Anti-nectin-4 antibody, conjugate containing same, and application thereof

Abstract

Provided are anti-Nectin-4 antibodies or antigen-binding fragments thereof, antibody conjugates comprising the same, and uses thereof in the manufacture of medicaments, particularly in the manufacture of medicaments for treating and / or preventing cancer. [Solution] Provided is an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4, which comprises three heavy chain CDRs, each containing a specific sequence defined according to the Chothia numbering system, and three light chain CDRs, each containing another specific sequence defined according to the Chothia numbering system.

Description

[Technical Field]

[0001] This application, filed on September 16, 2020, is a division of "Anti-Nectin-4 and tibody,conjugate including same,and appl Chinese Patent Application No. 20201097236 entitled "Contact Information Thereof" Priority of Patent Application No. 5.6 and "Anti-Nectin- 4 antibody,conjugate including same,and Chinese Patent Application No. 2020116 entitled "Application thereof" 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 its and a conjugate comprising the compound, in particular for 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 approximately 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 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 the PI3K-AKT signaling pathway Nectin-4 may promote the survival and proliferation of tumor cells by activating Nectin-4. , hepatocellular carcinoma, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer and edema 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 patient prognosis. Therefore, Nectin-4 is an ideal candidate for the development of anti-tumor drugs. Can be used as a target.

[0005] Currently, enfortumab vedotin (e.g., an ADC drug targeting nectin-4) is See, for example, WO2012 / 047724, which is already being used for the treatment of urothelial carcinoma. Enfortumab vedotin is a conjugate of a fully human anti-nectin-4 antibody. It is a gated VC-MMAE drug with a DAR value of 4. From the Phase III clinical trial data Four patients achieved a complete response (CR) and 41 patients achieved a partial response (PR). The objective response rate (ORR) was 41%, and the median OS was 13.6 months. The median duration of treatment was 5.75 months, and the median progression-free survival (PFS) was 5.4 months. In the checkpoint inhibitor treatment group, 89 patients The ORR was 40%, and in the checkpoint inhibitor-naïve group, the ORR was 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 oocytes. It was weakly to moderately expressed in the uterine lining, 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 urinary tract infections were anemia (8%), hyponatremia (7%), urinary tract infections (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 that specifically binds to Nectin-4 or an antigen-binding protein thereof. and a synthetic fragment thereof, wherein the antibody or antigen-binding fragment thereof is (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 its variants; and / or according to the Chothia numbering system 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 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 defined according to the Abm numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof, and the sequence set forth in SEQ ID NO: 10 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 5; H3 or its variants; and / or 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 barriers 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 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 its variants; 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, defined according to the Kabat numbering system: SEQ ID NO:1 CDR-H1 comprising the sequence set forth in 1 or a variant thereof, the sequence set forth in SEQ ID NO: 63 CDR-H2 or a variant thereof 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, 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 set forth in 8; 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 CDR-H2 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: CDR-L1 comprising the sequence set forth in SEQ ID NO: 16; a 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 CDR-H2 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 CDR-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, as 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 its variants; 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 its variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or variants thereof, CDR-L3 or a variant thereof comprising the sequence set forth in column number 26; 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, and 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 set forth in 6. or (2c) 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: 29, 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, as defined according to the Kabat numbering system: SEQ ID NO:4 CDR-H1 comprising the sequence set forth in SEQ ID NO: 30 or a variant thereof. CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 H3 or its variants; 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 its variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 25 or variants thereof, CDR-L3 or a variant thereof comprising the sequence set forth in column number 26; 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, as set forth in SEQ ID NO: 32 CDR-H2 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: CDR-L1 comprising the sequence set forth in SEQ ID NO: 34; or a variant thereof, a 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 defined according to the IMGT numbering system: SEQ ID NO: 54 CDR-H1 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 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 CDR-H2 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 defined according to: CDR- comprising the sequence set forth in SEQ ID NO: 42 CDR-L1 or a variant thereof, CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 43 a CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; 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 its variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or variants thereof, A CDR-L3 or 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, as defined according to the Kabat numbering system: SEQ ID NO:4 7 or a variant thereof, the sequence of SEQ ID NO: 64 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 H3 or its variants; 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 its variants, CDR-L2 comprising the sequence set forth in SEQ ID NO: 43 or variants thereof, 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 CDR-H2 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: CDR-L1 comprising the sequence set forth in SEQ ID NO: 52; or a variant thereof, a 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., substitution, deletion, or addition of one, two, or three amino acids), and the substitution is , preferably conservative substitutions.

[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 treatment of a cancer, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention and at least one therapeutic agent. Antibody conjugates containing a therapeutic agent are provided.

[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 an additional Fusion proteins comprising biologically active polypeptides are also contemplated.

[0012] The present invention relates to the antibody or antigen-binding fragment thereof, antibody conjugate, immune effector, and the like of the present invention. a pharmaceutically acceptable carrier and / or a fusion protein; The present invention also relates to pharmaceutical compositions comprising excipients.

[0013] In yet another general aspect, the invention provides a method for treating cancer comprising: 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 the use of the antibody or 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. The subject is administered the antibody or antigen-binding fragment thereof, antibody conjugate, or immune effector of the present invention. administering a therapeutically effective amount of the cells, 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, May be selected from pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, and esophageal cancer preferable. [Brief explanation of the drawings]

[0017] [Figure 1A] 1 is a graph showing the results of flow cytometry of A549-nectin-4 stable cell lines. [Figure 1B] 1 is a graph showing the results of flow cytometry of T24-nectin-4 stable cell lines. [Figure 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 74HZ, an anti-human nectin-4 humanized monoclonal antibody, 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 31HZ, an anti-human Nectin-4 humanized monoclonal antibody, and cynomolgus monkey Nectin-4 protein. [Figure 3E] 1 is a graph showing the results of ELISA detection of the affinity between 74HZ, an anti-human Nectin-4 humanized monoclonal antibody, and cynomolgus monkey Nectin-4 protein. [Figure 3F]1 is a graph showing the results of ELISA detection of the affinity between 56HZ, an anti-human Nectin-4 humanized monoclonal antibody, and cynomolgus monkey Nectin-4 protein. [Figure 4A] Fig. 10 is a graph showing the results of flow cytometry on 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 on 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 human Nectin-4 domains. [Figure 6B] 1 is a graph showing the detection results of 56HZ, an anti-human nectin-4 humanized monoclonal antibody that recognizes human nectin-4 domains. [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] Fig. 10 is a graph showing the results of killing of T24 cells 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 killing of MDA-MB-468 cells 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 in 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] 1 is a graph showing the change in tumor volume over time for each group of mice in a subcutaneous HuPrime® gastric cancer BL9200 PDX model in NOD / SCID mice. [Figure 14] Figure 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 INVENTION

[0018] definition In the present invention, unless otherwise specified, the technical and scientific terms used herein are understood by those skilled in 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" refers to 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" "including," "containing," and "having" ng) is open-ended and may include additional unrecited elements, steps, or Components are not excluded. The expression "consisting of" means Any element, step, or component not specified is excluded. The expression "consisting essentially of" Any element, step, or component specified, in addition to those fundamental to the claimed subject matter, Optional elements, steps or components that do not materially affect the fundamental and novel characteristics The word "comprising" means that the scope is limited to: "consisting essentially of" and " It should be understood to include the expression "consisting of" is.

[0021] The term "optional" or "optionally" refers to the event or situation that is described thereafter. The events or circumstances may or may not occur and this statement does not imply that the events or circumstances will occur. This means that the occurrence of an event or circumstance is included as well as the absence of that event or circumstance.

