino acid modifications. These back mutations have corresponding murine amino acid residues.

[0079] In a preferred embodiment, an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-4 The preferred embodiment includes a VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof. In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 has the sequence The VL has the sequence set forth in number 2 or a variant thereof.

[0080] In a preferred embodiment, an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-4 VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof, and The VL has the sequence or a variant thereof.

[0081] In a preferred embodiment, an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-4 The preferred VH comprises a VH having the sequence set forth in SEQ ID NO: 55 or a variant thereof. In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is It includes a VL having the sequence set forth in sequence number 56 or a variant thereof.

[0082] In a preferred embodiment, an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-4 The VH having the sequence set forth in SEQ ID NO: 55 or a variant thereof, and the VH having the sequence set forth in SEQ ID NO: 56 The present invention includes a VL having the sequence set forth above or a variant thereof.

[0083] In yet another preferred embodiment, an antibody that specifically binds to Nectin-4 or an antigen thereof The binding fragment comprises a VH having the sequence set forth in SEQ ID NO: 19 or a variant thereof. In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity is The fragment comprises a VL having the sequence set forth in SEQ ID NO: 20 or a variant thereof.

[0084] In yet another preferred embodiment, an antibody that specifically binds to Nectin-4 or an antigen thereof The binding fragments include a VH having the sequence set forth in SEQ ID NO: 19 or a variant thereof, and a VH having the sequence set forth in SEQ ID NO: No. 20 or a variant thereof.

[0085] In yet another preferred embodiment, an antibody that specifically binds to Nectin-4 or an antigen thereof The binding fragment comprises a VH having the sequence set forth in SEQ ID NO: 57 or a variant thereof. In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity is The fragment comprises a VL having the sequence set forth in SEQ ID NO:58 or a variant thereof.

[0086] In yet another preferred embodiment, an antibody that specifically binds to Nectin-4 or an antigen thereof The binding fragments include a VH having the sequence set forth in SEQ ID NO: 57 or a variant thereof, and a VH having the sequence set forth in SEQ ID NO: No. 58 or a variant thereof.

[0087] In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises a VH having the sequence set forth in SEQ ID NO: 37 or a variant thereof. In a preferred embodiment, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , VL having the sequence set forth in SEQ ID NO: 38 or a variant thereof.

[0088] In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments include a VH having the sequence set forth in SEQ ID NO: 37 or a variant thereof, and a VH having the sequence set forth in SEQ ID NO: 38 or a variant thereof.

[0089] In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises a VH having the sequence set forth in SEQ ID NO: 59 or a variant thereof. In a preferred embodiment, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 is , VL having the sequence set forth in SEQ ID NO: 60 or a variant thereof.

[0090] In another preferred embodiment, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments include a VH having the sequence set forth in SEQ ID NO: 59 or a variant thereof, and a VH having the sequence set forth in SEQ ID NO: 60 or a variant thereof.

[0091] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (1) A VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof and / or a sequence VL having the sequence described in No. 2 or a variant thereof; or (2) VH having the sequence set forth in SEQ ID NO: 19 or a variant thereof and / or a sequence A VL having the sequence set forth in no. 20 or a variant thereof; or (3) VH having the sequence set forth in SEQ ID NO: 37 or a variant thereof and / or a sequence A VL having the sequence set forth in no. 38 or a variant thereof; or (4) VH having the sequence set forth in SEQ ID NO: 55 or a variant thereof and / or a sequence VL having the sequence set forth in no. 56 or a variant thereof; or (5) VH having the sequence set forth in SEQ ID NO: 57 or a variant thereof and / or a sequence A VL having the sequence set forth in no. 58 or a variant thereof; or (6) VH having the sequence set forth in SEQ ID NO: 59 or a variant thereof and / or a sequence VL having the sequence set forth in No. 60 or a variant thereof Includes.

[0092] In certain embodiments of the above, the variant has at least 8 amino acids compared to the sequence from which it is derived. 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or A variant may have one or more amino acid substitutions, deletions, or deletions compared to the sequence from which it is derived. or additions (e.g., substitutions, deletions or additions of one, two, three, four or five amino acids) Preferably the substitutions are conservative substitutions.

[0093] Constant region In some embodiments, the anti-Nectin-4 antibody further comprises a heavy chain constant region. In one embodiment, the anti-Nectin-4 antibody further comprises a light chain constant region. The regions can be independently linked to heavy and light chain constant regions of any species of immunoglobulin. In a preferred embodiment, the heavy chain constant region and the light chain constant region can each independently be derived from A heavy chain constant region or a variant thereof and a light chain constant region or In a more preferred embodiment, the heavy chain constant region and the light chain constant region are derived from a variant thereof. Each region is independently a heavy chain constant region of a human immunoglobulin or a variant thereof, and The variant is derived from the wild-type light chain constant region or a variant thereof. Substitution, deletion or addition of one or more amino acids compared to the sequence (e.g., up to 20, Substitutions, deletions, or additions of up to 15, up to 10, or up to 5 amino acids; e.g., 1, It has 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0094] The heavy chain constant region may comprise at least one of a hinge region, a CH1, a CH2, a CH3, or a combination thereof. In one embodiment, the heavy chain constant region may comprise an amino acid sequence selected from any of the following: In one embodiment, the heavy chain constant region comprises CH1-hinge region-CH2. In one embodiment, the heavy chain constant region comprises CH1-hinge region-CH2-CH3. In embodiments, the heavy chain constant region further comprises a CH4. The heavy chain constant region may be any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), any class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b), or combinations thereof. The heavy chain constant region can be derived from an IgG (e.g., IgG1, IgG2, IgG3, In a preferred embodiment, the heavy chain constant region is derived from the IgG4 heavy chain constant region. The region is derived from the human IgG1 heavy chain constant region.

[0095] The light chain constant region can be derived from a λ (lambda) light chain or a κ (kappa) light chain constant region. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region.

[0096] In a particular embodiment, the heavy chain constant region comprises the sequence set forth in SEQ ID NO: 61 or a variant thereof. In certain embodiments, the light chain constant region comprises the sequence set forth in SEQ ID NO: 62, or a variant thereof. In these embodiments, the variant includes a variant that is less abundant than the sequence from which it is derived. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99%, or 100% sequence identity. Alternatively, the variant may have up to 20 amino acid changes compared to the sequence from which it is derived. Conservative substitutions (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; For example, one, two, three, four or five amino acid conservative substitutions.

[0097] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: (a) the heavy chain comprises a VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof, and the sequence 61 or a variant thereof, and the light chain comprises a heavy chain constant region having the sequence VL having the sequence set forth in sequence number 2 or a variant thereof and the sequence set forth in SEQ ID NO: 62 or a variant thereof; or (b) the heavy chain is a VH having the sequence set forth in SEQ ID NO: 19 or a variant thereof; 61 or a variant thereof, and the light chain comprises VL having the sequence set forth in SEQ ID NO: 20 or a variant thereof and or a variant thereof; or (c) the heavy chain is a VH having the sequence set forth in SEQ ID NO: 37 or a variant thereof and 61 or a variant thereof, and the light chain comprises VL having the sequence set forth in SEQ ID NO: 38 or a variant thereof and or a variant thereof; or (d) the heavy chain is a VH having the sequence set forth in SEQ ID NO: 55 or a variant thereof and 61 or a variant thereof, and the light chain comprises VL having the sequence set forth in SEQ ID NO: 56 or a variant thereof and or a variant thereof; or (e) the heavy chain comprises a VH having the sequence set forth in SEQ ID NO: 57 or a variant thereof and a sequence 61 or a variant thereof, and the light chain comprises VL having the sequence set forth in SEQ ID NO: 58 or a variant thereof and or a variant thereof; or (f) the heavy chain comprises a VH having the sequence set forth in SEQ ID NO: 59 or a variant thereof and a sequence 61 or a variant thereof, and the light chain comprises VL having the sequence set forth in SEQ ID NO: 60 or a variant thereof and or a variant thereof.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an scFv, Fab, Fab ', F(ab')2, Fv fragment, disulfide stabilized Fv (dsFv), diabody, The antibody is selected from the group consisting of a bispecific antibody and a multispecific antibody.

[0099] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention include murine antibodies, chimeric antibodies, or a humanized antibody.

[0100] Mouse Antibody In one embodiment, the anti-Nectin-4 antibody of the present invention is a mouse antibody and comprises a VH and a VL. wherein VH comprises the heavy chain CDRs and heavy chain framework regions described above and VL comprises the heavy chain CDRs and heavy chain framework regions described above. The light chain CDR and the light chain framework region are included. The framework regions are each independently derived from a mouse immunoglobulin framework region. In one embodiment, the anti-Nectin-4 antibody of the present invention comprises a mouse hybrid It is a monoclonal antibody produced by doma cells.

[0101] In certain embodiments, the murine antibody comprises the CDR-H1, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD19, The VH including R-H2, CDR-H3, and mouse immunoglobulin framework regions In a preferred embodiment, the heavy chain framework region comprises an amino acid sequence derived from The antibody comprises a VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof. In certain embodiments, the murine antibody comprises the CDR-L1, CDR-L2, CDR-L3, CDR-L4, CDR-L5, CDR-L6, CDR-L7, CDR-L8, CDR-L9, CDR-L10, CDR-L11, CDR-L12, CDR-L13, CDR-L14, CDR-L15, CDR-L16, CDR-L17, CDR-L18, -L2, VL including CDR-L3, and derived from mouse immunoglobulin framework regions In a preferred embodiment, the method further comprises a light chain framework region comprising an amino acid sequence comprising: The murine antibody comprises a VL having the sequence set forth in SEQ ID NO:2 or a variant thereof.

[0102] In another specific embodiment, the murine antibody comprises a CDR-H1 as set forth in group b of Table 1, VH containing CDR-H2, CDR-H3, and mouse immunoglobulin framework regions In a preferred embodiment, the heavy chain framework region comprises an amino acid sequence derived from The mouse antibody comprises a VH having the sequence set forth in SEQ ID NO: 19 or a variant thereof. In another particular embodiment, the murine antibody comprises a CDR-L1 as set forth in group b of Table 2, VL and mouse immunoglobulin framework regions including CDR-L2 and CDR-L3 In a preferred embodiment, the light chain further comprises a light chain framework region comprising an amino acid sequence derived from The murine antibody comprises a VL having the sequence set forth in SEQ ID NO: 20 or a variant thereof.

[0103] In yet another specific embodiment, the murine antibody is a CDR- VH containing H1, CDR-H2, CDR-H3, and mouse immunoglobulin framework The heavy chain framework region comprises an amino acid sequence derived from a heavy chain framework region. The mouse antibody comprises a VH having the sequence set forth in SEQ ID NO: 37 or a variant thereof. In yet another specific embodiment, the murine antibody is a CDRL listed in group c of Table 2. 1. VL including CDR-L2, CDR-L3, and mouse immunoglobulin framework The preferred embodiment further comprises a light chain framework region comprising an amino acid sequence derived from the region. In one embodiment, the murine antibody comprises a VL having the sequence set forth in SEQ ID NO: 38 or a variant thereof. include.

[0104] In certain embodiments of the above, the variant has at least 8 amino acids compared to the sequence from which it is derived. 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or A variant may have one or more amino acid substitutions, deletions, or deletions compared to the sequence from which it is derived. or additions (e.g., substitutions, deletions or additions of one, two, three, four or five amino acids) Preferably the substitutions are conservative substitutions.

[0105] In a particular embodiment, the murine antibody comprises a VH having the sequence set forth in SEQ ID NO:1 and a VL having the sequence set forth in SEQ ID NO: The VL has the sequence described in No. 2.

[0106] In another particular embodiment, the murine antibody comprises a VH having the sequence set forth in SEQ ID NO: 19 and It comprises a VL having the sequence set forth in SEQ ID NO:20.

[0107] In yet another specific embodiment, the murine antibody has the sequence set forth in SEQ ID NO:37. H and VL having the sequence set forth in SEQ ID NO:38.

[0108] Chimeric antibodies In one embodiment, the anti-Nectin-4 antibody of the present invention is a chimeric antibody. In some cases, the chimeric antibody contains the heavy chain CDRs and / or the light chain CDRs of the above-mentioned mouse antibody. In embodiments, the chimeric antibody comprises the heavy and / or light chain variable regions of the murine antibodies described above. In some embodiments, the chimeric antibody comprises the heavy chain variable region and the light chain variable region of the murine antibody described above. A variable region, and a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is , the heavy chain constant region is derived from a non-mouse immunoglobulin, and / or the light chain constant region is derived from a non-mouse immunoglobulin. It is derived from the light chain constant region of a mouse immunoglobulin. In a preferred embodiment, the heavy chain constant region The constant region of the heavy chain of the human immunoglobulin is derived from a human immunoglobulin heavy chain constant region, and / or the constant region of the light chain of the human immunoglobulin is derived from a human immunoglobulin light chain constant region. In a preferred embodiment, the heavy chain constant region is derived from a human immunoglobulin light chain constant region. The constant region of the light chain is derived from the human immunoglobulin light chain constant region. Coming.

[0109] In some embodiments, the chimeric antibody comprises the CDRs of a murine antibody listed in group a of Table 1. In some embodiments, the chimeric antibody comprises a CDR-H1, a CDR-H2, and a CDR-H3. Further, the CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody described in group a of Included.

[0110] In certain embodiments, the chimeric antibody comprises a VH having the sequence set forth in SEQ ID NO:1 or a variant thereof. In certain embodiments, the chimeric antibody has the sequence set forth in SEQ ID NO:2. VL or a variant thereof.

[0111] In another particular embodiment, the chimeric antibody comprises a VH having the sequence set forth in SEQ ID NO: 19 or In certain embodiments, the chimeric antibody has the sequence set forth in SEQ ID NO: 20, including variants thereof. or a variant thereof.

[0112] In yet another specific embodiment, the chimeric antibody has the sequence set forth in SEQ ID NO:37. H or a variant thereof. In certain embodiments, the chimeric antibody comprises the or a variant thereof.

[0113] In certain embodiments of the above, the variant has at least 8 amino acids compared to the sequence from which it is derived. 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or A variant may have one or more amino acid substitutions, deletions, or deletions compared to the sequence from which it is derived. or additions (e.g., substitutions, deletions or additions of one, two, three, four or five amino acids) Preferably the substitutions are conservative substitutions.

[0114] In some embodiments, the chimeric antibody further comprises a heavy chain constant region and / or a light chain constant region. In a preferred embodiment, the heavy chain constant region is derived from a human immunoglobulin heavy chain constant region. In one embodiment, the light chain constant region is derived from a human immunoglobulin light chain constant region. The chain constant region is derived from a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region. In one embodiment, the light chain constant region is derived from a human lambda or kappa light chain constant region. The constant region is derived from the human kappa light chain constant region. In a particular embodiment, the heavy chain constant region is In certain embodiments, the light chain constant comprises the amino acid sequence set forth in SEQ ID NO:61, or a variant thereof. The constant region comprises the amino acid sequence set forth in SEQ ID NO: 62 or a variant thereof. In morphology, a variant has at least 80% similarity to the sequence from which it is derived, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least or 98%, at least 99%, or 100% sequence identity. A sequence that is a conserved substitution of up to 20 amino acids compared to the sequence from which it is derived (e.g., up to 1 Conservative substitutions of 5, up to 10, or up to 5 amino acids; e.g., 1, 2, 3, Conservative substitutions of 4 or 5 amino acids.

[0115] In certain embodiments, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain is set forth in SEQ ID NO:1. and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61; and a light chain 2 and a VL having the sequence set forth in SEQ ID NO: 2 and a light chain constant having the sequence set forth in SEQ ID NO: 62. Includes the area.

[0116] In another specific embodiment, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain is and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61, The light chain has the VL having the sequence set forth in SEQ ID NO: 20 and the VL having the sequence set forth in SEQ ID NO: 62 Contains the light chain constant region.

[0117] In yet another specific embodiment, the chimeric antibody comprises a heavy chain and a light chain, wherein the heavy chain is No. 37 and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61. and the light chain comprises a VL having the sequence set forth in SEQ ID NO: 38 and a VL having the sequence set forth in SEQ ID NO: 62. The light chain constant region comprises

[0118] Humanized antibodies In one embodiment, the anti-Nectin-4 antibody of the present invention is a humanized antibody and comprises a VH and a VL. wherein VH comprises the heavy chain CDRs and heavy chain framework regions described above, and VL comprises the heavy chain CDRs and heavy chain framework regions described above. The light chain CDR and light chain framework region of the present invention are The framework regions each independently comprise a human immunoglobulin heavy chain framework region and In one embodiment, the heavy chain comprises an amino acid sequence derived from a light chain framework region. The framework regions and / or the light chain framework regions may be derived from one or more non-human (e.g. and / or or the light chain framework region may contain one or more amino acid backmutations, There are mouse amino acid residues that correspond to these back mutations. For example, a mouse or chimeric antibody can be generated. In one embodiment, a humanized antibody can be generated. The antibody is prepared from the anti-Nectin-4 mouse antibody of the present invention as described in the Examples of the present invention.

[0119] In certain embodiments, the humanized antibody comprises the CDR-H1 or barrier of the above-mentioned murine antibody. CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof. In one embodiment, the humanized antibody comprises the CDR-L1 of the above-mentioned murine antibody or a variant thereof, It further includes CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof.

[0120] In certain embodiments, the humanized antibody comprises the CDR-H1 or barrier of the above-mentioned murine antibody. a VH comprising a CDR-H2 or a variant thereof, a CDR-H3 or a variant thereof, and a heavy chain framework comprising an amino acid sequence derived from a human immunoglobulin heavy chain framework region. In yet another specific embodiment, the humanized antibody comprises a humanized antibody having a structure similar to that of the murine CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, CDR-L 3 or a variant thereof, and a VL derived from a human immunoglobulin light chain framework region. The light chain framework region further comprises an amino acid sequence as follows:

[0121] In a particular embodiment, the humanized antibody comprises CDR-H1 as set forth in SEQ ID NO:3, CDR-H2 as set forth in SEQ ID NO:4, CDR-H2 as set forth in SEQ ID NO:5, CDR-H3 as set forth in SEQ ID NO:6, and a human immunoglobulin heavy chain fragment A heavy chain framework region comprising an amino acid sequence derived from the framework region. In yet another particular embodiment, the VH is defined by the thia numbering system. The humanized antibody has CDR-L1 as set forth in SEQ ID NO:6, CDR-L2 as set forth in SEQ ID NO:7, 8, and a CDR-L3 of a human immunoglobulin light chain framework region. The light chain framework regions are derived from the Chothia numbering sequence. The VL further comprises a VL defined by the stem.

[0122] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 9, the CDR-H2 set forth in SEQ ID NO: 10 CDR-H2 as set forth in SEQ ID NO:5, CDR-H3 as set forth in SEQ ID NO:6, and a human immunoglobulin heavy chain A heavy chain framework region comprising an amino acid sequence derived from the framework region. In yet another specific embodiment, the VH comprises a VH defined by the hM numbering system. The antibody comprises CDR-L1 as set forth in SEQ ID NO:6, CDR-L2 as set forth in SEQ ID NO:7, and CDR-L3 as set forth in SEQ ID NO:8. CDR-L3 as set forth in No. 8, and a CDR-L4 derived from a human immunoglobulin light chain framework region. The light chain framework region comprises an amino acid sequence represented by the Abm numbering system. Further included are VLs as defined above.

[0123] In a particular embodiment, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 11, the CDR-H2 set forth in SEQ ID NO: 1 2 or 63, CDR-H3 as set forth in SEQ ID NO: 5, and a human immunoglobulin G A heavy chain framework region comprising an amino acid sequence derived from a purine heavy chain framework region. VH as defined by the Kabat numbering system. In an embodiment, the humanized antibody comprises a CDR-L1 as set forth in SEQ ID NO:6, a CDR-L2 as set forth in SEQ ID NO:7, R-L2, CDR-L3 as set forth in SEQ ID NO:8, and a human immunoglobulin light chain framework The light chain framework region includes an amino acid sequence derived from the light chain framework region, as determined by Kabat numbering. The VL further includes a VL defined by the VL system.

[0124] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 13, the CDR-H2 set forth in SEQ ID NO: 1 4, CDR-H3 as set forth in SEQ ID NO: 15, and a human immunoglobulin a heavy chain framework region comprising an amino acid sequence derived from a heavy chain framework region; In yet another particular embodiment, the VH is as defined by the IMGT numbering system. The humanized antibody has CDR-L1 as set forth in SEQ ID NO: 16, CDR-L2 as set forth in SEQ ID NO: 17, L2, CDR-L3 set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework region The light chain framework region comprises an amino acid sequence derived from the IMGT numbering system. The VL further includes a VL defined by the system.

[0125] In certain embodiments, the humanized antibody comprises a VL having the sequence set forth in SEQ ID NO:56 or its In one embodiment, the humanized antibody has the sequence set forth in SEQ ID NO:55. VH or a variant thereof.

[0126] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 21 or 18, CDR-H2 as set forth in sequence number 22, CDR-H3 as set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a heparin heavy chain framework region Further specific examples include VH as defined by the Chothia numbering system, including In certain embodiments, the humanized antibody comprises a CDR-L1 set forth in SEQ ID NO:24, a CDR-L2 set forth in SEQ ID NO:25, CDR-L2 as set forth in SEQ ID NO: 26, CDR-L3 as set forth in SEQ ID NO: 27, and a human immunoglobulin light chain A light chain framework region comprising an amino acid sequence derived from the framework region, Further included are VLs as defined by the Othia numbering system.

[0127] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 27 or 36, CDR-H2 as set forth in sequence number 28, CDR-H3 as set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a heparin heavy chain framework region In yet another specific embodiment, the VH is defined by the Abm numbering system, In one embodiment, the humanized antibody comprises a CDR-L1 as set forth in SEQ ID NO: 24, a CDR-L2 as set forth in SEQ ID NO: 25, DR-L2, CDR-L3 as set forth in SEQ ID NO: 26, and a human immunoglobulin light chain framework Abm numbering, including light chain framework regions containing amino acid sequences derived from framework regions. The VL further includes a VL defined by the VL system.

[0128] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 29 or 44, CDR-H2 as set forth in sequence number 30, CDR-H3 as set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a heparin heavy chain framework region In yet another specific embodiment, the VH is as defined by the Kabat numbering system, In embodiments, the humanized antibody comprises CDR-L1 as set forth in SEQ ID NO:24, CDR-L2 as set forth in SEQ ID NO:25 CDR-L2 of SEQ ID NO: 26, CDR-L3 of SEQ ID NO: 27, and a human immunoglobulin light chain fragment a light chain framework region comprising an amino acid sequence derived from a Kaba framework region; The VLs are further defined by the t numbering system.

[0129] In certain embodiments, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 31 or 54, CDR-H2 as set forth in sequence number 32, CDR-H3 as set forth in sequence number 33, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a heparin heavy chain framework region Further specific embodiments include VH as defined by the IMGT numbering system, including In an embodiment, the humanized antibody comprises a CDR-L1 as set forth in SEQ ID NO: 34, a CDR-L2 as set forth in SEQ ID NO: 35, CDR-L2, CDR-L3 as set forth in SEQ ID NO:26, and a human immunoglobulin light chain frame A light chain framework region comprising an amino acid sequence derived from the IMGT number It further includes a VL defined by the naming system.

[0130] In certain embodiments, the humanized antibody has a VL having the sequence set forth in SEQ ID NO:58 or its In one embodiment, the humanized antibody has the sequence set forth in SEQ ID NO:57. VH or a variant thereof.

[0131] In a particular embodiment, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 39, the CDR-H2 set forth in SEQ ID NO: 4 0, CDR-H2 as set forth in SEQ ID NO: 41, and a human immunoglobulin a heavy chain framework region comprising an amino acid sequence derived from a heavy chain framework region; In yet another specific embodiment, the VH numbering system is In one embodiment, the humanized antibody comprises a CDR-L1 as set forth in SEQ ID NO: 42, a CDR-L2 as set forth in SEQ ID NO: 43, 8, CDR-L2, CDR-L3 as set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework a light chain framework region comprising an amino acid sequence derived from a Chothia Further included are VLs defined by a numbering system.

[0132] In a particular embodiment, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 45, the CDR-H2 set forth in SEQ ID NO: 4 6, CDR-H3 as set forth in SEQ ID NO: 41, and a human immunoglobulin a heavy chain framework region comprising an amino acid sequence derived from a heavy chain framework region; In yet another particular embodiment, the VH comprises a VH as defined by the Abm numbering system. The humanized antibody comprises CDR-L1 as set forth in SEQ ID NO: 42, CDR-L2 as set forth in SEQ ID NO: 43, 2. CDR-L3 set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework region The light chain framework region comprises an amino acid sequence derived from the Abm numbering system. The VL further includes a VL defined by:

[0133] In a particular embodiment, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 47, the CDR-H2 set forth in SEQ ID NO: 4 8 or 64, CDR-H3 as set forth in SEQ ID NO: 41, and a human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a heparin heavy chain framework region In yet another specific embodiment, the VH is as defined by the Kabat numbering system, In embodiments, the humanized antibody comprises CDR-L1 as set forth in SEQ ID NO: 42, CDR-L2 as set forth in SEQ ID NO: 43 CDR-L2 of SEQ ID NO: 8, CDR-L3 of SEQ ID NO: 9, and a human immunoglobulin light chain frame a light chain framework region comprising an amino acid sequence derived from a Kabat Further included are VLs defined by a numbering system.

[0134] In a particular embodiment, the humanized antibody comprises the CDR-H1 set forth in SEQ ID NO: 49, the CDR-H2 set forth in SEQ ID NO: 5 0, CDR-H3 as set forth in SEQ ID NO:51, and a human immunoglobulin a heavy chain framework region comprising an amino acid sequence derived from a heavy chain framework region; In yet another particular embodiment, the VH is as defined by the IMGT numbering system. The humanized antibody comprises CDR-L1 as set forth in SEQ ID NO: 52, CDR-L2 as set forth in SEQ ID NO: 53, L2, CDR-L3 set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework region The light chain framework region comprises an amino acid sequence derived from the IMGT numbering system. The VL further includes a VL defined by the system.

[0135] In one embodiment, the humanized antibody has a VL having the sequence set forth in SEQ ID NO: 60 or a variant thereof. In one embodiment, the humanized antibody comprises a VH having the sequence set forth in SEQ ID NO:59. or variants thereof.

[0136] In certain embodiments of the above, the variant has at least 8 amino acids compared to the sequence from which it is derived. 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or A variant may have one or more amino acid substitutions, deletions, or deletions compared to the sequence from which it is derived. or additions (e.g., substitutions, deletions or additions of one, two, three, four or five amino acids) Preferably the substitutions are conservative substitutions.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (V H) and / or light chain variable regions (VL), as described above with respect to the humanized antibody. At least one CDR and a CDR sequence different from those of the heavy chain variable region and / or the light chain variable region of the and / or the framework regions contain amino acid mutations, wherein the amino acid mutations are one or more A substitution, deletion or addition of a number of amino acids, or any combination thereof (e.g., one, two or more The substitutions are conserved. It is preferred that the substitution is conservative.