[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 covalently or non-covalently bonded to the peptide. 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 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 "traditional 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. It contains a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)" 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 contains CH2 and CH3 and can bind to an Fc receptor. The "hinge region" refers to the fragment that provides the site of synthesis between the Fab fragment and the Fc fragment of an immunoglobulin. The part of an antibody that connects the constant region and the transmembrane fragment. In some cases, the hinge region 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 using known algorithms. and the applicable algorithms and software. For a description of 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 C-terminus to the C-terminus, it is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 In the present specification, the light chain variable region CDRs (CDRLs) are 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 The heavy chain variable region CDRs (CDRHs) 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 the antigen-binding site can be determined by the six CDRs that comprise the CDRs. In some cases, other fragments (e.g., Single domain antibodies, also known as nanobodies, are generally considered to have antigen-binding ability. Those skilled in the art can perform the analysis using methods well known in the art, for example, by 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 identical to the antigenic end It is understood by those skilled in the art that the term "antigen binding site" represents an efficient antigen-binding site capable of binding to a target antigen. The description of the CDR numbering system may be found, for example, in the Kabat numbering system. For more information, see Kabat, EA et al. (1991) Sequences of Proteins eins of Immunological Interest, Fifth Edi tion, USDapartment of Health and Human Ser 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 vonheim: 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 considered to be the smallest antigen-binding fragment that contains the original binding site. as single domain antibodies (VHH, sdAb or nanobodies) containing only the variable regions of A "single-chain Fv (scFv)" is a Fv consisting of VH and VL bound to a peptide. Disulfide-stabilized Fv(d "disulfide-stabilized single-chain Fv (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 Preferably, the 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 molecule that connects the CL and CH1 of Fab with a peptide linker. "F(ab')2" is basically a fragment of the hinge region. It contains two Fab fragments linked by a disulfide bond at F It is 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 antibody or antigen-binding fragment may be "monovalent," "bivalent," "trivalent," or "tetravalent," or more can also be multivalent, i.e., multiple (e.g., one, two, three, 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. This represents a multispecific antibody capable of

[0031] As used herein, a "diabody" refers to a combination of two scFv and refers to a (bivalent) antibody having two antigen-binding sites, each comprising a VH and a VL of each scFv. 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 shorter than the full length, but contains the same antigen-binding fragment as the full length antibody. and / or a portion of a variable region of the target gene (e.g., one or more CDRs and / or (containing one or more antigen-binding sites) and therefore specifically bind to the antigen of a full-length antibody. 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, synthetic antibodies, and the like. These may be synthetic or recombinantly produced derivatives. Examples of synthetic fragments include, but are not limited to, Fv, scFv, dsFv, scD sFv, Fab, scFab, Fab', F(ab')2, diabody, Fd and Fd Antigen-binding fragments include fragments of the nucleotide sequence of the nucleotide sequence of the nucleotide, fragments ... 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. It refers to a population of antibodies, which may contain small amounts of antibody molecules. Monoclonal antibodies are generally made of a variety of antibodies, each of which is substantially identical except for certain naturally occurring mutations. Monoclonal antibodies useful in the present invention are For example, transgenic animals using recombinant DNA techniques (e.g., transgenic 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 isolation from a The antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody. The antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

[0034] A "chimeric antibody" is a chimeric antibody that is a chimeric antibody consisting of a portion (e.g., CDR, FR, variable region, constant region, or any of these) the remaining sequences are identical or homologous to the corresponding sequences of antibodies derived from a particular species, 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, "chimeric antibody" includes antibodies that contain portions belonging to different antibody classes or subclasses. In certain embodiments, chimeric antibodies are 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 both non-human and human antibody sequences. Thus, a humanized antibody refers to an antibody that contains minimal sequence derived from non-human immunoglobulin. A non-human antibody may be an antibody derived from any non-human species. In addition, the antibody may be an antibody that contains portions derived from non-human species (e.g., a chimeric antibody). Species may include, for example, mice, rats, rabbits, alpacas, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. A chimeric 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 CDR sequences of a human antibody (e.g., a murine antibody) into a human antibody framework region. In some cases, the antigen-binding ability and / or stability of the humanized antibody may be improved. In order to maintain the essential amino acid sequence of the framework of a non-human antibody (e.g., a murine antibody), The amino acid residues can be retained in the framework regions of the human antibody, i.e., Perform "reverse mutation" (see, e.g., 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, "percent (%) sequence identity" of an amino acid sequence, "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, for example, 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 due to non-covalent interactions. The magnitude of "affinity" is usually expressed as the equilibrium dissociation constant K D or EC 50 It can be reported as K D is 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) radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), using conventional techniques known in the art, such as immunoassay, or flow cytometry. 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 specifically bound antibody and 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 Both are about 10 -7 M, at least about 10 -8 M, at least about 10 -9 I am 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 partially or completely identical to a reference amino acid sequence. For example, the amino acid sequence of the heavy chain constant region derived from a human immunoglobulin is identical or homologous to the amino acid sequence of the heavy chain constant region derived from a human immunoglobulin. The amino acid sequence has the following structure compared to the sequence 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 It has.

[0040] As used herein, a "variant" of a polypeptide or amino acid sequence refers to a variant thereof. one or more amino acid mutations or The amino acid mutations include substitutions, deletions, or additions of amino acids. Amino acids in non-essential regions of a polypeptide or protein are generally It will be apparent to 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., E. Tummings Pub. Co., p. 224). Suitable conservative substitutions are readily apparent to those skilled in the art. Some non-limiting examples of common conservative substitutions of amino acid residues are listed below. In certain cases, the amino acid substitutions are non-conservative substitutions. To alter the properties of the antibody fragment, for example, 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 encompassed within the scope of the antibodies or antigen-binding fragments 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 comprise 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 has been isolated from the environment. that the product (peptide) is separated from the source or environment in which it occurs, i.e., This means that the composition 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. The vehicle used to introduce the vector into a suitable host cell to transform the host cell. Upon transformation, the exogenous nucleic acid is amplified or expressed. Vectors are generally stored as episomes. However, genes or parts of genes 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 (e.g., 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 expressed in a regulatory sequence (e.g., Regulatory sequences include sequences that are operably linked to a gene (e.g., promoter, ribosome binding site). It may contain promoter and terminator sequences, and optionally may contain an origin of replication, a selection It may also contain 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. , 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 also includes expression vectors containing the vector.

[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 can be a eukaryotic cell or a prokaryotic cell.

[0046] As used herein, the term "treatment" refers to a disease / symptom. Amelioration of the condition, e.g., reduction or elimination of a disease / symptom, prevention of the onset, exacerbation and / or progression of a disease / symptom Treatment therefore includes prevention, treatment and and / or healing.

[0047] An "effective amount" is a dose that reduces the severity of symptoms of the disease, the frequency and duration of asymptomatic periods of the disease, To increase time or prevent damage or disability resulting from contracting a disease An "effective amount" refers to a dose sufficient 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 size 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 at least about 60%, more preferably at least about 70%, more preferably Preferably, the inhibition is at least about 80%. Conventional animal tumor models in the art, such as spontaneous tumors, induced tumors, and The efficacy of the antibody can be assessed using animal models involving cell culture and transplanted tumors. The ability of the compound to inhibit growth is tested using in vitro assays well known in the art. The antibody or antigen-binding fragment thereof, antibody conjugate, and immunological effector of the present invention can also be used. An effective amount of the agent, such as a steroid or steroid drug, can reduce tumor size or otherwise improve the target tumor. It is possible to alleviate symptoms in patients with rheumatoid arthritis (e.g., prevent and / or treat metastasis or recurrence) Those skilled in the art will be able to determine the appropriate dosage based on, for example, the subject's age, health, sex, severity of symptoms, and specific The effective amount can be determined based on factors such as the composition or route of administration. It can be administered in one or more doses.

[0048] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" The active ingredient is 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 Pharmaceutical Sciences., edited by Gennaro AR, 19th ed., P Ennsylvania: Mack Publishing Company, 1995 (see, for example, JP 2004-2009022), which may 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 buffers. Although not required, 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. Antibacterial and antifungal agents include various antifungal agents such as chlorobutanol, phenol, and sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugars, 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 required, water, aqueous buffers (e.g., buffered saline), alcohols and polyols ( Examples of preservatives include, but are not limited to, glycerol. , e.g., 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 a drug. These 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 steroidal albumin 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 an organism, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject ( A "subject" is a cancer patient, human or animal. The terms "ct" 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 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 containing the amino acid sequence. Preferably, the nectin-4 is human nectin-4. Human nectin-4 is preferably identified by, for example, Uniprot identification number (ID): Q96NY8 The non-human Nectin-4 may have an amino acid sequence represented by the amino acid sequence of non-human Nectin-4. It may be a mammalian nectin-4, such as 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 amino acid sequence may be represented by the iprot identification number (ID): D3ZET1. The non-human nectin-4 may also be, for example, mouse nectin-4, e.g., Uni It may have the amino acid sequence shown by 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 that specifically binds to Nectin-4 or its antigen-binding activity 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, the sequence set forth in SEQ ID NO: 4 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 present invention also includes a CDR-L3 comprising the sequence or a variant thereof.

[0057] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of 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, and CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 10. 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: 6; 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 set forth in SEQ ID NO: 8 R-L3 or a variant thereof.

[0058] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises 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: 11, SEQ ID NO: 12 or 63 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 5 In another particular embodiment, the antibody specifically binds 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 barriers a CDR-L2 comprising the sequence set forth in SEQ ID NO: 7 or a variant thereof; a CDR-L2 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof; The present invention also includes a CDR-L3 comprising the sequence described above or a variant thereof.

[0059] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of the following three heavy chain CDRs, defined according to the IMGT numbering system: CDR-H1 or a variant thereof comprising the sequence set forth in SEQ ID NO: 13, the sequence set forth in SEQ ID NO: 14 CDR-H2 or a variant thereof 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 one of the following three antibodies defined according to the IMGT numbering system: two 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 SEQ ID NO: 17, and 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 , (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: CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 4, the sequence set forth in 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 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 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 CDs 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, 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: 6 CDR-L1 or a variant thereof, CDR-L2 or CDR-L3 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 (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 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 15 CDR-H3 or a variant thereof, and 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 that specifically binds to Nectin-4 or its antigen-binding activity 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, set forth in SEQ ID NO: 22 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 In another specific 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 used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof a CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 25, and the sequence It includes a CDR-L3 comprising the sequence set forth in No. 26 or a variant thereof.