[0138] In some embodiments, the anti-Nectin-4 humanized antibody further comprises a heavy chain constant region. In embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In one embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region. In one embodiment, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 61 or a variant thereof. Including Ant.

[0139] In some embodiments, the anti-Nectin-4 humanized antibody further comprises a light chain constant region. In embodiments, the light chain constant region is derived from a human lambda or kappa light chain constant region. The light chain constant region is derived from the human kappa light chain constant region. In one embodiment, the light chain constant region is It comprises the amino acid sequence set forth in sequence number 62 or a variant thereof.

[0140] In certain embodiments of the above, the variant has at least 8 amino acids compared to the sequence from which it is derived. 0%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; or A variant may contain up to 20 conservative amino acid substitutions ( For example, conservative substitutions of up to 15, up to 10, or up to 5 amino acids; , 2, 3, 4 or 5 amino acid conservative substitutions).

[0141] In certain embodiments, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain is set forth in SEQ ID NO:55. and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61; has a VL having the sequence set forth in SEQ ID NO:56 and a light chain having the sequence set forth in SEQ ID NO:62 Contains a constant region.

[0142] In certain embodiments, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain is set forth in SEQ ID NO:57. and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61; has a VL having the sequence set forth in SEQ ID NO:58 and a light chain having the sequence set forth in SEQ ID NO:62 Contains a constant region.

[0143] In certain embodiments, the humanized antibody comprises a heavy chain and a light chain, wherein the heavy chain is set forth in SEQ ID NO:59. and a heavy chain constant region having the sequence set forth in SEQ ID NO: 61; has a VL having the sequence set forth in SEQ ID NO: 60 and a light chain having the sequence set forth in SEQ ID NO: 62 Contains a constant region.

[0144] The anti-Nectin-4 antibody or antigen-binding fragment thereof of the present invention can be produced using methods known in the art. Such methods can be used, for example, to prepare and produce phage display. Immune libraries, yeast display libraries, immortalized B cells (e.g., mouse B cells Antibodies or antigen-binding fragments are prepared and isolated from the hybridoma cells or EBV-immortalized B cells. Immunizing an animal, for example by isolating an antigen or DNA encoding an antigen. Alternatively, a method for immunizing an animal (e.g., a mouse) using From the animal, antibody-expressing B cells can be isolated. It is preferred to prepare the cells as mortalized B cells, for example as hybridomas or EBV-immortalized B cells. It is preferable to use a nucleic acid molecule or expression vector encoding the antibody or antigen-binding fragment thereof of the present invention. and then transforming a host cell with the anti-Nectin-4 or antigen-binding fragment thereof of the present invention. In some embodiments, the anti-Nectin-4 of the present invention can be expressed from a host cell. Antibodies can be obtained from immunized animals, e.g., human Nectin-4 protein immunized with Obtained from mice that had been cultured.

[0145] multispecific antibodies In one aspect, the present invention relates to an anti-Nectin-4 antibody or an antigen-binding fragment thereof of the present invention, and At least one other antibody or antigen-binding fragment or antibody analogue thereof, thereby Multispecific, capable of binding not only to Chin-4 but also to at least one other antigen An antibody is provided.

[0146] In one embodiment, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. In one embodiment, the multispecific antibody comprises a first scFv (or scdsFv) and a second scFv (or scdsFv), The first scFv (or scdsFv) is a fusion protein of the present invention linked by a short peptide linker. The second scFv (or scdsFv) comprises a short peptide fragment and a VL of another antibody. The VL of the present invention and the VH of another antibody are linked by a tethered linker. The short peptide linker is a peptide having a length of 5 to 10 amino acids that binds to an antigen other than cutin-4. Contains acid residues.

[0147] In one embodiment, the other antigen is a cell surface protein, receptor, or receptor subunit. The cell surface protein can be, for example, an immune cell receptor. The immune cell can be, for example, , immune effector cells, e.g., natural killer (NK) cells, natural killer T (NKT) cells, cytotoxic T (CTL) cells, etc. In a preferred embodiment, The bispecific antibody can bind to both Nectin-4 and PD-1.

[0148] Multispecific antibodies are multivalent antibodies, e.g., bivalent, trivalent, tetravalent or higher. could be.

[0149] In a preferred embodiment, the multispecific antibody is selected from the group consisting of scFv, dsFv, scdsFv, Fa b, scFab, Fab', and F(ab')2. include.

[0150] The multispecific antibody may further comprise an antibody heavy chain constant region and / or a light chain constant region. The heavy chain constant region may comprise at least one of a hinge region, a CH1, a CH2, a CH3, or a combination thereof. The heavy chain constant region may comprise an amino acid sequence selected from any of the following: It is preferable.

[0151] Methods for constructing multispecific antibodies using antibodies or antibody fragments of interest are known to those skilled in the art. It is well known (e.g., International Patent Application Publication No. WO93 / 08829; Suresh et al., 1986)Methods in Enzymology, 121:210;Traun See ecker et al. (1991) EMBO 10:3655-3659).

[0152] Multispecific antibodies can be produced and isolated using a variety of techniques known in the art. For example, recombinant DNA techniques can be used to create nucleic acid molecules encoding multispecific antibodies. The nucleic acid molecule can be obtained by, optionally, cloning the nucleic acid molecule into an expression vector and then infecting a host Transforming a cell with the nucleic acid molecule or expression vector and culturing the transformed host cell under appropriate conditions. and finally, the multispecific antibody is produced in a host cell or It is isolated and purified from the culture medium.

[0153] Fusion proteins In yet another aspect, the present invention provides an antibody or antigen-binding fragment thereof of the present invention, and Fusion proteins comprising biologically active polypeptides are provided.

[0154] In a preferred embodiment, the additional biologically active polypeptide has a therapeutic activity, a binding activity or an enzyme activity. Non-limiting examples of biologically active polypeptides include polypeptides or proteins having biological activity. Examples include, but are not limited to, protein toxins (e.g., diphtheria toxin, enzymes (e.g., urease, horseradish peroxidase), and cytokines. It can be done.

[0155] Nucleic Acids, Vectors and Host Cells In another aspect, the present invention provides a nucleic acid encoding the anti-Nectin-4 antibody or antigen-binding fragment of the present invention. The nucleic acid can be prepared by any method known in the art. For example, phage display libraries, yeast display libraries, , immunized animals, immortalized cells (e.g., murine B cell hybridoma cells, EBV vectors) The antibody can be obtained by isolating it from human B cells (i.e., immortalized B cells) or by chemical synthesis. The nucleic acid molecules of the present invention can be codon-optimized for the host cell used for expression. The nucleic acid molecule can be isolated.

[0156] In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule. In one embodiment, the nucleic acid molecules of the invention are prepared as recombinant nucleic acids. The vector is cloned into an expression vector. The expression vector contains a regulatory sequence and an antibiotic resistance gene. The recombinant nucleic acid may further comprise any additional polynucleotide sequence. Acids can be synthesized by, for example, chemical synthesis, recombinant DNA techniques (e.g., polymerase chain reaction (PCR) The esters can be prepared using techniques well known in the art, such as the Sambroo K, J., EFFritsch, and T. Maniatis. (1989). Mol ecular cloning: a laboratory manual, 2nd e d.Cold Spring Harbor Laboratory,Cold Spr. (See, for example, Sting Harbor, NY). Expression vectors can be used to express a polypeptide or It may also include polynucleotide sequences that encode proteins, where a polypeptide or The protein facilitates detection and / or isolation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins include, but are not limited to, Without being limited thereto, affinity tags (e.g., biotin, polyhistidine tags (His 6), or glutathione S-transferase (GSH) tag), protease cleavage site and reporter proteins (e.g., fluorescent proteins). The nucleic acid molecules of the present invention can be present in one or more vectors. In embodiments, the expression vector is a DNA plasmid, e.g., an expression vector for use in bacterial cells, yeast cells, or mammalian cells. In another embodiment, the expression vector is a DNA plasmid for expression in mammalian cells. In another embodiment, the expression vector is a phage vector. or a phagemid vector.

[0157] The present invention also provides a host cell comprising at least one nucleic acid or vector as described above. In a preferred embodiment, the host cell of the present invention is used to produce the anti-Nectin-4 antibody of the present invention or The antigen-binding fragment is expressed in the host cell. Examples of host cells include, but are not limited to, prokaryotic cells. Cells (e.g., bacteria, e.g., E. coli), eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0158] Bacteria (e.g., E. coli BL21(DE3)) can produce Fv, scFv, Fab and Fab' In some embodiments, the expression of small antigen-binding fragments, such as heavy chain fragments, is particularly advantageous. The nucleic acid molecule encoding the light chain variable region and the light chain variable region is cloned into a single vector, The only mammalian host cells suitable for antibody expression are Examples of suitable cells include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK293 cells), Chinese hamster ovary (CHO) cells and other mammalian cells suitable for antibody expression are available. Some examples include:

[0159] The present invention relates to a method for producing the anti-Nectin-4 antibody or antigen-binding fragment of the present invention in a host cell. A method for producing a (I) Transforming a host cell with at least one nucleic acid or expression vector described herein. and (II) culturing the transformed host cell under appropriate conditions to induce expression of the nucleic acid or expression vector; and (III) isolating the antibody or antigen-binding fragment of the present invention from a host cell or its culture medium and A step of purifying A method is also provided, including:

[0160] In some embodiments, a single vector containing polynucleotide sequences encoding the heavy and light chains is In some embodiments, two vectors are used, one for the antibody. In some embodiments, the host cell encodes an antibody that encodes an antibody light chain and the other encodes an antibody heavy chain. or partners that may help increase the solubility, stability and / or folding of the antibody fragment. Techniques for isolating and purifying antibodies from host cells are known to those of skill in the art. is well known.

[0161] Antibody conjugates The present invention relates to an antibody of the present invention or an antigen-binding site thereof conjugated to at least one therapeutic agent. The antibody drug conjugates (ADCs) include antibody conjugates, including synthetic fragments thereof. A typical type of antibody conjugate, in which the therapeutic agent is, for example, a cytotoxic agent. It's fine.

[0162] In some preferred embodiments, the antibody conjugates of the invention target Nectin-4 positive targets. They are capable of targeting cells and tracking or killing the targeted cells. In a more preferred embodiment, the antibody conjugate of the present invention inhibits Nectin-4 positive cancer cells. In some embodiments, the cancer cells are selected from the group consisting of gastric cancer cells, breast cancer cells, and the like. cells, bladder cancer cells, lung cancer cells, liver cancer cells, colon cancer cells, rectal cancer cells, head and neck cancer cells, and ovarian cancer cells.

[0163] Therapeutic agents include cytotoxic agents, therapeutic antibodies (e.g., antibodies that specifically bind to an additional antigen), and the like. or antigen-binding fragments thereof), radioisotopes, oligonucleotides and their analogues (e.g. Interfering RNA), biologically active peptides, protein toxins (e.g., diphtheria toxins, ricin) and enzymes (e.g., urease). A cytotoxic agent is an agent that inhibits or reduces the activity or function of cells and / or damages cells. Cytotoxic agents refer to substances that kill cells. Examples of cytotoxic agents include, but are not limited to: Maytansinoids (e.g., maytansine), auristatins (e.g., MMAF, MMAE, MMAD), duostatin, cryptophycin, bin Alkaloids (e.g., vinblastine, vincristine), colchicine, dolastatin , taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, Cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihy methadone, doxorubicin), cytotoxic antibiotics (e.g., mitoxantrazole, actinomycin, duocarmycin (e.g., CC-1065), auromycin mycin, duomycin, calicheamicin, endomycin, phenomycin), Xorubicin, daunorubicin, calicheamicin, cisplatin, ethidium bromide, Rheomycin, mitomycin, mithramycin, pladienolide, podophyllotoxin , etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine , mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, cal Mustine, lomustine, methotrexate, melphalan, teniposide, glucocorticoids Examples include Ido.

[0164] Radioisotopes can be, for example: 212 Bi, 213 Bi, 131 I, 125 I, 111 I n, 177 Lu, 186 Re, 188 Re, 153 Sm, 90 You can choose from Y Antibodies labeled with radioisotopes are sometimes called radioimmunoconjugates. do.

[0165] In one embodiment, the therapeutic agent is a molecule with anti-tumor biological activity. Molecules that inhibit the proliferation of cells include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radioactive agents, and the like. Antibodies targeting tumor-specific antigens, immune checkpoint inhibitors, and other anti-tumor In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In a preferred embodiment, the therapeutic agent is a radioisotope. teeth, [ka] It is.

[0166] The therapeutic agent and the antibody or antigen-binding fragment of the invention are combined using any technique known in the art. The conjugation can be via a linker. The linker is a covalent conjugate. Reactive groups for coupling, e.g., amines, hydroxylamines, maleimides, carbo The alkyl group may include an alkyl, aryl, phenyl, thiol, sulfhydryl, or hydroxyl group. The linker may be cleavable or non-cleavable. A cleavable linker is, for example, For example, an enzymatically cleavable linker (e.g., a peptide containing a protease cleavage site). , pH-sensitive linkers (e.g., hydrazone-type linkers), or reducible linkers (e.g., For example, disulfide bonds.

[0167] In one embodiment, the linker is an amine, a hydroxylamine, a maleimide, a carboxyl aryl, phenyl, thiol, sulfhydryl, and hydroxyl groups. In one embodiment, the linker is a chemical bond. In one embodiment, the linker is a The carrier contains an amino acid or a peptide consisting of 2 to 10 amino acids. It may be a natural acid or a non-natural amino acid.

[0168] In a preferred embodiment, the linker is Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, [ka] (wherein one of positions 1 and 2 is a linker that is an antibody or antigen-binding fragment thereof of the present invention. and the other is the position where the linker is attached to the therapeutic agent. is selected from the group consisting of:

[0169] In some embodiments, the therapeutic agent and the linker are conjugated to form an intermediate, and then the In some embodiments, the antibody or antigen-binding fragment of the invention is conjugated to the The intermediate forms a thioether bond with a sulfhydryl group of the antibody or antigen-binding fragment of the present invention. The structures and preparation methods of such intermediates and Methods for preparing antibody conjugates using the same are described, for example, in the entire relevant section. No. WO2019 / 114666, which is incorporated herein by reference. It is listed.

[0170] In one embodiment, the antibody conjugate has formula (I):

number

[0171] In certain embodiments, the (LD) moiety has the structure of formula (1) A thioether bond is formed between the antigen-binding fragment of the compound of formula (1) and the sulfhydryl group of the compound of formula (1). The two are linked by the creation of [ka] The antibody conjugate of the present invention has the formula (2): [ka] (wherein A is an antibody or antigen-binding fragment of the invention, γ is a sulfhydryl group of A and The number of (LD) moieties that are linked to A by forming a thioether bond, from 1 to (an integer between 0 and 10) In a particular embodiment, A is an anti-human Nectin-4 humanized monoclonal antibody of the present invention. It is a local antibody.

[0172] In yet another aspect, the invention provides a composition comprising an antibody conjugate of the invention. In one embodiment, the therapeutic agent in the composition and the anti-Nectin-4 antibody or its antigen-binding activity of the present invention are The molar ratio of the fragments (DAR value) may be a decimal or integer value between 1 and 10, for example between 1 and 8. Decimals or integers, e.g., 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3 .3, 3.4, 3.5, 3.6, 3.7, 3.79, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0 , 7.03, 7.1, 7.12, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7 For example, .8, 7.9 or 8.0.

[0173] CAR and CAR-expressing immune effector cells Using the antibodies of the present invention or antigen-binding fragments thereof to construct chimeric antigen receptors (CARs) Thus, in another aspect, the present invention provides a chimeric antigen receptor (CAR) Also provided.

[0174] As used herein, the term "chimeric antigen receptor (CAR)" refers to, e.g., Targeting (e.g., cancer) by antigen-binding fragments that specifically bind to antigens on the surface of cancer cells It specifically recognizes and binds to the target antigen (antigen expressed on the surface of a cell) and attaches a CAR to its surface. can confer targeting specificity to the cells in which they are expressed (e.g., immune effector cells); CAR is an artificial receptor. When a target is recognized by CAR, immune effectors expressing CAR are activated. Preferably, this results in activation and / or expansion of the target cells.

[0175] In some embodiments, the chimeric antigen receptor is a chimeric antigen receptor of the invention that specifically binds Nectin-4. An antibody or an antigen-binding fragment thereof (e.g., scFv), a transmembrane domain, and one or more In such an embodiment, the invention provides a nucleic acid molecule comprising an intracellular T cell signaling domain of Also provided is a method for the preparation of a chimeric antigen receptor comprising the steps of: The nucleotide sequence of the chimeric antigen receptor can be used to identify the antibody of the present invention or its antigen-binding activity. It further includes a nucleotide sequence encoding a fragment (e.g., an ScFv). In one embodiment, the nucleic acid molecule of the present invention encodes an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. The nucleic acid molecule can be isolated that encodes a chimeric antigen receptor comprising the nucleic acid sequence of

[0176] In yet another aspect, the present invention provides a method for the preparation of a CAR of the present invention comprising administering to a subject a CAR-containing medicament ... on a surface of the subject a CAR-containing medicament. Also provided are immune effector cells that have been isolated from the human T lymphocytes. The immune effector cells include T lymphocytes (e.g., Cytotoxic T cells (CTL), natural killer cells (NK) and natural killer T Preferably, the immune effector cells are selected from the group consisting of NKT cells. Targeting Nectin-4 positive disease cells (e.g., Nectin-4 positive cancer cells) , which are activated and exert effector functions that, for example, cause the death of Nectin-4-positive cancer cells. Get started.

[0177] In yet another aspect, the present invention provides a nucleic acid molecule encoding a CAR, an expression vector comprising the nucleic acid molecule, The present invention also relates to CAR-expressing immune cells (e.g., CTLs), as well as CAR-expressing vectors.

[0178] Pharmaceutical Compositions The present invention relates to antibodies or antigen-binding fragments, antibody conjugates, compositions, CAR expressing Immune effector cells, multispecific antibodies or fusion proteins of the invention, and pharma- ceutical Pharmaceutical compositions comprising acceptable carriers and / or excipients are also provided.

[0179] The pharmaceutical compositions provided by the present invention may include, but are not limited to, solid, semi-solid, liquid, It can be in a variety of forms, including a powder or lyophilized form.

[0180] Depending on the dosage form, pharma- ceutically acceptable carriers and / or excipients include, but are not limited to, However, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, flow agents, etc. Enhancers, sweeteners, coatings, structure-forming excipients, preservatives, antioxidants (e.g., ascorbyl phosphate, Bis(2-methyl-2-phenylpropanediol), cysteine ​​hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite Sodium, Ascorbyl Palmitate, Butylated Hydroxyanisole (BHA), Butyl Hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, Citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.) , solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid, and syrups) , buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., non-ionic surfactants, e.g., polysorbate 80, polysorbate 20, poloxamer or polyethylene glycol), antibacterial agents, antifungal agents, isotonicity agents (e.g. For example, trehalose, sucrose, mannitol, sorbitol, lactose, glucose ), absorption delaying agents, chelating agents and emulsifying agents.

[0181] The pharmaceutical compositions may be administered intravenously, intramuscularly, subcutaneously, parenterally, spinally or epidermally (e.g., by injection or Preferably, the antibody or antigen-binding fragment thereof is suitable for administration via injection. For compositions containing antibody conjugates, preferred dosage forms are generally, for example, injectable solutions, The composition may be a lyophilized powder. For compositions, suitable carriers and / or excipients include buffers (e.g., citrate buffer, acetate buffer, etc. buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonicity agent (e.g. trehalose, sucrose, mannitol, sorbitol, lactose, glucose), Non-ionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer) -) or a combination thereof.

[0182] Pharmaceutical compositions are generally sterile and stable under the conditions of manufacture and storage. An active substance (e.g., an antibody or an antibody conjugate of the present invention) is mixed in a suitable solvent, and the above Add one or a combination of the above carriers and / or excipients, and then sterilize, if desired. Sterile injection solutions can be prepared by microfiltration using a sterile filter. The active ingredient is then mixed with a sterile carrier containing a basic dispersion medium and the required other ingredients from those enumerated above. and / or by incorporating it into an excipient. Sterile powder for preparing a sterile injection solution. In the latter case, the preferred method of preparation is vacuum drying and freezing the previously sterile filtered solution. Drying (lyophilization) to obtain a powder of the active ingredient, in combination with any additional desired ingredients. This includes obtaining.

[0183] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art. For example, it can be administered by systemic or local administration. Parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity) , topical, epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local administration) The exact dosage to be administered may vary depending on, for example, the metabolic characteristics of the pharmaceutical composition, the duration of treatment, and other factors. the duration, the rate of excretion of the particular compound, the purpose of the treatment, the route of administration, as well as the age, health, and weight of the patient. The various factors that may be considered include the condition of the subject, such as sex, diet, medical history, and other factors well known in the medical field. As will be appreciated by those of skill in the art, the method of administration will depend on factors such as injection or infusion. There is a match.

[0184] As a general guideline, the antibody conjugate of the present invention may be administered in a concentration of about 0.0001 to 100 mg / mL. The drug is administered at a dosage ranging from 0.01 to 20 mg / kg, or more commonly, 0.01 to 20 mg / kg of subject body weight. For example, the dosage administered can be 0.3 mg / kg body weight, 1mg per g, 3mg per kg, 5mg per kg, 10 mg or 20 mg per kg of body weight, or in the range of 1 to 20 mg per kg of body weight Exemplary treatment regimens required include once a week, once every two weeks, once every three weeks, once a week, once every 4 weeks, once a month, once every 3 months, once every 3-6 months, or once after the first dose. There may be a slight shortening of the drug interval and then a longer drug interval. There is a match.

[0185] Nectin-4-related diseases The antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, The multispecific antibody, fusion protein or pharmaceutical composition, or a medicament prepared therefrom, is used to treat a disease. The present invention can be used for the treatment and / or prevention of diseases associated with Nectin-4. The disease is preferably associated with abnormal expression of Nectin-4. The term "normal expression" refers to the expression of a protein in a sample that is in a normal state (or a target state). Subsamples, e.g., from subjects not suffering from a disease associated with abnormal expression of Nectin-4 This refers to a level that is too high or too low compared to the reference value (sample).

[0186] Preferably, the disease is characterized by high expression of Nectin-4. For example, the disease of a subject having or suspected of having a disease (e.g., bladder cancer) Nectin-4 is highly expressed in tissues (e.g., bladder) whereas it is not expressed in non-diseased counterparts. Nectin-4 is not expressed or is expressed at low levels in elephant tissues.

[0187] The disease is preferably cancer. The cancer is preferably stomach cancer, liver cancer, hepatocellular carcinoma, or bladder cancer. , urothelial cancer, urethral cancer, renal pelvis cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, ductal cancer, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, and rectal cancer and esophageal cancer.

[0188] Cancer treatments include, but are not limited to, measures of tumor regression, tumor weight or size. change in size, time to progression, time to survival, progression-free survival, response rate, duration of response, quality of life , protein expression and / or activity, etc.

[0189] Diagnostic and Treatment Uses The anti-Nectin-4 antibody or its antigen-binding fragment or antibody conjugate of the present invention is For detecting, diagnosing or monitoring diseases and / or disorders associated with chin-4, For example, the anti-Nectin-4 antibody or its The antigen-binding fragment or antibody conjugate can be detected in situ, in vivo, ex vivo, For use in ivo and in vitro diagnostic or imaging assays It is possible.

[0190] In some embodiments, the method comprises detecting the expression level of Nectin-4 in a subject, and / or Alternatively, the antibody or antigen-binding fragment of the present invention can be used to track cells expressing Nectin-4. It can be conjugated with at least one label to obtain a labeled antibody. Such labeled antibodies are also included within the scope of the antibodies or antigen-binding fragments of the present invention. These include enzymes (e.g., horseradish peroxidase), fluorescent dyes, radioisotopes, biotin, Preferably, the label is selected from the group consisting of fluorine, fluorine-containing polymer, and colloidal gold. Techniques for testing are well known to those skilled in the art.

[0191] In one aspect, the present invention provides a method for detecting the presence of Nectin-4 in a sample or detecting the expression of Nectin-4. 1. A method for determining a current level, comprising: (a) contacting a sample with the antibody or antigen-binding fragment of the present invention, or a labeled antibody; Top and (b) detecting the formation of an immune complex between the antibody or antigen-binding fragment and Nectin-4 in the sample; or the amount thereof to detect the presence of Nectin-4 in the sample or to detect the expression of Nectin-4. Steps to determine the current situation The present invention provides a method comprising:

[0192] The present invention provides a method for diagnosing a disease associated with Nectin-4 in a subject, comprising: (a) obtaining a sample of a subject; (b) detecting nephrons in a sample using an antibody or antigen-binding fragment or a labeled antibody of the present invention; detecting the expression level of tyrosine-4; (c) comparing the expression level of Nectin-4 with the expression level of a reference protein; and detecting an increase or decrease in the expression level of Nectin-4 compared to the expression level of a reference protein. Also provided is a method, wherein abnormal expression of Nectin-4 is demonstrated.

[0193] The reference protein may be nectin-4 in a standard sample, and the standard sample may be, for example, The sample may be derived from a subject who is not affected by a disease associated with abnormal expression of Nectin-4. The sample may be in any form, such as cells, tissues, or body fluids. These include, but are not limited to, Western blotting, flow cytometry, and immunohistochemistry. using techniques known in the art, including immunohistochemistry (IHC), immunohistochemistry (ELISA), and immunohistochemistry (ELISA). This can be implemented.

[0194] The present invention relates to the use of an antibody or an antigen-binding fragment thereof of the present invention in the treatment and / or prevention of a disease. , antibody conjugates, immune effector cells, multispecific antibodies, fusion proteins or pharmaceuticals The invention also relates to the use of pharmaceutical compositions.

[0195] The present invention relates to a method for the preparation of a medicament for use in the treatment and / or prevention of a disease, comprising administering to said patient a compound of the present invention. Antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, multispecific The present invention also relates to the use of the antibody, fusion protein or pharmaceutical composition of the present invention. The cancer is as described in the specification.

[0196] The antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, The multispecific antibody, fusion protein or pharmaceutical composition may be administered with any of the therapeutic agents described herein. The combination of the agent can be administered in combination with at least one other agent, or in combination with at least one other agent. For example, the above therapeutic agents can be administered all at the same time, or separately. If administered separately (when the dosing regimens differ from each other), administer consecutively without interruption. The doses can be administered either intravenously or at predetermined intervals.

[0197] Treatment The present invention relates to a method for treating a disease associated with Nectin-4 in a subject in need thereof. In one embodiment, the disease is a cancer as described herein.