[0062] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of 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 specific embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three distinct classes of antibodies 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, and a CDR-L2 or variant thereof comprising the sequence set forth in SEQ ID NO: 26. The present invention also includes a CDR-L3 comprising the sequence or a variant thereof.

[0063] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises 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: 29, 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 In another particular embodiment, the antibody specifically binds to Nectin-4. The antibodies or antigen-binding fragments thereof are as follows, defined according to the Kabat 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, The present invention also includes a CDR-L3 comprising the sequence described above or a variant thereof.

[0064] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of the following three heavy 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, 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 or a variant thereof. In another particular embodiment, the antibody specifically binds to Nectin-4. The antibody or antigen-binding fragment thereof may be one of the following three antibodies defined according to the IMGT numbering system: two 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 present invention also includes a CDR-L3 comprising the sequence or a variant thereof.

[0065] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 , (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 or a variant thereof comprising the sequence set forth in SEQ ID NO: 24 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; 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: 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 or variant thereof comprising the sequence set forth in SEQ ID NO: 26; or (c) the following six heavy and light chain CDs 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 CDR-H2 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof CDR-H3 or a variant thereof comprising the sequence set forth in SEQ ID NO: 24 CDR-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 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 33 CDR-H3 or a variant thereof, and 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 that specifically binds to Nectin-4 or its antigen-binding activity 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, set forth in SEQ ID NO: 22 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 23 In another specific 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 used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof a CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 25, and the sequence It includes a CDR-L3 comprising the sequence set forth in No. 26 or a variant thereof.

[0067] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of 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 specific embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three distinct classes of antibodies 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, and a CDR-L2 or variant thereof comprising the sequence set forth in SEQ ID NO: 26. The present invention also includes a CDR-L3 comprising the sequence or a variant thereof.

[0068] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 44 or a variant thereof, 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 In another particular embodiment, the antibody specifically binds to Nectin-4. The antibodies or antigen-binding fragments thereof are as follows, defined according to the Kabat numbering system: Three light chain CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 24 or a variant thereof , CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 25, and SEQ ID NO: 26 or a variant thereof.

[0069] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity 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 antibody 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: 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 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 , (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 or a variant thereof comprising the sequence set forth in SEQ ID NO: 24 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; 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: 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 or variant thereof comprising the sequence set forth in SEQ ID NO: 26; or (c) the following six heavy and light chain CDs 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 CDR-H2 comprising the sequence set forth in SEQ ID NO: 23 or a variant thereof CDR-H3 or a variant thereof comprising the sequence set forth in SEQ ID NO: 24 CDR-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 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 33 CDR-H3 or a variant thereof, and 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 that specifically binds to Nectin-4 or its antigen-binding activity 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, set forth in SEQ ID NO: 40 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 In another specific 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 used: CDR-L1 comprising the sequence set forth in SEQ ID NO: 42 or a variant thereof a CDR-L2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 43, and the sequence It includes a CDR-L3 comprising the sequence set forth in No. 8 or a variant thereof.

[0072] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragments consist of the following three heavy chain CDRs defined according to the Abm numbering system: SEQ ID NO: 45 or a variant thereof, and the sequence set forth in SEQ ID NO: 46 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41, In another specific embodiment, an antibody that specifically binds to Nectin-4 is The antibody or antigen-binding fragment thereof has three distinct classes of antibodies 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 also includes a CDR-L3 or a variant thereof comprising the sequence.

[0073] In certain embodiments, an antibody that specifically binds to Nectin-4 or its antigen-binding activity The fragment comprises the following three heavy chain CDRs, defined according to the Kabat numbering system: CDR-H1 comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof, CDR-H2 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, 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 that specifically binds to Nectin-4 or its antigen-binding activity 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 SEQ ID NO: 50 CDR-H2 or a variant thereof 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 one of the following three antibodies defined according to the IMGT numbering system: two 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 SEQ ID NO: 8 or a variant thereof.

[0075] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 , (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, comprising the sequence set forth in SEQ ID NO: 41 CDR-H3 or a variant thereof comprising the sequence set forth in SEQ ID NO: 42 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 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 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41 CDR-H3 or a variant thereof, and 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 CDs 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 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 41. CDR-H3 or a variant thereof comprising the sequence set forth in SEQ ID NO: 42 CDR-L1 or a variant thereof, CDR-L1 comprising the sequence set forth in SEQ ID NO: 43 2 or a variant thereof, CDR-L3 comprising the sequence set forth in SEQ ID NO: 8 or a variant thereof 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 CDR-H2 or a variant thereof comprising the sequence set forth in SEQ ID NO: 51 CDR-H3 or a variant thereof, and 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 case, the variant must be 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 from any species of immunoglobulin. The heavy chain framework regions and the light chain framework regions can be derived from the 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 regions and the light chain framework regions comprise Independently, the heavy and light chain framework regions of a human germline antibody It contains 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 comprises a human and / or a heavy chain framework region derived from a human immunoglobulin, 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 embodiments, the antibody or antigen-binding fragment thereof 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 may contain one or more non-human (e.g., murine) amino acid residues, e.g., heavy chain fragments The framework region and / or the light chain framework region may contain one or more amino acid reversions. 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 VH comprises a VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof. In another 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 VH having the sequence set forth in SEQ ID NO: 2 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 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 column 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 VH having the sequence set forth in SEQ ID NO: 55 or a variant thereof, and VH having the sequence set forth in SEQ ID NO: 56 The present invention also 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, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 , 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, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 , 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) VH having the sequence set forth in SEQ ID NO: 1 or a variant thereof and / or SEQ ID NO: VL having the sequence set forth 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 sequence 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 sequence 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 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 sequence 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 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 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 1, 2, 3, 4, or 5 amino acids) Preferably, the substitution is a conservative substitution.

[0093] constant region In some embodiments, the anti-Nectin-4 antibody further comprises a heavy chain constant region. In some embodiments, the anti-Nectin-4 antibody further comprises a light chain constant region. The regions can independently be linked to the 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 variants thereof. The regions each independently comprise 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, 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 portion of: 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 of 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 a combination thereof. The heavy chain constant region can be derived from an IgG (e.g., IgG1, IgG2, IgG3, Preferably, the heavy chain constant region is derived from an 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 specific 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 may have a low or no mutation compared to 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, a 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 is 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 set forth in SEQ ID NO: 62 or a variant thereof. 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 comprises a VH having the sequence set forth in SEQ ID NO: 19 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: 20 or a variant thereof and VL having the sequence set forth in SEQ ID NO: 62 or a variant thereof; or (c) The heavy chain comprises a VH having the sequence set forth in SEQ ID NO: 37 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: 38 or a variant thereof and VL having the sequence set forth in SEQ ID NO: 62 or a variant thereof; or (d) The heavy chain comprises a VH having the sequence set forth in SEQ ID NO: 55 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: 56 or a variant thereof and VL having the sequence set forth in SEQ ID NO: 62 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 VL having the sequence set forth in SEQ ID NO: 62 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 VL having the sequence set forth in SEQ ID NO: 62 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 are murine antibodies, chimeric antibodies, or or a humanized antibody.

[0100] Mouse antibodies 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. light chain CDRs and light chain framework regions, 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 a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18, 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 a mouse 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 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, C -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 containing CDR-L2 and CDR-L3 In a preferred embodiment, the present invention 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. In the mouse antibody, the VH has 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 present invention 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 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 1, 2, 3, 4, or 5 amino acids) Preferably, the substitution is a conservative substitution.

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

[0106] In another specific 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 V 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 light chain CDRs of the above-mentioned murine antibody. In embodiments, the chimeric antibody comprises the heavy and / or light chain variable regions of the above-described murine antibodies. In some embodiments, the chimeric antibody comprises the heavy chain variable region and the light chain variable region of the above-described murine antibody. a variable region, and a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is , derived from a heavy chain constant region of 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 heavy chain constant region is derived from a human immunoglobulin heavy chain constant region, and / or the light chain constant region is derived from a human immunoglobulin heavy 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 constant region of a human immunoglobulin light chain. 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, 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 a V H or a variant thereof. In certain embodiments, the chimeric antibody comprises the antibody set forth in SEQ ID NO: 38 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 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 1, 2, 3, 4, or 5 amino acids) Preferably, the substitution is a conservative substitution.