[0198] The method includes administering to a subject an antibody or antigen-binding fragment thereof, an antibody conjugate, a composition of the invention, , immune effector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions in a therapeutically effective amount. The method includes administering

[0199] In certain embodiments, the methods of the present invention include surgery, radiation therapy, chemotherapy, targeted therapy, and the like. a therapeutic approach selected from the group consisting of chemotherapy, immunotherapy, hormone therapy, anti-angiogenic therapy, and palliative care; The method further includes administering a therapeutic regimen.

[0200] In certain embodiments, the methods of the present invention include administering to a subject a chemotherapeutic agent, a radioisotope, an immune checkpoint inhibitor, or a combination thereof. and selected from the group consisting of cross-linking inhibitors, antibodies targeting other antigens, and other anti-tumor drugs. The method further includes administering one or more therapeutic agents to the patient, such as, for example, chemotherapeutic agents. For example, antimetabolites, alkylating agents, cytotoxic agents, topoisomerase inhibitors, microtubule inhibitors Other antitumor drugs include, for example, angiogenesis inhibitors, deacetylase inhibitors, HDAC inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors , p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors (e.g., bortezomib imib, carfilzomib, ixazomib, marizomib, oprozomib) and tyrosinequinone Examples of chemotherapeutic agents include: These include, but are not limited to, cyclophosphamide, ifosfamide, melphalan, and busulfan. Rufan, chlormethine, chlorambucil, lomustine, carmustine (BCNU), C CNU, cisplatin (DDP), carboplatin (CBP), oxaliplatin (OX A), methotrexate (MTX), 6-mercaptopurine (6-MP), 5-fluoro Uracil (5-FU), cytarabine, gemcitabine, vinblastine, vincristine, Vindesine, camptothecin, irinotecan, topotecan, rubitecan, etoposide, tetanus Niposide, Paclitaxel, Taxane, Docetaxel, Paclitaxel liposome, Ac Tinomycin D, idarubicin, doxorubicin, epirubicin, mitomycin, ble These include ramycin, doxorubicin, and epirubicin. Immune checkpoint inhibitors These include, but are not limited to, PD-1, PD-L1, CTLA4, and LAG-3. and an antibody or an antigen-binding fragment thereof that specifically binds to TIM-3 or VISTA. Examples of radioisotopes include: 212 Bi, 213 Bi, 131 I, 125 I, 111 In, 177 Lu, 186 Re, 188 Re, 153 Sm, 90 Y mentions the following: can.

[0201] In some embodiments, the antibodies or antigen-binding fragments thereof, antibody conjugates, immunoglobulins, and the like of the present invention are The immunoeffector cells, multispecific antibodies, fusion proteins or pharmaceutical compositions are combined with chemotherapeutic agents. In one embodiment, the chemotherapeutic agent is cisplatin, oxaliplatin , gemcitabine, cyclophosphamide, 5-fluorouracil, and combinations thereof In another embodiment, the antibody conjugate of the present invention is selected from the group consisting of: In one embodiment, the present invention is used in combination with an immune checkpoint inhibitor. anti-PD-L1 antibody or antigen-binding fragment thereof, anti-PD-1 antibody or antigen-binding fragment thereof In yet another embodiment, the antibody of the present invention is selected from the group consisting of antibody or antigen-binding fragment thereof, antibody conjugate, immune effector cell, multispecific antibody The antibody, fusion protein or pharmaceutical composition is used in combination with a radioisotope.

[0202] kit The present invention relates to the antibodies or antigen-binding fragments, antibody conjugates, labeled antibodies, polyclonal antibodies, and the like of the present invention. Bispecific antibodies, fusion proteins, pharmaceutical compositions or immune effector cells, and uses The kit may also include a suitable container. In some embodiments, the kit further comprises a device for administration. This also includes labels used to indicate the intended use and / or method of use of a container. The term "kit" refers to any item on or accompanying the kit, or otherwise supplied with the kit. "Contract" includes any written or recorded material relating to the proceedings.

[0203] Beneficial effects In a particular aspect, the present invention relates to an anti-Nectin-4 antibody or an antigen-binding fragment thereof, particularly an anti-Nectin-4 antibody or an antigen-binding fragment thereof. The present invention provides a humanized monoclonal antibody against Nectin-4. High affinity: in the examples, ELISA, biofilm interference technique and flow The binding of the antibodies of the present invention to Nectin-4 can be measured using various methods, such as cytometry. The affinity was detected, and the results showed that the anti-Nectin-4 antibody of the present invention has an in vitro affinity. It has high affinity for both engineered nectin-4 and cell surface nectin-4. Surprisingly, the humanized monoclonal antibodies 31HZ and 56H The dynamic affinity of Z and 74HZ for human nectin-4 and cynomolgus nectin-4 was All were better than the prior art Enfortumab, which demonstrated that the antibodies of the present invention are in v It was shown that ivo-binding activity has a higher affinity for nectin-4, This also indicates that the endocytic activity of the antibodies 31HZ, 56HZ and 74HZ is greater. Furthermore, the antibody of the present invention has high specificity for Nectin-4. Isomerism: 31HZ, 56HZ and 74HZ all specifically recognize human nectin-4 However, it does not recognize nectin-1, nectin-2, or nectin-3. The antibody blocked the binding of nectin-4 to nectin-1, and the 31HZ, 56HZ, and 74HZ The competitive binding ability of for nectin-4 is equivalent to that of enfortumab.

[0204] In another specific embodiment, the present invention provides an anti-Nectin-4 antibody capable of targeting Nectin-4 positive cells. Also provided is a Cutin-4 antibody conjugate. The antibody conjugate of the present invention has high affinity It can target Nectin-4 positive cells with high affinity and specificity. This reduces off-target toxic effects and side effects, This is very useful for cancer treatment or in vivo diagnosis. This point is also confirmed in the Examples. The ADC of the present invention is capable of targeting human breast ductal cancer cells and human gastric cancer cells that endogenously express human nectin-4. It has potent killing activity against various tumor cell lines, including human breast cancer cells and human ovarian cancer cells. Furthermore, in a mouse xenograft model of non-small cell lung cancer, the ADC of the present invention showed significant tumor inhibition. It is active and has a good safety profile. EXAMPLES

[0205] The following examples are merely illustrative of the invention and therefore should not be construed as limiting the scope of the invention. The following examples are not to be construed as limiting the scope of the present invention. Test methods were performed according to conventional methods and conditions or according to the product instructions.

[0206] Example 1: Preparation of anti-human Nectin-4 humanized monoclonal antibody 1.1 Construction of cell lines overexpressing human nectin-4 To verify the specificity and function of the human nectin-4 antibody, the complete human nectin-4 antibody was Coding sequence (Uniprot ID: Q96NY8-1) (Nanjing GenSc (synthesized by Ript Biotechnology Co., Ltd.) The vector was cloned into the antiviral vector pLVX-IRES-puro and then analyzed by the Moha mmadi Z et al. Mol Biotechnol. 2015 Sep;57(9):79 Virus was prepared using the lentiviral packaging system described in (3-800). The resulting virus was used to infect A549 non-small cell lung cancer cells (ATCC) and T24 Human bladder transitional cell carcinoma cells (ATCC) were infected with each of these vectors and subjected to puromycin screening. By gene expression and monoclonal selection, we isolated a monoclonal A clone overexpressing human nectin-4. 549-nectin-4 and T24-nectin-4 stable cell lines were obtained.

[0207] 1.2 Detection of cell lines overexpressing human nectin-4 A549-nectin-4 stable cell line and T24-nectin-4 stable cell line were analyzed by flow cytometry. Flow cytometry (Flow cytometer: Beckman, CytoFlex; detection antibody enzyme Nfortumab, a sequence described in International Patent Application Publication No. WO2012 / 047724 These cells were identified by the expression of human nectin-4. As shown in Figures 1A and 1B, the flow cytometry results showed that A Both 549-nectin-4 and T24-nectin-4 had high positive rates (almost 100%). Thus, both A549-nectin-4 and T24-nectin-4 have It was shown to be a sufficiently homogeneous and stable cell line that can be used for further experiments.

[0208] 1.3 Preparation of anti-human nectin-4 mouse monoclonal antibody Wild-type mice were immunized with the protein and were then treated with anti-human nectin-4 mouse antibodies. The monoclonal antibody was obtained by emulsifying human IgG1 in complete Freund's adjuvant (CFA). Nectin-4 protein (Sino Biological, Cat:19771-H0 8H) were used to culture two 5-6 week-old Balb / c mice and two C57 mice. Specifically, 50 μg of protein was used for subcutaneous primary immunization, followed by Every 2 weeks, the mice were given nectin-4 protein emulsified with incomplete Freund's adjuvant (IFA). The mice were boosted intraperitoneally with 25 μg of protein, and after three boosts, Tumor cells T47D (a type of human breast cancer cell line) that endogenously express 6 Use individually The final intraperitoneal booster was administered using the same antibody, and spleen cells were obtained 3 to 5 days later and hybridized. Mouse spleen cells were used as the vector for fusion. Sp2 / 0 cells (ATCC, Cat# CRL-1581) were used as the vector for fusion. ) mouse myeloma cell line was fused with PEG using standard fusion procedures, and then HAT Perform pressure screening using ELISA and ELISA screening after 10-14 days. As a result, about 200 positive clones that bound to the Nectin-4 protein were obtained. Further screening by cytometry revealed that T47D tumor cells were recognized. Three positive hybridoma clones capable of carrying out the above-mentioned steps were obtained for subcloning, and finally Then, subcloning selection was performed by limiting dilution to obtain single clones, and their numbers were were 31#, 56#, and 74#, respectively.

[0209] 1.4 Affinity detection of anti-human nectin-4 mouse antibody The affinity of the candidate molecules to human nectin-4 was detected by ELISA. Lymphoblastoid clones 31#, 56# and 74# were cultured without serum, and the supernatants were 0 mL was obtained and then purified with Protein A (MabSelect SuRe, GE). The mouse antibody was obtained by purification.

[0210] Isolated and purified from the culture supernatants of hybridoma cell lines 31#, 56# and 74# The affinity of the mouse monoclonal antibody was determined by ELISA. Sino Biological, Cat:19771-H08H) was diluted with carbonate (CBS). Dilute to 1 μg / mL in buffer and add to 96-well plate in a volume of 100 μL / well The plates were then placed at 4°C for 16-20 hours. The CBS buffer in the 96-well plate was then pipetted. and replace the plate with PBST (pH 7.4, containing 0.05% Tween 20). Wash once with PBST (blocking solution) and once with 2% nonfat dry milk (PBST blocking solution). ) at 200 μL / well and incubate at room temperature for 1 hour for blocking. The blocking solution was removed and the plate was washed three times with PBST buffer. Nectin-4 mouse antibody was diluted to an appropriate concentration in PBST / 2% skim milk powder and then plated. The plates were incubated at room temperature for 1.5 hours. The reaction mixture was removed, the plate was washed three times with PBST, and diluted with PBST / 1% nonfat dry milk ( Dilution ratio 1:5000) HRP-conjugated goat anti-mouse IgG secondary antibody (Jackson (purchased from Laboratory) was added at 50 μL / well and incubated at room temperature for 1 hour. The plate was washed three times with PBST, and then TMB was added at 100 μL / well. The mixture was incubated at room temperature for 5-10 minutes for color development. Color development was stopped by adding 1 μL / well of 100 mM NaCl. The optical density (OD) was detected at dual wavelengths of 450 / 620 nm.

[0211] The detection results are shown in Figure 2 and Table 3. All three anti-human Nectin-4 mouse antibodies can bind to human nectin-4 protein with high affinity and is involved in EC 50 is 30~ It ranged from 70 pM. [Table 4]

[0212] 1.5 Amplification of the variable region of anti-Nectin-4 mouse monoclonal antibody The hybridoma cells are cultured to approximately 8,000 to 10,000 cells, and then the cells are The first strand cDNA was dissolved and purified using a cDNA reverse transcription kit (Thermo Fisher Scientific) The VH and VK genes were synthesized using cDNA. Amplify by PCR using primers from A, and purify the PCR product using a DNA purification kit (Q The DNA was purified by ELISA (Cat. No. 28104) and ligated into the PUC57 vector. Approximately five clones were picked from each ligation reaction and sequenced. The sequences were analyzed using Vector NTI 11.5 (Thermo Fisher Sci. ) and Sequencer 5.4.6 (Genecodes). The variable region sequences and CDR sequences of the Tonectin-4 mouse antibody were submitted to IMGT and abYsis. The results are shown in Table 4 below. [Table 5]

[0213] 1.6 Humanization of anti-human nectin-4 mouse antibody Murine antibodies 31#, 56#, and 74# were humanized by CDR grafting. In summary, humanization involves the following steps: 1. Introduction of mouse monoclonal antibodies The amino acid sequence was aligned with the amino acid sequence of a human germline antibody to determine high homology and We found sequences with better physicochemical properties as human germline framework sequences. We analyzed and investigated HLA-DR affinity and identified human germline variants with low affinity. A framework sequence was selected; then the six CDRs of the mouse antibody were cloned into the selected heavy and The light chain framework sequences were grafted into each other.

[0214] Furthermore, using computer simulation techniques and using molecular docking The framework amino acid sequences of the variable region and its surrounding regions were analyzed, and the spatial arrangement of the three regions was determined. The dimensional binding modes were investigated: electrostatic forces, van der Waals forces, hydrophilic and hydrophobic forces, and elastomeric forces. By calculating the entropy value, the spatial fractal dynamics of the Nectin-4 protein was determined. The framework can be maintained by resolving important amino acids in the mouse antibody amino acid sequence. The mouse antibody was analyzed and these mouse amino acids were retained in the grafted antibody. In order to maximize the retention of the antigen-binding ability of the humanized antibody, A series of back mutations were performed on the amino acid residues.

[0215] According to the above method, humanized antibodies based on the CDRs of mouse antibodies 31#, 56# and 74# were prepared. The humanized antibodies were named 31HZ, 56HZ, and 74HZ, respectively. The heavy chain constant region of each antibody is a human IgG1 heavy chain constant region (SEQ ID NO: 61), and each antibody The light chain constant region of was a human kappa light chain constant region (SEQ ID NO: 62). The light chain CDR amino acid sequences of 31HZ, 56HZ and 74HZ are 31#, 56# and 74HZ, respectively. The light chain CDRs of the constructed humanized antibodies 31HZ, 56HZ and 74HZ were identical to those of the humanized antibodies 31HZ, 56HZ and 74HZ. The amino acid sequences of the heavy chain CDRs of 4HZ are shown in Table 5.1: [Table 6]

[0216] Furthermore, the amino acids in the variable and constant regions of the humanized antibodies 31HZ, 56HZ, and 74HZ The acid sequences are shown in Table 5.2: [Table 7]

[0217] 1.7 Preparation of anti-human Nectin-4 humanized antibody The heavy and light chain amino acid sequences of the above-mentioned anti-human Nectin-4 humanized antibody were codon-optimized. The plasmid ptt5 (Nanjing GenScript Biotec The heavy chain and The plasmids corresponding to the light chain were co-transfected into CHO-E cells, and the supernatant was purified using Purified by InA (MabSelect SuRe, GE) and anti-human nectin-4 A humanized antibody was obtained.

[0218] Example 2: Detection of the affinity of anti-human Nectin-4 humanized monoclonal antibodies 2.1 Anti-human nectin-4 humanized monoclonal antibodies against nectin-4 proteins from different species Detection of affinity of local antibodies Anti-human nectin-4 humanized monoclonals against nectin-4 proteins of different species The affinity of the antibody was detected by ELISA. The specific steps were as follows: Human nectin-4 or cynomolgus nectin-4 antigen was coated with CBS coating solution (Na 0.32 g of 2CO3 and 0.59 g of NaHCO3 were weighed and dissolved in deionized water. Dilute to 1 μg / mL with 200 mL of PBS (maximum of 200 mL) and place on ELISA plate per well. The wells were coated with 100 μL of ... Wash once with 300 μL of PBS; PBS (containing 2% BSA, purchased from BOVOGEN) 100 μL of BSAS 1.0 (Cat. No.) was added and blocked at 37° C. for 2 hours. 31HZ, 56HZ, 74HZ and enfortumab antibody were diluted in PBS (containing 2% BSA). Dilute (starting at 1500ng / mL, 3-fold dilutions, 11 concentration points) and take 100μL. , added to corresponding wells and incubated at 37°C for 2 hours; 300 μL PBST and washed three times with HRP-conjugated goat anti-human secondary antibody (purchased from Jackson, 10 9-035-00) in PBS (containing 2% BSA) at a ratio of 1:10000, and 100 μL PBST 30 was added to the corresponding wells and incubated at 37°C for 1 hour; 0 μL; TMB color development solution (purchased from InnoReagents, TMB- S-004) Add 100 μL to the corresponding wells and allow to develop for 5 to 20 minutes at room temperature. To stop the reaction, 450 μL of 2N H2SO was added, and the OD The readings were collected using a microplate reader (purchased from MD, SpectraMaxM2). The data were collected and imported into GraphPad Prism for curve fitting.

[0219] The experimental results are shown in Figures 3A to 3F and Table 6. From these results, The affinity of the enzyme to nectin-4 protein and cynomolgus monkey nectin-4 protein was Fortumab against human nectin-4 protein and cynomolgus monkey nectin-4 protein and that the affinity of 56HZ and 74HZ human nectin-4 was better than that of The affinity of enfortumab for the cynomolgus monkey nectin-4 protein was Affinity for human nectin-4 protein and cynomolgus monkey nectin-4 protein It is shown that they were basically equivalent. [Table 8]

[0220] 2.2 Anti-human nectin-4 humanized monoclonal antibodies against nectin-4 proteins of different species Detection of dynamic affinity of local antibodies The dynamic affinity of anti-human nectin-4 humanized monoclonal antibody was analyzed using Octetforteva. Detection was performed using Octet Fortebio®. The pool included the following: First, anti-human nectin-4 humanized monoclonal antibody (concentration 5 μg / mL) g / mL) was immobilized on a ProA sensor, and then human or cynomolgus nectin-4 (400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25 The antibody was subjected to a binding reaction with 100 nM of 100 mM of 100 mM of 100 mM of 0 nM ... The products fixed in the solid phase were subjected to a dissociation reaction in PBST buffer. After the experiment was completed, the results were , using Data Analysis 11.0 software in 1:1 mode. The antibody was analyzed by performing a bar fitting, thereby determining the relative affinity of the antibody to the antigen. The sum constant was obtained.

[0221] The experimental results are shown in Table 7. Human nectin-4 protein at 31HZ, 56HZ and 74HZ The dynamic affinity of enfortumab for human thyroid protein and cynomolgus monkey nectin-4 protein was Dynamic affinity for tonectin-4 protein and cynomolgus monkey nectin-4 protein was better than [Table 9]

[0222] 2.3 Anti-human nectin-4 humanized monoclonal antibody against human nectin-4 on the surface of tumor cell membrane Clonal antibody affinity detection Using T47D cells, 31HZ and 74HZ were detected on human nectin-4 on the cell membrane surface. The affinity of HZ was detected. The specific steps were as follows: T47D cells were digested. The cells were then washed twice with PBS (containing 1% BSA). Resuspend cells in 0.1% CO and plate 3 x 10 cells in 96-well chip-bottom plates. 5 Pieces / well 50 μL of the antibody to be tested was added at a final concentration of 15 μg / mL. Starting from 0.01 mg / mL, 4-fold serial dilutions were added to each well for a total of 10 concentration points; human IgG was used as a negative control. Used as a control; after thorough mixing, the resulting mixture was incubated in the dark at 4°C for 1 h. After washing three times with PBS, the cells were incubated with FITC-labeled anti-human Fc secondary antibody. Add 100 mg of 10 ... The plate was incubated for 0.5 h at 4 °C for 1 h; washed three times with PBS and then run on a flow cytometer. - (Flow cytometer brand: Beckman, model: Cytoflex) Detection was performed using

[0223] Using the same method, the membrane surface of NCI-N87 cells (a type of human gastric cancer cell line, ATCC) The affinity of 56HZ to human nectin-4 was detected at 3.33 μg / mL. Starting from 0.01, 3-fold dilutions were made, for a total of 10 concentration points.

[0224] The experimental results are shown in Figures 4A and 4B and Table 8. From these results, The affinity of 6HZ for nectin-4 on the cell membrane surface was higher than that of enfortumab for nectin-4 on the cell membrane surface. It is shown that the affinity for Nectin-4 was equivalent. [Table 10]

[0225] Example 3: Detection of endocytosis activity of anti-human nectin-4 humanized monoclonal antibody The endocytosis activity of anti-human nectin-4 humanized monoclonal antibody was evaluated using flow cytometry. The specific steps were as follows: T47D, MDA -MB-468 (a type of human breast cancer cell line, ATCC) or A549-Nectin-4 cells The cells were digested with trypsin, counted, and resuspended in PBS (containing 1% BSA) and microdissected. Cell density is 3×10 6 The 31HZ, 56HZ, or 74HZ antibodies to be tested were adjusted to 100 / mL. The antibodies were added to 100 μL of resuspended cells at a final concentration of 10 μg / mL and the isotype hCG was Antibody IgG was used as a negative control and incubated on ice for 1 hour; After washing three times with pre-chilled PBS after incubation, the cells were resuspended in cell culture medium and divided into two portions. One part was placed in a 37°C cell incubator and incubated for 1, 2 and 4 hours. The other part was kept on ice and incubated for 1 h, 2 h, and After incubation, the cells were washed with pre-chilled PBS for 4 hours. The cells were washed three times and resuspended in 50 μL of PBS (containing 1% BSA) and incubated with anti-human fluorescent antibody. The secondary antibody (BioLegend) was added and incubated at 4°C for 30 minutes; After incubation, the cells were washed three times with pre-chilled PBS, and then the fluorescent signal in the cells was detected. The results (expressed as mean fluorescence intensity (MFI)) were analyzed using a flow cytometer (Beckman ) and the antibody endocytosis rate was calculated according to the following formula: Endocytosis Cis ratio (%) = [1-(MFI antibody group at 37°C-MFI control group at 37°C) / (MFI antibody group on ice - MFI control group on ice)] × 100%.

[0226] The endocytosis activity of 56HZ in T24-nectin-4 cells was examined by the same method. It was detected.

[0227] The experimental results are shown in Figures 5A to 5D. The endocytosis activity of 74HZ was better than that of enfortumab. It is shown that there was.

[0228] Example 4: Binding of anti-human nectin-4 humanized monoclonal antibody to human nectin-4 Synthetic domain detection Human nectin-4 contains one IgV domain and two IgC domains. To determine the binding domain of the humanized monoclonal antibody to human Nectin-4, A549 stable cell line A549-Nectin-4-I overexpressing the IgV domain of Nectin-4 A549 stable cell line A5 overexpressing gV and two IgC domains of nectin-4 49-Nectin-4-IgCC was constructed and the binding domains of the antibodies to be tested were analyzed by flow cytometry. The specific steps were as follows: A549-negative chromosome 1 (ANC) was detected by ELISA. A549-nectin-4-IgV cells and A549-nectin-4-IgCC cells were digested and centrifuged. The cells were resuspended in PBS (containing 1% BSA) and washed twice with PBS. 300.000 cells / well in 96-well tip-bottom plates, 50μL per well 1. Add 50 μL of the antibody to be tested to each well at a final concentration of 40 μg / mL. IgG was used as a negative control; after thorough mixing, the resulting mixture was incubated at 4°C in the dark. The reaction was allowed to proceed for 1 hour; the cells were washed three times with PBS, and then FITC-labeled anti-human leukocytes were added. Add Fc secondary antibody (brand: BioLegend, product number: 409322) and Incubated for 0.5 h at 4°C in the dark; washed three times with PBS and flow cytometer. Cytometer (Flow cytometer brand: Beckman, model: Cytof Detection was performed using the lex.

[0229] The experimental results are shown in Figures 6A and 6B. 74HZ and enfortumab bind only to the IgV domain of human nectin-4, and Ig No binding to the C domain is shown.

[0230] Example 5: Detection of competitive activity of anti-human nectin-4 humanized monoclonal antibody Human nectin-4 binds to other nectin-4 molecules or to nectin-1 through the IgV domain. 31HZ, 56HZ and 74HZ all bind to the IgV domain of nectin-4. The competitive activity of these three antibodies was detected by competitive ELISA. The general steps were as follows: Nectin-4 protein was added to the CBS-coated solution. Dilute to 2 μg / mL with 100 μL per well and incubate at 37°C for 2 hours. Wash once with 300 μL of PBST, then rinse with PBS (2% BS A) was added at 100 μL per well and incubated at 37° C. for 2 hours. Biotin-labeled nectin-1-hFc was diluted to 20 μg / mL in PBS (2% BSA). The detecting antibody was diluted in PBS (2% BSA) at 31Hz and 74Hz. The dilution of HZ was 3-fold dilution starting from 3.73 μg / mL, with 9 concentration points, and the concentration of 56HZ The dilutions were 2-fold dilutions starting from 1.25 μg / mL, resulting in 10 concentration points; The diluted solutions were mixed thoroughly in equal volumes and then added to the corresponding wells at 100 μL / well. and incubated at room temperature for 2 hours; wash with 300 μL of PBST. was performed three times; HRP-streptavidin was diluted 1:1 with PBS (2% BSA) The antibody was diluted 1:10000 and added to the corresponding wells at 100 μL / well. The antibody was incubated at room temperature for 1 hour. Incubation was performed; washing was performed five times with 300 μL of PBST; TM Add 100 μL of B to the corresponding wells and allow color development at room temperature for 10 minutes, then The reaction was terminated by adding 450 μL of 2N H2SO4 at 450 nm. A microplate reader was used to collect readings.

[0231] The experimental results are shown in Figures 7A and 7B. and 74HZ could competitively inhibit the binding of human nectin-4 to human nectin-1. Competitive ICs for 31HZ, 56HZ and 74HZ50 The values ​​were 27.62 ng / mL, respectively. , 26.90ng / mL, and 27.22ng / mL. 50 Value is Holtumab IC 50 It is shown that the value was equivalent to that of 27.11 ng / mL.