[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 certain embodiments, 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% and 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 conservative substitution of up to 20 amino acids (e.g., up to 1 Five, up to ten, or up to five amino acid conservative substitutions; e.g., one, two, three, Conservative substitutions of four or five 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 , 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 are included, and the heavy chain framework region and the light chain framework region are included. The framework regions each independently comprise a human immunoglobulin heavy chain framework region and In such embodiments, the heavy chain comprises an amino acid sequence derived from a light chain framework region. The framework regions and / or light chain framework regions may be derived from one or more non-human (e.g., and / or Alternatively, the light chain framework region may contain one or more amino acid backmutations, There are mouse amino acid residues corresponding 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 its barrier of the above-mentioned murine antibody. CDR-H2 or its variants, CDR-H3 or its variants. In this 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 its 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 framework region 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 further comprises a light chain framework region comprising an amino acid sequence as follows:

[0121] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 3, a CDR-H2 set forth in SEQ ID NO: 4, a CDR-H3 set forth in SEQ ID NO: 5, a CDR-H4 set forth in SEQ ID NO: 6, a CDR-H5 set forth in SEQ ID NO: 7, a CDR-H6 set forth in SEQ ID NO: 8, a CDR-H7 set forth in SEQ ID NO: 9 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 comprises a VH as defined by the thia numbering system. The humanized antibody has CDR-L1 set forth in SEQ ID NO: 6 and CDR-L2 set forth in SEQ ID NO: 7. , CDR-L3 set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework region The Chothia numbering sequence includes the light chain framework region containing the amino acid sequence derived from It further comprises a VL defined by the stem.

[0122] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 9, a CDR-H2 set forth in SEQ ID NO: 10 CDR-H2 set forth in SEQ ID NO: 5, CDR-H3 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 VH numbering system. The antibody comprises CDR-L1 set forth in SEQ ID NO: 6, CDR-L2 set forth in SEQ ID NO: 7, and CDR-L3 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 includes VL as defined.

[0123] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 11, a CDR-H2 set forth in SEQ ID NO: 1 2 or 63, CDR-H3 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; and VH as defined by the Kabat numbering system. In one embodiment, the humanized antibody comprises a CDR-L1 set forth in SEQ ID NO: 6, a CDR-L2 set forth in SEQ ID NO: 7, and a CDR-L3 set forth in SEQ ID NO: 8. R-L2, CDR-L3 set forth in SEQ ID NO: 8, and a human immunoglobulin light chain framework Kabat numbering, including light chain framework regions containing amino acid sequences derived from the light chain framework regions. It further includes a VL defined by the system.

[0124] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 13, a CDR-H2 set forth in SEQ ID NO: 1 CDR-H2 set forth in SEQ ID NO: 4, CDR-H3 set forth in SEQ ID NO: 15, and 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 set forth in SEQ ID NO: 16, CDR-L2 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 a CDR-H1 set forth in SEQ ID NO: 21 or 18, a CDR-H1 set forth in SEQ ID NO: 22 or 23, a CDR-H2 set forth in SEQ ID NO: 24 or 25, a CDR-H3 set forth in SEQ ID NO: 26 or 27, a CDR-H4 set forth in SEQ ID NO: 27 or 28, a CDR-H5 set forth CDR-H2 set forth in sequence number 22, CDR-H3 set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a globulin heavy chain framework region; Further specific VHs include those 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 a CDR-H1 set forth in SEQ ID NO: 27 or 36, a CDR-H2 set forth in SEQ ID NO: 28 or 39, a CDR-H3 set forth in SEQ ID NO: 39 or 40, a CDR-H4 set forth in SEQ ID NO: 41 or 42, a CDR-H5 set forth in SEQ ID NO: 43 or 44, a CDR-H6 set forth CDR-H2 set forth in sequence number 28, CDR-H3 set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a globulin heavy chain framework region; In yet another specific embodiment, the VH is defined by the Abm numbering system, including In one embodiment, the humanized antibody comprises CDR-L1 set forth in SEQ ID NO: 24, CDR-L2 set forth in SEQ ID NO: 25, DR-L2, CDR-L3 set forth in SEQ ID NO: 26, and a human immunoglobulin light chain framework Abm numbered light chain framework regions containing amino acid sequences derived from framework regions. It further includes a VL defined by the system.

[0128] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 29 or 44, a CDR-H2 set forth in SEQ ID NO: 29 or 44, a CDR-H3 set forth in SEQ ID NO: 29 or 44, a CDR-H4 set forth in SEQ ID NO: 29 or 44, a CDR-H5 set forth in SEQ ID NO: 29 or 44, a CDR-H6 set forth CDR-H2 set forth in sequence number 30, CDR-H3 set forth in sequence number 23, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a globulin heavy chain framework region;

[0039] In yet another specific embodiment, the VH is defined by the Kabat numbering system, including In embodiments, the humanized antibody comprises CDR-L1 set forth in SEQ ID NO: 24, CDR-L2 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 Further included are VLs as defined by the t numbering system.

[0129] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 31 or 54, a CDR-H2 set forth in SEQ ID NO: 32 or 54, a CDR-H3 set forth in SEQ ID NO: 33 or 54, a CDR-H4 set forth in SEQ ID NO: 34 or 54, a CDR-H5 set forth in SEQ ID NO: 35 or 54, a CDR-H6 set forth CDR-H2 set forth in sequence number 32, CDR-H3 set forth in sequence number 33, and human immunoglobulin G a heavy chain framework region comprising an amino acid sequence derived from a globulin heavy chain framework region; Further specific embodiments include VHs as defined by the IMGT numbering system, including: In some embodiments, the humanized antibody comprises a CDR-L1 set forth in SEQ ID NO: 34, a CDR-L2 set forth in SEQ ID NO: 35, and a CDR-L3 set forth in SEQ ID NO: 36. CDR-L2, CDR-L3 set forth in SEQ ID NO: 26, and a human immunoglobulin light chain frame IMGT number 1001, including a light chain framework region containing an amino acid sequence derived from the framework region. 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 certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 39, a CDR-H2 set forth in SEQ ID NO: 4 CDR-H2 set forth in SEQ ID NO: 0, CDR-H3 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 CDR-L1 set forth in SEQ ID NO: 42, CDR-L2 set forth in SEQ ID NO: 43, CDR-L2, CDR-L3 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 It further includes a VL defined by a numbering system.

[0132] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 45, a CDR-H2 set forth in SEQ ID NO: 4 6, CDR-H2 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 set forth in SEQ ID NO: 42, CDR-L2 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 and a light chain framework region comprising an amino acid sequence derived from the Abm numbering system. Further included is a VL defined by:

[0133] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 47, a CDR-H2 set forth in SEQ ID NO: 4 CDR-H2 set forth in SEQ ID NO: 8 or 64, CDR-H3 set forth in SEQ ID NO: 41, and a human immunoglobulin a heavy chain framework region comprising an amino acid sequence derived from a globulin heavy chain framework region;

[0039] In yet another specific embodiment, the VH is defined by the Kabat numbering system, including In embodiments, the humanized antibody comprises CDR-L1 set forth in SEQ ID NO: 42, CDR-L2 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 It further includes a VL defined by a numbering system.

[0134] In certain embodiments, the humanized antibody comprises a CDR-H1 set forth in SEQ ID NO: 49, a CDR-H2 set forth in SEQ ID NO: 5 CDR-H2 set forth in SEQ ID NO: 0, CDR-H3 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 has CDR-L1 set forth in SEQ ID NO: 52, CDR-L2 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 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 1, 2, 3, 4, or 5 amino acids) Preferably, the substitution is a conservative substitution.

[0137] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (V 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 light chain variable region of interest. and / or the framework regions contain amino acid mutations, wherein the amino acid mutations are one or more Substitution, deletion or addition of several amino acids, or any combination thereof (e.g., one, two or more The substitutions are conserved amino acid substitutions, deletions or additions of three amino acids, or any combination thereof. Preferably, it is a conservative substitution.

[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 a human kappa light chain constant region. 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 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 the antigen. The method can also be used to immunize animals (e.g., mice) using Antibody-expressing B cells can be isolated from the animals. Preferably, the cells are mortalized, for example, prepared as hybridomas or EBV-immortalized B cells. It is preferable to use a nucleic acid molecule or expression vector encoding the antibody of the present invention or an antigen-binding fragment thereof. 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, for example, by immunizing with human Nectin-4 protein. Obtained from mice that have been exposed to the virus.

[0145] multispecific antibodies In one aspect, the present invention provides 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 thereof or antibody analogue, thereby Multispecific, capable of binding not only to tyrosine-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), wherein The first scFv (or scdsFv) is a fragment of the present invention linked by a short peptide linker. The second scFv (or scdsFv) contains a VH of one antibody and a VL of another antibody, and a short peptide fragment. The VL of the present invention and the VH of another antibody are linked by a tethered linker, and the other antibody is The short peptide linker is a peptide that binds to an antigen other than cutin-4 and is 5 to 10 amino acids long. 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, such as natural killer (NK) cells, natural killer T cells, (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 comprises 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 a group consisting of a CH1, a CH2, a CH3, a CH4, a CH5, a CH6, a CH7, a CH8, a CH9, a CH10, a CH11, a CH12, a CH13, a CH14, a CH15, a CH16, a CH17, a CH18, a CH19, a CH20, a CH21, a CH22, a CH23, a CH24 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, nucleic acid molecules encoding multispecific antibodies can be produced using recombinant DNA techniques. The nucleic acid molecule can be obtained by optionally cloning it into an expression vector and then inoculating it into a host Transforming a cell with the nucleic acid molecule or expression vector and culturing the transformed host cell under appropriate conditions. to express the nucleic acid or expression vector, and finally to produce the multispecific antibody 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 an additional 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) It can be obtained by isolation from human B cells (i.e., immortalized B cells) or by chemical synthesis. The nucleic acid molecules of the present invention may 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, which contains regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid comprising the nucleic acid of the present invention 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 hydroxybenzoates can be prepared using techniques well known in the art, such as those described in Sambroo et al. 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, Wood Harbor, NY). Expression vectors can be used to express polypeptides 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 limitation, affinity tags (e.g., biotin, polyhistidine tags (His 6), or glutathione S-transferase (GSH) tag), protease cleavage site polypeptides containing the sequence, 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., a vector expressed in bacterial cells, yeast cells, or mammalian cells. In another embodiment, the expression vector is a DNA plasmid for expression in mammalian cells. In other embodiments, the expression vector is a viral 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 its The antigen-binding fragment is expressed in a host cell, for example, but not limited to, a prokaryotic cell. cells (e.g., bacteria, e.g., E. coli), eukaryotic cells (e.g., yeast, insects, etc.) 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 molecules encoding the variable region and the light chain variable region are cloned into a single vector, and the BL This is the only mammalian host cell suitable for antibody expression. Although not specified, 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. 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 making (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 the expression of the nucleic acid or expression vector; expressing the (III) isolating and denaturing the antibody or antigen-binding fragment of the present invention from a host cell or its culture medium. a step of purifying Also provided is a method comprising:

[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 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. The antibody further comprises a plasmid. Techniques for isolating and purifying antibodies from host cells are known to those skilled 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. Antibody drug conjugates (ADCs) are also provided, including synthetic fragments thereof. A typical type of antibody conjugate, in which the therapeutic agent is, for example, a cytotoxic agent. It's okay to have it.

[0162] In some preferred embodiments, the antibody conjugates of the invention bind to 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 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. antibodies 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, vin Alkaloids (e.g., vinblastine, vincristine), colchicine, dolastatin amines, taxanes, paclitaxel, docetaxel, cabazitaxel, enediyne antibiotics, Cytochalasins, camptothecins, anthracyclines (e.g., daunorubicin, dihydrochloride), hydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g., mitochondrial mycin, actinomycin, duocarmycin (e.g., CC-1065), auromycin mycin, duomycin, calicheamicin, endomycin, phenomycin), Xorubicin, daunorubicin, calicheamicin, cisplatin, ethidium bromide, Rheomycin, mitomycin, mithramycin, pladienolide, podophyllotoxin thiamin, etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine , mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, cal Mustine, lomustine, methotrexate, melphalan, teniposide, glucocorticoid 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 Radiolabeled antibodies are sometimes called radioimmunoconjugates. do.

[0165] In one embodiment, the therapeutic agent is a molecule with anti-tumor biological activity. Molecules that inhibit the growth 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] is.

[0166] Therapeutic agents and antibodies or antigen-binding fragments of the invention can be 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., amine, hydroxylamine, maleimide, carbo The alkyl group may contain 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 phenyl, thiol, sulfhydryl, and hydroxyl groups. In one embodiment, the linker is a chemical bond. In one embodiment, the linker is a phosphorus The carrier contains an amino acid or peptide consisting of 2 to 10 amino acids. It may be a natural amino acid or an unnatural amino acid.

[0168] In a preferred embodiment, the linker is selected from the group consisting of 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, followed by 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 structure and preparation method of such intermediates and the Methods for preparing antibody conjugates using the same are described, for example, in the entire relevant section. The invention described in International Patent Application Publication 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). They are connected by being created, [ka] The antibody conjugate of the present invention has the formula (2): [ka] (wherein A is an antibody or antigen-binding fragment of the present 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 1 and 10) In a specific embodiment, A is an anti-human Nectin-4 humanized monoclonal antibody of the present invention. It is a monoclonal 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 of the present invention or its antigen-binding activity The molar ratio of the fragments (DAR value) can be a decimal or integer between 1 and 10, for example, between 1 and 8. Decimal or integer numbers, 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 Constructing chimeric antigen receptors (CARs) using the antibodies of the present invention or antigen-binding fragments thereof 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 a chimeric antigen receptor (CAR) that is ... Targeting by antigen-binding fragments that specifically bind to antigens on the surface of cancer cells (e.g., cancer It specifically recognizes and binds to the antigen (antigen expressed on the surface of a cell) and also attaches a CAR to its surface. can confer targeting specificity to expressing cells (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 to Nectin-4. an antibody or an antigen-binding fragment thereof (e.g., scFv), a transmembrane domain, and one or more In such embodiments, the invention provides a nucleic acid molecule comprising an intracellular T cell signaling domain of Also provided is a method for producing a chimeric antigen receptor comprising the steps of: The nucleotide sequence of the chimeric antigen receptor can be used in combination with the antibody of the present invention or its antigen-binding activity. It further includes nucleotide sequences encoding fragments (e.g., 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. A nucleic acid molecule encoding a chimeric antigen receptor comprising the nucleic acid molecule of interest can be isolated.

[0176] In yet another aspect, the present invention provides a method for producing a CAR of the present invention, comprising administering to a mammalian animal, comprising administering to the mammalian animal a CAR of the present invention, a mammalian animal, or a mammalian target animal, comprising administering to the mammalian animal a CAR of the present invention, a mammalian target animal, or a mammalian target animal, ... a mammalian target animal, a mammalian Also provided are immune effector cells derived from the human T lymphocytes (e.g., T lymphocytes). 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 cause, for example, 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 Composition The present invention relates to antibodies or antigen-binding fragments, antibody conjugates, compositions, and CAR-expressing Immune effector cells, multispecific antibodies or fusion proteins of the invention, and pharmaceutically acceptable carriers thereof Pharmaceutical compositions comprising acceptable carriers and / or excipients are also provided.

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

[0180] Depending on the dosage form, pharmaceutically acceptable carriers and / or excipients may include, but are not limited to: However, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, flow agents Enhancers, sweeteners, coatings, structure-forming excipients, preservatives, antioxidants (e.g., ascorbic acid, Acid, Cysteine ​​Hydrochloride, Sodium Bisulfate, 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 syrup) , 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 may also be included.

[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 comprising antibody conjugates, preferred dosage forms are generally, for example, injectable solutions, The composition containing an antibody or an antigen-binding fragment thereof or an antibody conjugate may be a lyophilized powder. For compositions, suitable carriers and / or excipients include buffers (e.g., citrate buffer, acetate buffer, etc.). buffers, phosphate buffers, histidine buffers, histidine salt buffers), tonicity agents (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. The active substance (e.g., an antibody or an antibody conjugate of the present invention) is mixed in a suitable solvent, and the Add one or a combination of the above carriers and / or excipients, and then, if necessary, sterilize. Sterile injection solutions can be prepared by bacterial microfiltration. 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 desired additional 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, administration can be by systemic or local administration. Parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity) topical, local, epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local administration) The exact dosage to be administered will depend, for example, on the metabolic characteristics of the pharmaceutical composition, the duration of treatment, and the like. the duration, the rate of excretion of the particular compound, the purpose of treatment, the route of administration, and the age, health, and weight of the patient. , gender, diet, medical history, and other factors well known in the medical field. Those skilled in the art will appreciate that the method of administration will depend on factors such as whether the drug is injected or infused. There is a match.

[0184] As a general guideline, the antibody conjugates of the present invention may be administered in a concentration of about 0.0001 to 100 mg / mL. kg, more commonly in a dosage range of 0.01 to 20 mg / kg of subject body weight. For example, the dosage administered can be 0.3 mg per kg of body weight, 1mg per g, 3mg per kg of body weight, 5mg per kg of body weight, 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 after the first dose There may be a slight shortening of the dosing interval and then a prolongation of the dosing 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). Subsamples, e.g., from subjects not suffering from a disease associated with abnormal expression of Nectin-4. This refers to a value 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, such as stomach cancer, liver cancer, hepatocellular carcinoma, and 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 Preferably, the cancer is selected from the group consisting of esophageal cancer and esophageal cancer.

[0188] Cancer treatment may include, but is not limited to, the degree 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 therapeutic uses The anti-Nectin-4 antibody of the present invention, or its antigen-binding fragment, or antibody conjugate is administered to Nectin-4. diagnostics for detecting, diagnosing, or monitoring diseases and / or disorders associated with tyrosine-4 For example, the anti-Nectin-4 antibody provided by the present invention or its The antigen-binding fragment or antibody conjugate can be administered in situ, in vivo, ex vivo, For use in diagnostic or imaging assays in vivo and in vitro It is possible.