[0232] Example 6: Detection of specificity of anti-human Nectin-4 humanized monoclonal antibody The nectin family includes nectin-1, nectin-2, nectin-3 and nectin- 4, which share approximately 25% sequence identity. The anti-human nectin-4 humanized monoclonal antibody of the present invention has the same structure and function. The body recognizes only nectin-4, but not nectin-1, nectin-2, or nectin-3. To detect whether the antibody was specific for the antibody tested, the specificity was detected by ELISA. The specific steps were as follows: human nectin-1, nectin-2, nectin- 3 and nectin-4 antigens were diluted to 1 μg / mL in CBS coating solution and analyzed by ELISA. A plate was coated with 100 μL per well and incubated at 4° C. overnight; the next day, The liquid in the wells was discarded, and the wells were washed once with 300 μL of PBS; 100 μL of 100% BSA was added and blocking was carried out at 37°C for 2 hours. 31HZ, 56HZ, 74HZ and enfortumab antibody were diluted in PBS (containing 2% BSA). Dilute, take 100 μL and add to corresponding wells and incubate for 2 hours at 37 °C. Wash with 300 μL of PBST three times; HRP-labeled goat anti-human 2 Dilute the primary antibody in PBS (containing 2% BSA) at a ratio of 1:10,000 and add 100 μL. PB was added to the corresponding wells and incubated at 37°C for 1 hour. Washing with 300 μL of ST was performed five times; 100 μL of TMB color development solution was added to the corresponding The color was developed at room temperature for 5-20 minutes; Add 450 μL of H2SO and collect the OD reading at 450 nm. A chromatographic plate reader was used and the data was analyzed using GraphPad for curve fitting. Imported into d Prism.

[0233] The experimental results are shown in Figures 8A, 8B, and 8C. Both 6HZ and 74HZ specifically recognized human nectin-4 and inhibited nectin-1 and nectin-2. It is shown that Nectin-2 and Nectin-3 did not recognize the IL-1 marker.

[0234] Example 7: Preparation of anti-human nectin-4 ADC By coupling TL001 with anti-human Nectin-4 humanized monoclonal antibody, Thus, an anti-human nectin-4 ADC was prepared. Here, the structure of TL001 is represented by formula (1): : [ka] As shown in.

[0235] The preparation method of TL001 is incorporated herein by reference in its entirety. Please refer to WO2019 / 114666.

[0236] The method for preparing anti-human nectin-4 antibody conjugate (anti-human nectin-4 ADC) , which were as follows: (1) Coupling: Take 30 mg of anti-human nectin-4 humanized monoclonal antibody, Dilute with diluent (20 mM PB + 105 mM NaCl, pH 7.7) and prepare sodium edetate. sodium solution was added to a final concentration of 5 mM and mixed thoroughly; TCEP solution was added (TCE The molar ratio of P to antibody was 4.4:1, and the mixture was thoroughly mixed and allowed to stand at room temperature for 30 minutes; TL001 dissolved in methyl sulfoxide (the molar ratio of TL001 to antibody was 9:1) (a) was added to the above solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours to obtain the coupled reagent. The samples were divided into 31HZ-TL001, 56HZ-TL001, and 74HZ-TL001. The anti-human nectin-4 ADCs were named TL001 and enfortumab-TL001. It was subjected to buffer exchange and then aliquoted and stored at 4°C.

[0237] (2) Detection: The molecular weight of the anti-human nectin-4 ADC was analyzed by LC-MS under the following conditions: Chromatography conditions: Liquid chromatography column: Thermo MAbPac RP 3.0×100 mm; Mobile phase A: 0.1%FA / 98%H2O / 2%ACN; Mobile phase B: 0.1%FA / 2%H2O / 98%ACN; Flow rate: 0.25 mL per minute; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Sample Size: 1μL; [Table 11] Switching valve: 0~3 minutes for disposal, 3~22 minutes for MS, 22~3 minutes for disposal 0 minutes Mass spectrometry conditions: Mass spectrometer model: AB Sciex Triple TOF 5600+; Parameters: GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500;DP 200;CE 10;m / z 600-4000;Up to 40 total Im Bin.

[0238] The theoretical molecular weights and measured molecular weights of the coupled anti-human 31HZ-TL001 light and heavy chains The molecular weights determined (heavy chain calculated according to the predominant glycoform G0F) are shown in the table below: [Table 12]

[0239] The light chains (LC and DAR1) of 31HZ-TL001 coupled to 0 to 1 toxin The percentages were 0.1% and 99.9%, respectively), and coupling with 0 to 4 toxins. The percentage of the heavy chains (HC, DAR1, DAR2, DAR3 and DAR4) that were , 2.0%, 10.2%, 86.1%, and 1.8%, respectively), thus the The drug-antibody ratio (DAR) was calculated using the formula: DAR = light chain DAR1 × 2 + heavy chain (DAR1 × 1 + According to the formula: DAR2 x 2 + DAR3 x 3 + DAR4 x 4) x 2, the calculated value is 7.75. Ta.

[0240] 56HZ-TL001 and 74HZ-TL00 were detected using the same method. The DAR values ​​for 1 and enfortumab-TL001 were 7.71, 7.98, and 8. The number was 01.

[0241] According to the above mass spectrometry analysis results, the structure of the anti-human nectin-4 ADC was determined to be as follows: should be set to: [ka] (In the formula, A represents an anti-human nectin-4 humanized monoclonal antibody, and γ represents a thiol group of A. The number of TL001 linked to A by forming a thioether bond with TL001 is 1 to 1 (It is an integer with a value of 0.)

[0242] By coupling VC-MMAE with enfortumab, enfortumab-V The preparation and detection methods were as described above. The molar ratio of was 1:5. The DAR value of this batch of enfortumab-VC-MMAE was The figure was 5.09.

[0243] Example 8: Anti-human nectin-4 A against tumor cell lines with high expression of human nectin-4 Detection of DC killing activity 31HZ-TL001, 74HZ-T on cell lines with high expression of human nectin-4 To detect the killing activity of L001 and enfortumab-TL001, A549-nec The Chin-4 stable cell line was selected. The specific experimental steps were as follows: A549 - Digest Nectin-4 cells, collect cells by centrifugation, and re-culture in DMEM + 4% FBS medium. Resuspend the cells in 100,000 / mL culture medium and plate 100 μL / well of cells onto a 96-well plate. ADC molecules were spread on the plate at final concentrations of 150 μg / mL to 100 μg / mL in DMEM basal medium. Starting with 4-fold dilutions, dilute the gradient to 11 concentration points and add them to the corresponding wells per well. Add 100 μL of serum to give a final serum concentration of 2%; incubate at 37°C, 5% CO2 The incubation was carried out for 72 hours in a 200-mL PBS-MS / mL PBS containing CCK8 (Rhinogen) for 2 h. Add 100 μL / well and incubate at 37℃ in a 5% CO2 incubator for 0.5 to 2.5 hours. Incubate and read OD450nm every 30 min using a microplate reader ( MD) and imported into GraphPad Prism for curve fitting. did.

[0244] The experimental results are shown in Figures 9A and 9B. , 74HZ-TL001 and enfortumab-TL001 all showed a similar binding pattern to A549-nectin. -4 cells, and EC 50 The values ​​were 1390ng / mL and 1 364ng / mL and 2049ng / mL, respectively. and 74HZ-TL001 killing activity against tumor cell lines with high expression of human nectin-4 Efficacy of Enfortumab-TL001 against tumor cell lines with high expression of human nectin-4 The killing activity of human Nectin-4 was better than that of human Nectin-4. 31HZ-TL001, 74HZ-TL001 and Enhol Killing activity of Tumab-TL001EC 50 The values ​​were 5982ng / mL and 6251ng / mL, respectively. Overall, the A549-Nectin-4 cells and Regarding the killing activity against A549 cells, 31HZ-TL001, 74HZ-TL00 1 and enfortumab-TL001 are nectin-4 specific.

[0245] 31HZ-TL on T24-nectin-4 cell line with high expression of human nectin-4 The killing activity of 56HZ-TL001 and 56HZ-TL001 was detected by the same experimental method as above. The experimental results are shown in Figures 9C and 9D. Both 56HZ-TL001 and 56HZ-TL001 were able to effectively kill T24-nectin-4 cells. K, EC 50The values ​​were 1277ng / mL and 1568ng / mL, respectively. , indicating that the killing activities of the two ADCs are essentially equivalent; 31HZ-TL001 and 5HZ-TL001 on T24 cells that do not overexpress human nectin-4 Killing activity of 6HZ-TL001 EC 50 The values ​​were 2884ng / mL and 3665ng / mL, respectively. These killing activities were significantly higher than those against T24-nectin-4 cells. The killing effects of both 31HZ-TL001 and 56HZ-TL001 were weak against nectin. It was shown that the -4 was specific.

[0246] Example 9: Anti-human nectin-4 against tumor cell lines that endogenously express human nectin-4 Detection of killing activity of ADC 31HZ-TL001, 74 against tumor cell lines that endogenously express human nectin-4 To detect the killing activity of HZ-TL001 and Enfortumab-TL001, T47 The specific experimental steps were as follows: T47D cells were cultured in 100% ethanol-free medium. The digested cells were collected by centrifugation and diluted in 1640+4% FBS medium at 100,000 / ml. The cells were resuspended in 100 mL of water and spread into a 96-well plate at 100 μL / well; ADC The molecules were diluted 4-fold into 1640 basal medium starting at a final concentration of 150 μg / mL, with 11 The concentration points were diluted in a gradient and added to the corresponding wells at 100 μL per well. The normal serum concentration was 2%; the mice were incubated at 37°C in a 5% CO2 incubator for 72 hours. CCK8 (Rhinogen) was added at 20 μL / well, and incubation was performed for 3 h. Incubate at 7°C in a 5% CO2 incubator for 0.5 to 2.5 hours, then rinse every 30 minutes. OD450nm readings were collected on a microplate reader (MD) and curve-fitted. The data were imported into GraphPad Prism for plotting.

[0247] The experimental results are shown in FIG. 10A. -TL001 and enfortumab-TL001 effectively killed T47D cells. Can, EC 50 The values ​​were 206.1ng / mL, 611.3ng / mL and 229. The killing activity of 31HZ-TL001 against T47D cells was shown to be 2ng / mL. The activity was equivalent to that of enfortumab-TL001 against T47D cells. is shown.

[0248] 31H on the tumor cell line MDA-MB-468, which endogenously expresses human nectin-4 The killing activity of Z-TL001, 74HZ-TL001 and Enfortumab-TL001 was The results are shown in FIG. 10B. , 31HZ-TL001, 74HZ-TL001 and enfortumab-TL001 were MD It can effectively kill A-MB-468 cells and EC 50 The values ​​are 1030n g / mL, 2277 ng / mL, and 1190 ng / mL. The killing activity of 31HZ-TL001 against MDA-MB-468 cells was The killing activity of TA-TL001 against MDA-MB-468 cells was shown to be equivalent to that of TA-TL001. can be.

[0249] 31HZ-T on the tumor cell line NCI-N87, which endogenously expresses human nectin-4 The killing activity of L001 and 56HZ-TL001 was detected by the same experimental method as above. The experimental results are shown in FIG. 10C. Both HZ-TL001 and HZ-TL001 were able to effectively kill NCI-N87 cells, with an EC5 The initial values ​​were 9376 ng / mL and 10935 ng / mL, respectively. As a result, the killing activities of 31HZ-TL001 and 56HZ-TL001 were essentially equal. It is shown that.

[0250] In conclusion, 31HZ-TL001 and 56HZ-TL001 express human nectin-4 in vivo. The killing activity of enfortumab-TL001 against tumor cell lines expressing The killing activity of this antibody is essentially equivalent to that of tumor cell lines that endogenously express tyrosine-4.

[0251] Example 10: In vivo pharmacodynamics of anti-human nectin-4 ADCs in the CDX model nature of By constructing a CDX model of the human non-small cell lung cancer cell line NCI-H322M, The antitumor effects of ADC molecules were evaluated. The specific steps were as follows: NCI- H322M cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS). The cells were incubated at 37°C and 5% CO2. The cells in the exponential growth phase were harvested and resuspended in PBS. The mice were then cultured in a 100-mL flask using a 10-mL syringe and incubated with 100 mL of ... e Biotechnology Co., Ltd.) 1×10 7 Pieces / mouse (PBS The subcutaneous xenograft tumor model was established by subcutaneous inoculation with 0.01 mL of 0.01% IgG4. Width: 70~100mm 3 Once tumor volume reached 100 μg / kg, mice were randomized according to tumor volume, with 5 mice per group. The mice were divided into groups. The day of grouping was recorded as day 0. The groups were divided into a vehicle group (normal saline ) (negative control), 31HZ-TL001 group (3 mg / kg and 10 mg / kg), 56H Z-TL001 group (3mg / kg and 10mg / kg), Enfortumab-TL001 group (10mg / kg) and enfortumab-VC-MMAE group (10mg / kg) All samples were administered by tail vein injection twice weekly for a total of six doses.

[0252] The diameter of the tumor was measured twice a week after administration using a caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 (wherein a and b represent the length and short diameter of the tumor, respectively). Mortality was recorded daily.

[0253] The tumor growth inhibition rate TGI (%) was calculated using the following formula. %) was used to evaluate tumor inhibition efficacy:

number

[0254] The experimental results are shown in Table 9 and Figures 11 and 12. L001, 56HZ-TL001, Enfortumab-TL001 and Enfortumab-V C-MMAE significantly inhibited tumor growth in the NCI-H322M xenograft tumor model It is shown that the inhibitory effect was dose-dependent. After six doses (21 days), the negative control group At the 10 mg / kg dose level, 31HZ-TL001, 56HZ-TL The TGI values ​​of enfortumab-TL001, enfortumab-VC-MMAE, and enfortumab-TL001 were The results were 155.79%, 163.56%, 156.31% and 173.79%, respectively. During the observation period, no significant weight loss was observed in any of the treatment groups, which indicates that the animals It has been shown that this ADC was well tolerated. [Table 13]

[0255] From the above results, 31HZ-T at the dose levels of 3mg / kg and 10mg / kg Considering the TGI values ​​of L001 and 56HZ-TL001, Both 6HZ-TL001 and 6HZ-TL001 were able to effectively inhibit tumor growth in a dose-dependent manner. At the 10 mg / kg dose level, 31HZ-TL001 and 56HZ-T Both L001 and L002 were able to partially regress tumors in all five mice in the group. The efficacy of enfortumab-TL001 and enfortumab-VC-MMAE was essentially equal. Furthermore, in mice bearing 31HZ-TL001 and 56HZ-TL001 tumors, The drug was well tolerated.