[0190] In some embodiments, for detecting the expression level of Nectin-4 in a subject, and / or Alternatively, the antibody or antigen-binding fragment of the present invention may 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 encompassed 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 colloidal gold 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) A step of contacting a sample with the antibody or antigen-binding fragment or labeled antibody of the present invention. 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 a sample or to detect the expression of Nectin-4. Steps to determine the current The present invention provides a method comprising:

[0192] The present invention provides a method for diagnosing a Nectin-4-associated disease in a subject, comprising: (a) obtaining a sample of interest; (b) Using the antibody or antigen-binding fragment or 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 the 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. Techniques include, but are not limited to, Western blotting, flow cytometry, and immunohistochemistry. using techniques known in the art, including immunohistochemistry (IHC), ELISA assays, 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 treatment and / or prevention of a disease, comprising administering to a subject a compound of the present invention in the manufacture of a medicament for use in the treatment and / or prevention of a disease. Antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies The present invention also relates to the use of a specific antibody, fusion protein or pharmaceutical composition. 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 in combination with at least one of the therapeutic agents described herein. The combination of the compounds can be administered in combination with at least one or more other compounds. For example, all of the above therapeutic agents can be administered simultaneously, or they can be administered separately. If administered separately (when the administration regimens are different from each other), administer them continuously without interruption. The doses can be administered in a single dose or at predetermined intervals.

[0197] Treatment method The present invention provides a method for treating a Nectin-4-associated disease 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, antibody conjugate, or composition of the present invention. a therapeutically effective amount of an immune effector cell, a multispecific antibody, a fusion protein, or a pharmaceutical composition The method includes administering the

[0199] In certain embodiments, the methods of the present invention include surgery, radiation therapy, chemotherapy, targeted therapy, and the like. a treatment 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 the use of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and the like. and other anti-tumor drugs. The method further includes administering one or more therapeutic agents to treat the tumor. Chemotherapeutic agents include, for example, 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 mib, carfilzomib, ixazomib, marizomib, oprozomib) and tyrosinequinone Examples of chemotherapeutic agents include: These include, but are not limited to, cyclophosphamide, ifosfamide, melphalan, and busulfan. Lufan, chlormethine, chlorambucil, lomustine, carmustine (BCNU), C CNU, cisplatin (DDP), carboplatin (CBP), oxaliplatin (OX A), methotrexate (MTX), 6-mercaptopurine (6-MP), 5-fluorouracil Uracil (5-FU), cytarabine, gemcitabine, vinblastine, vincristine, Vindesine, camptothecin, irinotecan, topotecan, rubitecan, etoposide, Niposide, paclitaxel, taxane, docetaxel, paclitaxel liposome, ac Tinomycin D, idarubicin, doxorubicin, epirubicin, mitomycin, ble These include cyclosporine, doxorubicin, and epirubicin. Immune checkpoint inhibitors These include, but are not limited to, PD-1, PD-L1, CTLA4, and LAG-3. and antibodies or antigen-binding fragments thereof that specifically bind 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 can.

[0201] In some embodiments, the antibodies or antigen-binding fragments thereof, antibody conjugates, immunoglobulins, and the like of the present invention are The immunological effector 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 its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment In yet another embodiment, the antibodies of the present invention are selected from the group consisting of: antibodies or antigen-binding fragments thereof, antibody conjugates, immune effector cells, multispecific antibodies 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 Kits containing instructions for using the kits are also provided. The kits may also include suitable containers. 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 or otherwise supplied with the kit. "(a)" includes any written or recorded material that is

[0203] Beneficial effects In a particular aspect, the present invention provides 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 examples, ELISA, biofilm interference techniques 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 affinity for in vitro 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 monkey nectin-4 was All were better than the prior art enfortumab, demonstrating that the antibodies of the present invention are effective in It has been 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 higher. Furthermore, the antibody of the present invention has high specificity for Nectin-4. 31HZ, 56HZ, and 74HZ all specifically recognize human nectin-4 However, it does not recognize nectin-1, nectin-2, and nectin-3. The antibody blocked the binding of nectin-4 to nectin-1, and the concentrations of 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. The present invention also provides a cutin-4 antibody conjugate. 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 inhibiting human breast ductal carcinoma cells that endogenously express human Nectin-4, human gastric cancer cells, and It has potent killing activity against various tumor cell lines, including human breast cancer cells and human ovarian cancer cells. Furthermore, in a mouse non-small cell lung cancer xenograft model, the ADC of the present invention showed significant tumor inhibition. It is active and has a good safety profile. [Example]

[0205] The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. The following examples are intended to be illustrative and not restrictive. Testing methods were performed according to conventional methods and conditions or according to the manufacturer's instructions.

[0206] Example 1: Preparation of anti-human nectin-4 humanized monoclonal antibody 1.1 Construction of a cell line overexpressing human nectin-4 To verify the specificity and function of the human nectin-4 antibody, the complete human nectin-4 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 analyzed as described in the literature (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 (ATCC) cells were infected with each virus and subjected to puromycin screening. Monoclonal A overexpressing human nectin-4 was isolated by cloning and monoclonal selection. 549-nectin-4 stable cell lines 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 rfentumab, 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 confirmed that A Both 549-nectin-4 and T24-nectin-4 had high positive rates (almost 100%) This allows both A549-nectin-4 and T24-nectin-4 to It was shown to be a sufficiently homogeneous 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 then treated with anti-human nectin-4 mice. The monoclonal antibody was obtained from human IgG1-positive mice emulsified with complete Freund's adjuvant (CFA). Nectin-4 protein (Sino Biological, Cat: 19771-H0 Two 5- to 6-week-old Balb / c mice and two 5- to 6-week-old C57 mice were used. Specifically, 50 μg of protein was used for subcutaneous primary immunization, followed by , and nectin-4 protein emulsified with incomplete Freund's adjuvant (IFA) every two weeks. After three boosts, the mice were given an intraperitoneal boost of 25 μg of Nectin-4. T47D tumor cells (a type of human breast cancer cell line) that endogenously express 6 Used individually A final intraperitoneal booster was administered using the antibody, and spleen cells were obtained 3 to 5 days later and used for hybridization. Mouse spleen cells were cultured in Sp2 / 0 (ATCC, Cat# CRL-1581) and then fused to the spleen of the mouse. ) mouse myeloma cell line was fused with PEG using standard fusion procedures, and then HAT Perform pressure screening using ELISA followed by ELISA screening 10-14 days later. As a result, approximately 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 were obtained for subcloning, and finally 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 for human nectin-4 was detected by ELISA. The lysoma monoclonal 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 the preparation of the antibody.

[0210] Isolated and purified from the culture supernatant of hybridoma cell lines 31#, 56# and 74# The affinity of the mouse monoclonal antibody was determined by ELISA. Sino Biological, Cat: 19771-H08H) with carbonate (CBS) buffer. Dilute to 1 μg / mL in buffer and add 100 μL / well to a 96-well plate The plate was then placed at 4°C for 16 to 20 hours. The CBS buffer in the 96-well plate was then pipetted. and resuspend the plate in PBST (pH 7.4, containing 0.05% Tween 20). Wash once with PBS (blocking solution) and once with 2% non-fat dry milk (PBST). ) 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 with PBST / 2% nonfat dry milk, and then The plate was added at a volume of 100 μL / well and incubated at room temperature for 1.5 hours. The reaction mixture was removed, and the plates were washed three times with PBST and diluted with PBST / 1% non-fat dry milk ( HRP-conjugated goat anti-mouse IgG secondary antibody (Jackson) at a dilution ratio of 1:5000 (purchased from the 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 100 μL / well of TMB was added. The mixture was added at 50°C and incubated at room temperature for 5-10 minutes for color development. The color development was stopped by adding 1 μL / well of PBS. 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 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 Hybridoma cells are cultured to approximately 8,000 to 10,000 cells, and then the cells are The first strand cDNA was lysed and purified using a cDNA reverse transcription kit (Thermo Fisher Scientific) The VH and VK genes were synthesized using cDNA. Amplified by PCR using primers from A, and purified the PCR product with a DNA purification kit (Q (Imagine, Cat#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 involved the following steps: 1. Enzyme synthesis of a mouse monoclonal antibody; The amino acid sequences were aligned with the amino acid sequences of human germline antibodies 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 fragments with low affinity. A framework sequence was selected; then, the six CDRs of the mouse antibody were selected for the heavy chain and The light chain framework sequences were grafted into each.

[0214] Furthermore, using computer simulation techniques and using molecular docking The framework amino acid sequences of the variable region and its surrounding region were analyzed, and the spatial distribution of the three Dimensional bonding modes were investigated: electrostatic forces, van der Waals forces, hydrophilic and hydrophobic forces, and elastomeric forces. By calculating the entropy value, the spatial fluctuating ratio of the molecules interacting with the nectin-4 protein was calculated. 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. These humanized antibodies were named 31HZ, 56HZ, and 74HZ, respectively. The heavy chain constant region of each antibody was a human IgG1 heavy chain constant region (SEQ ID NO: 61). The light chain constant region of the humanized antibody was the 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 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 amino 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) was synthesized using the The heavy chain and The plasmids corresponding to the light chain were co-transfected into CHO-E cells, and the supernatant was purified using the proteinase inhibitor ELISA. Purified by InA (MabSelect SuRe, GE) and anti-human nectin-4 A humanized antibody was obtained.

[0218] Example 2: Detection of affinity of anti-human nectin-4 humanized monoclonal antibody 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 monoclonal antibodies against nectin-4 proteins from different species The affinity of the antibody was detected by ELISA. The specific steps were as follows: Human nectin-4 or cynomolgus monkey 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 distribute per well on an ELISA plate. The plate was coated with 100 μL of the 100 μL plate and left to stand overnight at 4°C. The next day, the liquid in the well was discarded and Wash once with 300 μL of PBS (containing 2% BSA, purchased from BOVOGEN) 100 μL of BSAS 1.0 (Cat. No.) 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 (starting from 1500 ng / mL, 3-fold dilutions, 11 concentration points) and take 100 μL. , added to the corresponding wells and incubated at 37°C for 2 hours; 300 μL of PBST and washed three times with HRP-labeled goat anti-human secondary antibody (purchased from Jackson, 10 9-035-00) was diluted 1:10000 with PBS (containing 2% BSA) and added to 100 μL was taken and added to the corresponding wells and incubated at 37°C for 1 hour; Washed 5 times with 0 μL of TMB color development solution (purchased from InnoReagents, TMB- Add 100 μL of S-004 to the corresponding wells and allow to develop for 5-20 minutes at room temperature. To stop the reaction, 450 μL of 2N H2SO was added, and the OD450nm was 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, it is clear that the 31 Hz human The affinity for nectin-4 protein and cynomolgus monkey nectin-4 protein is Fortumab's response to human nectin-4 protein and cynomolgus monkey nectin-4 protein and 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 from different species Detection of dynamic affinity of a monoclonal antibody The dynamic affinity of anti-human nectin-4 humanized monoclonal antibody was analyzed using Octetforteva Detection was performed using Octet Fortebio®. The pool included: first, anti-human nectin-4 humanized monoclonal antibody (concentration 5 μg); g / mL) was immobilized on a ProA sensor, followed by human or cynomolgus nectin-4 (400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25 The resulting binding reaction was performed with 100 nM of ATP (serial diluted to 0 nM). The product fixed in the solid phase was 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 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 31 Hz, 56 Hz and 74 Hz The dynamic affinity of enfortumab for human thyroid cancer and cynomolgus monkey nectin-4 protein Dynamic affinity for monkey nectin-4 protein and cynomolgus monkey nectin-4 protein was better than [Table 9]

[0222] 2.3 Humanized anti-human nectin-4 monoclonal antibody against human nectin-4 on tumor cell membrane surface Clonal antibody affinity detection Using T47D cells, 31HZ and 74HZ were used to detect human nectin-4 on the cell membrane surface. The affinity of HZ was detected by the following steps: digesting T47D cells. The cells were then resuspended by centrifugation and washed twice with PBS; Resuspend 3 x 10 cells in PBS (preservative) and plate in a 96-well tip-bottom plate. 5 Pieces / well 50 μL of the antibody to be tested was added to a final concentration of 15 μg / mL. Starting from 0.01, 4-fold serial dilutions were added to each well for a total of 10 concentration points; After thorough mixing, the resulting mixture was incubated in the dark at 4°C for 1 hour. After washing three times with PBS, the cells were incubated with FITC-labeled anti-human Fc secondary antibody. The cells were incubated in the dark at 4°C with 100µL of PBS (BioLegend brand, product number 409322). After incubation for 0.5 hours at 4°C, the plate was washed three times with PBS and then analyzed by flow cytometry. (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, ATCC) The affinity of 56HZ for 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, it can be seen that 31 Hz and 5 The affinity of 6HZ for nectin-4 on the cell membrane surface was higher than that of enfortumab It is shown that the affinity for nectin-4 was equivalent to that for nectin-4. [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 by 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 antibody to be tested was adjusted to 1000pg / mL. The antibody was added to 100 μL of resuspended cells at a final concentration of 10 μg / mL and the isotype Antibody IgG was used as a negative control and was incubated on ice for 1 hour; After washing three times with pre-chilled PBS, the cells were resuspended in cell culture medium and divided into two portions. One part was placed in a cell incubator at 37°C and incubated for 1, 2 and 4 hours. The other part was kept on ice and incubated for 1 hour, 2 hours, and and incubated for 4 hours (affinity group); after incubation, the cells were washed with pre-cooled PBS. The cells were washed three times, resuspended in 50 μL of PBS (containing 1% BSA), and incubated with an anti-human fluorescent antibody. The primary 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 values ​​(expressed as mean fluorescence intensity (MFI)) were analyzed by 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 endocytic 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 antibodies 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 domain of the antibody to be tested was analyzed by flow cytometry. The specific steps were as follows: 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. 96-well tip-bottom plate, 300,000 cells / well, 50 μL per well 50 μL of the antibody to be tested was added to each well at a final concentration of 40 μg / mL. IgG was used as a negative control; after thorough mixing, the resulting mixture was incubated in the dark at 4°C. The reaction was allowed to proceed for 1 hour; after washing with PBS three times, FITC-labeled anti-human leukocytes were added. Add the Fc secondary antibody (Brand: BioLegend, Product Number: 409322) Incubated in the dark at 4°C for 0.5 h; washed three times with PBS and Cytometer (Flow cytometer brand: Beckman, model: Cytof Detection was performed using 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 It is shown that there was no binding to the C domain.

[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 nectin-1 through the IgV domain. 31HZ, 56HZ, and 74HZ are all IgV domains 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. Washing was performed once with 300 μL of PBST, followed by 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), where 31 Hz and 74 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; washed with 300 μL of PBST. was performed three times; HRP-streptavidin was diluted 1:1 with PBS (2% BSA). The solution was diluted 1:1000 and added to the corresponding wells at 100 μL / well. The solution was incubated at room temperature for 1 hour. Incubation was performed; washing was performed five times with 300 μL of PBST; TM B 100 μL was added to the corresponding wells and color development was carried out at room temperature for 10 minutes, and then The reaction was terminated by adding 450 μL of 2N H2SO4 and the measurement at 450 nm was A microplate reader was used to collect the 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, and their IC 50 The value is Holtumab IC 50 It is shown that the value was equivalent to that of the control (27.11 ng / mL).

[0232] Example 6: Detection of the specificity of anti-human nectin-4 humanized monoclonal antibodies The nectin family includes nectin-1, nectin-2, nectin-3 and nectin- 4, which share approximately 25% sequence identity. However, they have similar structures and functions. The body recognizes only nectin-4, but not nectin-1, nectin-2, or nectin-3. To detect whether or not the antibody had any specificity, the specificity of the tested antibody was determined 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 used for ELISA. A plate was coated with 100 μL per well and left to stand at 4°C overnight; the next day, The liquid in the wells was discarded and 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, add to the corresponding wells, and incubate for 2 hours at 37°C. Washing was performed three times with 300 μL of PBST. HRP-labeled goat anti-human IgG was used. Dilute the primary antibody with 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 was performed five times with 300 μL of ST; 100 μL of TMB color development solution was added to the corresponding washes. The color was developed at room temperature for 5-20 minutes; Add 450 μL of H2SO and collect the OD reading at 450 nm. Using a plate reader, the data was analyzed using GraphPad for curve fitting. d imported into Prism.

[0233] The experimental results are shown in Figures 8A, 8B, and 8C. Both 6HZ and 74HZ specifically recognize human nectin-4 and nectin-1. It is shown that the IgG1A-specific ...

[0234] Example 7: Preparation of anti-human nectin-4 ADC By coupling TL001 with anti-human nectin-4 humanized monoclonal antibody, Therefore, 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. Reference can be made to WO2019 / 114666.

[0236] The method for preparing anti-human nectin-4 antibody conjugate (anti-human nectin-4 ADC) , 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 edetate sodium A 5 mM sodium solution was added to the solution and mixed thoroughly; a 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) (The above solution was added to the above solution, mixed thoroughly, and allowed to stand at room temperature for 2 hours to obtain the coupled reagent. These samples were classified into 31HZ-TL001, 56HZ-TL001, and 74HZ-TL002, respectively. 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 x 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 to 3 minutes for disposal, 3 to 22 minutes for MS, 22 to 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 Lim Bin.