[0256] Example 11: In vivo pharmacodynamics of anti-human nectin-4 ADCs in PDX models nature of HuPrime® Bladder Cancer Xenograft Model BL9200 (Crown Bi oscience (Taicang) Co., Ltd., with high expression of nectin-4, Tumor tissue was collected from tumor-bearing mice (derived from a 63-year-old male patient) and cultured in a 3×3×3 mm diameter tube. The tissue was cut into small pieces and then cut into pieces from a NOD / SCID mouse (Jiangsu Jicui Yaokan The mice were inoculated subcutaneously into the right anterior scapula of mice (Gibco Biotechnology Co., Ltd.). The average tumor volume of tumor-bearing mice was approximately 150–250 mm 3 Once tumor size reached 100 μg / mL, mice were cultured at 100 μg / mL for 1 h. The mice were randomly divided into groups according to their size, with seven mice in each group. The day of grouping was defined as day 0. There were four groups: human IgG1 isotype control antibody group (IgG1 3 mg / kg, negative control), human IgG1-TL001 group (abbreviated as IgG1-TL001, 3 mg / kg; see Example 7 for preparation method), 1 kg for 56HZ-TL001 All samples were administered twice a week for a total of six doses via the tail vein. After administration, the tumor volume and body weight of the mice were observed and measured periodically. The measurement and calculation methods were as described in Example 10.

[0257] The experimental results are shown in Table 10 and Figures 13 and 14. After six doses (day 20), The 56H group at 3 mg / kg was compared with the 56H group at 3 mg / kg human IgG1 (hIgG1) negative control. Z-TL001 showed an inhibitory TGI of 105.55% (P<0.05) on tumor growth. As shown, partial regression of tumors was observed in four mice in the 3 mg / kg 56HZ-TL001 group. At 3 mg / kg, 56HZ-TL001 reduced the expression of the isotype Significantly improved tumor growth compared to the control ADC (hIgG1-TL001, 3 mg / kg) An inhibitory effect against hIgG1 was observed (p<0.001). One mouse in the hIgG1 group died. No deaths were observed in the other groups, which had normal activity. Body weight was slightly decreased, with the greatest decrease in the IgG1 group due to the lack of effective treatment. The ADC was also significantly reduced in tumor-bearing mice, indicating that the ADC was well tolerated by tumor-bearing mice. It is shown that:

[0258] The above results indicate that 56HZ-TL001 is a promising candidate for the treatment of bladder cancer in BL9200 PDX xenograft models. It was shown that the compound showed a significant inhibitory effect on the tumor growth of tumor-bearing mice. 56HZ-TL001 was well tolerated. [Table 14]

[0259] Although specific embodiments of the present invention have been described in detail, in light of all the teachings disclosed It will be understood that various modifications and variations may be made to the details and that all such changes are within the scope of the present invention. Those skilled in the art will understand that the scope of the present invention is as defined by the appended claims. The range is indicated by the formula:

[0260] Array List [Table 15] TIFF2025029065000027.tif223148TIFF2025029065000028.tif215148

Claims

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

2. (a) Six heavy and light chains defined according to the Chothia numbering system: CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO:3 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 4 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 10 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 11 or a variant thereof; CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 12 or 63; CDR-H3 comprising the sequence set forth in SEQ ID NO:5 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO:6 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (d) the six heavy and light chain CDRs defined according to the IMGT numbering system: ： CDR-H1 comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 14 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 15 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 16 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof Including, A variant may have one or more amino acid substitutions, deletions or deletions compared to the sequence from which it is derived. has an addition (e.g., a substitution, deletion or addition of one, two or three amino acids), , which are preferably conservative substitutions. The antibody or antigen-binding fragment thereof according to claim 1.

3. (a) Six heavy and light chains defined according to the Chothia numbering system: CDRs: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 21 or 18; CDR-H2 comprising the sequence set forth in SEQ ID NO: 22 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof; or (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 27 or 36; CDR-H2 comprising the sequence set forth in SEQ ID NO: 28 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 29 or 44; CDR-H2 comprising the sequence set forth in SEQ ID NO: 30 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof; or (d) the six heavy and light chain CDRs defined according to the IMGT numbering system: ： CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 31 or 54; CDR-H2 comprising the sequence set forth in SEQ ID NO: 32 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 33 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 34 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 35 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:26 or a variant thereof; Including, A variant may have one or more amino acid substitutions, deletions or deletions compared to the sequence from which it is derived. has an addition (e.g., a substitution, deletion or addition of one, two or three amino acids), , which are preferably conservative substitutions. The antibody or antigen-binding fragment thereof according to claim 1.

4. (a) Six heavy and light chains defined according to the Chothia numbering system: CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 39 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 40 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 41 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof; CDR-L3 comprising SEQ ID NO:8 or a variant thereof; or (b) the following six heavy and light chain CDRs defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 45 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 46 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO: 41 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (c) the following six heavy and light chain CD4s defined according to the Kabat numbering system: R: CDR-H1 comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof; CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 48 or 64; CDR-H3 comprising the sequence set forth in SEQ ID NO: 41 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof; or (d) the six heavy and light chain CDRs defined according to the IMGT numbering system: ： CDR-H1 comprising the sequence set forth in SEQ ID NO: 49 or a variant thereof; CDR-H2 comprising the sequence set forth in SEQ ID NO: 50 or a variant thereof; CDR-H3 comprising the sequence set forth in SEQ ID NO:51 or a variant thereof; CDR-L1 comprising the sequence set forth in SEQ ID NO:52 or a variant thereof; CDR-L2 comprising the sequence set forth in SEQ ID NO: 53 or a variant thereof; CDR-L3 comprising the sequence set forth in SEQ ID NO:8 or a variant thereof Including, A variant may have one or more amino acid substitutions, deletions or deletions compared to the sequence from which it is derived. has an addition (e.g., a substitution, deletion or addition of one, two or three amino acids), , which are preferably conservative substitutions. The antibody or antigen-binding fragment thereof according to claim 1.

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

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

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

8. Human immunoglobulin heavy chain constant region or variant thereof and light chain constant region or variant thereof and further including variants of A variant has one or more amino acid substitutions compared to the wild-type sequence from which it is derived. , deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acids) Substitutions, deletions or additions of acids; for example, substitutions of one, two, three, four or five amino acids; deletion or addition), The heavy chain constant region is an IgA heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. Preferably, the antibody has a G heavy chain constant region and the light chain constant region is selected from a κ or λ light chain constant region. Desirably, More preferably, the heavy chain constant region is an IgG1 heavy chain constant region; The heavy chain constant region has the sequence set forth in SEQ ID NO:61, and the light chain constant region is a κ light chain constant region. and / or the light chain constant region has the sequence set forth in SEQ ID NO:

62. Shii, An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

9. comprising a heavy chain and a light chain, (a) the heavy chain has a VH having the sequence set forth in SEQ ID NO: 1 and a VH having the sequence set forth in SEQ ID NO: 61 and a light chain having a VL having the sequence set forth in SEQ ID NO: 2 and a light chain having a VL having the sequence set forth in SEQ ID NO: 62; or (b) the heavy chain has a VH having the sequence set forth in SEQ ID NO: 19 and a VH having the sequence set forth in SEQ ID NO: 61 and a light chain comprising a VL having the sequence set forth in SEQ ID NO: 20 and a VL having the sequence set forth in SEQ ID NO: 21, or comprising a light chain constant region having the sequence set forth in SEQ ID NO:62; (c) the heavy chain has a VH having the sequence set forth in SEQ ID NO: 37 and a VH having the sequence set forth in SEQ ID NO: 61 and a light chain comprising a VL having the sequence set forth in SEQ ID NO: 38 and a VL having the sequence set forth in SEQ ID NO:

39. or comprising a light chain constant region having the sequence set forth in SEQ ID NO:62; (d) the heavy chain has a VH having the sequence set forth in SEQ ID NO: 55 and a VH having the sequence set forth in SEQ ID NO: 61 and a light chain comprising a VL having the sequence set forth in SEQ ID NO:56 and a VL having the sequence or comprising a light chain constant region having the sequence set forth in SEQ ID NO:62; (e) the heavy chain has a VH having the sequence set forth in SEQ ID NO:57 and a VH having the sequence set forth in SEQ ID NO:61 and a light chain comprising a VL having the sequence set forth in SEQ ID NO:58 and a VL having the sequence set forth in SEQ ID NO:

59. or comprising a light chain constant region having the sequence set forth in SEQ ID NO:62; (f) the heavy chain has a VH having the sequence set forth in SEQ ID NO:59 and a sequence set forth in SEQ ID NO:61 and a light chain comprising a VL having the sequence set forth in SEQ ID NO: 60 and a VL having the sequence set forth in SEQ ID NO: 61, comprising a light chain constant region having the sequence set forth in SEQ ID NO:62; An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

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

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

12. Conjugated to at least one label, preferably an enzyme, a fluorescent dye, , a radioisotope, biotin and colloidal gold.

2. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

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

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

15. Formula (I), [0010] (In the formula, A represents an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, L includes an amino acid or peptide consisting of 2 to 10 amino acids, and L includes Val, Cit , Phe, Lys, D-Val, Leu, Gly, Ala, Asn, 【number】 and one of positions 1 and 2 is selected from the group consisting of a group where L and A are connected. the other represents the position where L and D are connected; D is a cytotoxic agent, a therapeutic antibody, a radioisotope, an oligonucleotide, and the like. D is selected from the group consisting of analogs, biologically active peptides, protein toxins and enzymes; is preferably a molecule having antitumor biological activity, and D is 【number】 It is more preferable that γ is an integer from 1 to 10.

15. The antibody conjugate of claim 13 or 14, having the structure shown in

16. The (LD) moiety forms a thioether bond with the sulfhydryl group of A. having a structure of formula (1) 【Chemistry 3】 The antibody conjugate has the structure of formula (2): 【Chemistry 4】 (wherein γ is an integer from 1 to 10, and A is any one of claims 1 to 11. (representing an antibody or antigen-binding fragment thereof described in having The antibody conjugate of claim 15.

17. the therapeutic agent is a radioisotope; Radioactive isotopes are ２１２ Bi, ２１３ Bi, １３１ I, １２５ I, １１１ In, １７ ７ Lu, １８６ Re, １８８ Re, １５３ Sm, ９０ Y is selected from the group consisting of preferable, The antibody conjugate of claim 13.

18. A composition comprising the antibody conjugate of any one of claims 13 to 17, The molar ratio (DAR value) of the therapeutic agent to the antibody or antigen-binding fragment thereof in the composition is from 1 to 10. A decimal or integer between 1 and 8 (e.g., a decimal or integer between 1 and 8, e.g., 1.0, 1.5, 2 ．０、２．５、３．０、３．１、３．２、３．３、３．４、３．５、３．６、３．７、３ ．７９、３．８、３．９、４．０、４．１、４．２、４．３、４．４、４．５、４．６、 ４．７、４．８、４．９、５．０、５．１、５．２、５．３、５．４、５．５、５．６、 ５．７、５．８、５．９、６．０、６．１、６．２、６．３、６．４、６．５、６．６、 ６．７、６．８、６．９、６．９５、７．０、７．０３、７．１、７．１２、７．２、７ 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, etc. thing.

19. The antibody or antigen-binding fragment thereof, or the transmembrane domain according to any one of claims 1 to 11. and one or more intracellular T cell signaling domains. A.R.

20. An immune effector cell expressing the CAR according to claim 19 on its surface, and a cytotoxic T cell, ... Preferably, an immune effector cell.

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

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

23. A host cell comprising the nucleic acid molecule of claim 21 or the expression vector of claim 22. So, Preferably, the cell is a eukaryotic cell, more preferably a mammalian cell, Most preferably, the cell is a human hamster ovary cell; or Preferably, the cell is a prokaryotic cell, such as Escherichia coli. More preferably, the host cell.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and at least one other antibody or an antigen-binding fragment or antibody analog thereof, thereby and at least one other antigen, and are capable of binding bispecific or trispecific or a multispecific antibody, which is preferably a tetraspecific antibody.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and an additional A fusion protein comprising a biologically active polypeptide, The additional biologically active polypeptide may be a polypeptide having therapeutic activity, binding activity or enzymatic activity. A fusion protein, which is a tide or protein.

26. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, claims 13 to 1 The antibody conjugate according to any one of claims 7 to 18, the composition according to claim 20, 25. An immune effector cell according to claim 24, a multispecific antibody according to claim 25 or A pharmaceutical composition comprising the fusion protein as described above and a pharma- ceutical acceptable carrier and / or excipient. Composition.

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

28. Medicines, from antineoplastics, chemotherapeutics, radioisotopes, and immune checkpoint inhibitors 28. The method of claim 27, for use in combination with one or more therapeutic agents selected from the group consisting of: Use of.

29. Medicine, surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, vascular and palliative care. The use according to claim 27, wherein the use is performed in combination with

30. Method for detecting the presence of nectin-4 in a sample or determining the expression level of nectin-4 - Patent application And, (a) administering to a sample the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11; contacting; (b) detecting the formation of an immune complex between the antibody or antigen-binding fragment and Nectin-4 in the sample; or the amount thereof to detect the presence of Nectin-4 in a sample or to detect the expression of Nectin-4. Steps to determine the current level A method comprising:

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