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

[0239] 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%), and therefore, the effects of toxins and antibodies 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, it is calculated to be 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. It 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] (wherein A represents an anti-human nectin-4 humanized monoclonal antibody, γ represents a thiol group of A) The number of TL001 molecules linked to A via a thioether bond is 1 to 1. 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 enfortumab-VC-MMAE was 1:5. The DAR value of this batch of enfortumab-VC-MMAE was It 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 A549 stable cell line was selected. The specific experimental steps were as follows: -Nectin-4 digested cells, collected cells by centrifugation, and resuspended in DMEM + 4% FBS medium. Resuspend cells in 100,000 / mL of medium and place 100 μL / well of cells in a 96-well plate. ADC molecules were added in DMEM basal medium at final concentrations of 150 μg / mL to 100 μg / mL. Starting with 11 concentration points, dilute the gradient as follows: 100 μL was added to give a final serum concentration of 2%. Incubate at 37°C, 5% CO The incubation was carried out in a 24-well plate at 4°C for 72 hours; Add 0 μL / well and incubate at 37°C in a 5% CO2 incubator for 0.5 to 2.5 hours. Incubate and read the OD at 450 nm every 30 minutes 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 response to A549-nectin. -4 cells, and EC 50 The values ​​were 1390ng / mL and 1 The results were 364ng / mL and 2049ng / mL, respectively. and 74HZ-TL001 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-TL001 EC 50 The values ​​were 5982ng / mL and 6251n Overall, the A549-nectin-4 cells and Regarding the killing activity against A549 cells, 31HZ-TL001 and 74HZ-TL00 1 and enfortumab-TL001 are nectin-4 specific.

[0245] 31HZ-TL against 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 effectively killed T24-nectin-4 cells. EC 50The values ​​were 1277ng / mL and 1568ng / mL, respectively. This indicates 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 EC of 6HZ-TL001 50 The values ​​were 2884ng / mL and 3665n 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. -4 was shown to be specific.

[0246] Example 9: Anti-human nectin-4 against tumor cell lines that endogenously express human nectin-4 Detection of killing activity of ADCs 31HZ-TL001, 74 against tumor cell lines endogenously expressing 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 a 5% CO2-free medium. The digested cells were collected by centrifugation and diluted to 100,000 / ml in 1640 + 4% FBS medium. The cells were resuspended to 100 μL / well and spread into a 96-well plate; ADC The molecules were diluted 4-fold into 1640 basal medium, starting from a final concentration of 150 μg / mL, 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 cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. CCK8 (Rhinogen) was added at 20 μL / well, and 3 Incubate at 7°C in a 5% CO2 incubator for 0.5 to 2.5 hours, and then resuspend every 30 minutes. OD450nm readings were collected on a microplate reader (MD) and curve fit was performed. The data was imported into GraphPad Prism for mapping.

[0247] The experimental results are shown in Figure 10A. -TL001 and enfortumab-TL001 effectively killed T47D cells. Can be done, EC 50 The values ​​were 206.1ng / mL, 611.3ng / mL, and 229. The killing of T47D cells by 31HZ-TL001 was shown to be 2ng / mL. The activity was comparable 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 Figure 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 The results showed that the serum creatine phosphate concentrations were 2277 ng / mL, 2277 ng / mL, and 1190 ng / mL. The killing activity of 31HZ-TL001 against MDA-MB-468 cells was confirmed. 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 Figure 10C. Both HZ-TL001 and HZ-TL001 were able to effectively kill NCI-N87 cells, with an EC5 The 0 values ​​were 9376 ng / mL and 10935 ng / mL, respectively. As a result, the killing activity of 31HZ-TL001 and 56HZ-TL001 was substantially equal. It is shown that

[0250] In conclusion, human nectin-4 was detected in 31HZ-TL001 and 56HZ-TL001. The killing activity of enfortumab-TL001 against tumor cell lines expressing the IL-1 receptor agonist (IL-1) was confirmed by the human The killing activity is essentially equivalent to that against tumor cell lines that endogenously express tyrosine-4.

[0251] Example 10: In vivo pharmacodynamics of anti-human nectin-4 ADC 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 cultured at 37°C and 5% CO2. The cells in the exponential growth phase were harvested and resuspended in PBS. The cells were then suspended in a suspension of female NOD / SCID mice (Biocytogen Jiangsu Gen e Biotechnology Co., Ltd.) 1×10 7 pcs / mouse (PBS The tumor mass was measured by subcutaneous inoculation with 0.1 mL of PBS (suspended in 0.1 mL of PBS). Depth 70~100mm 3 Once tumor volume reached 1000 mg / kg, mice were randomized according to tumor volume, with 5 mice per group. The day of grouping was recorded as day 0, and 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 (3 mg / kg and 10 mg / kg), Enfortumab-TL001 group (10 mg / kg) and enfortumab-VC-MMAE group (10 mg / kg) All samples were administered via tail vein injection twice a week for a total of six doses.

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

[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 The inhibitory effect was shown to be 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 indicated that the animals This indicates that the ADC was well tolerated. [Table 13]

[0255] From the above results, 31HZ-T at the dose levels of 3 mg / kg and 10 mg / 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 is essentially the same. 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 ADC 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 measured in a 3 x 3 x 3 mm diameter tube. Cut into small pieces and place in a NOD / SCID mouse (Jiangsu Jicui Yaokan The mice were subcutaneously inoculated into the right anterior scapula of mice (Gibberish 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 for 10 min. The mice were randomly divided into groups of 7 mice per group, and the day of grouping was defined as day 0. There were four groups: a 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 6 doses (day 20), 3 56H at 3 mg / kg compared with the 56H at 3 mg / kg human IgG1 (hIgG1) negative control group Z-TL001 showed 105.55% (P<0.05) inhibitory TGI on tumor growth. Partial tumor regression was observed in four mice in the 3 mg / kg 56HZ-TL001 group. At 3 mg / kg, 56HZ-TL001 significantly reduced the isotype-specific Significantly improved tumor growth compared to the control ADC (hIgG1-TL001, 3 mg / kg) One mouse in the hIgG1 group died, and No deaths were observed in the other groups, which had normal activity. There was a slight decrease in body weight, with the IgG1 group losing the most weight due to the lack of effective treatment. The tumor-bearing mice also showed a significant decrease in the number of tumors present in the ADC, demonstrating that the ADC was well tolerated by the tumor-bearing mice. It is shown that:

[0258] The above results indicate that 56HZ-TL001 is effective in treating BL9200 bladder cancer PDX xenograft models. Furthermore, it was shown that the compound showed a significant inhibitory effect on tumor growth in 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 can 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 in the appended claims. ranges and any equivalents thereof.

[0260] Array List [Table 15] TIFF2026041845000027.tif223148TIFF2026041845000028.tif215148

Claims

1. An antibody that specifically binds to nectin-4 or an antigen-binding fragment thereof, (a) The following three heavy chain CDRs: CDR-H1 containing the sequence described in Sequence ID No. 39, CDR-H2 containing the sequence described in Sequence ID No. 40, CDR-H3 containing the sequence described in Sequence ID No. 41, and / or The following three light chain CDRs: CDR-L1 containing the sequence described in Sequence ID No. 42, CDR-L2 containing the sequence described in Sequence ID No. 43, CDR-L3 containing Sequence ID No. 8; or (b) The following three heavy chain CDRs: CDR-H1 containing the sequence described in Sequence ID No. 45, CDR-H2 containing the sequence described in Sequence ID No. 46, CDR-H3 containing the sequence described in Sequence ID No. 41, and / or The following three light chain CDRs: CDR-L1 containing the sequence described in Sequence ID No. 42, CDR-L2 containing the sequence described in Sequence ID No. 43, CDR-L3 containing the sequence described in Sequence ID No. 8; or (c) The following three heavy chain CDRs: CDR-H1 containing the sequence described in Sequence ID No. 47, CDR-H2 containing the sequence described in Sequence ID No. 48 or 64, CDR-H3 containing the sequence described in Sequence ID No. 41, and / or The following three light chain CDRs: CDR-L1 containing the sequence described in Sequence ID No. 42, CDR-L2 containing the sequence described in Sequence ID No. 43, CDR-L3 containing the sequence described in Sequence ID No. 8; or (d) The following three heavy chain CDRs: CDR-H1 containing the sequence described in Sequence ID No. 49, CDR-H2 containing the sequence described in Sequence ID No. 50, CDR-H3 containing the sequence described in Sequence ID No. 51, and / or The following three light chain CDRs: CDR-L1 containing the sequence described in Sequence ID No. 52, CDR-L2 containing the sequence described in Sequence ID No. 53, CDR-L3 containing the sequence described in Sequence ID No. 8 including, An antibody or its antigen-binding fragment.

2. VH or a variant thereof having the sequence described in SEQ ID NO: 37 or SEQ ID NO: 59 and / or VL or a variant thereof having the sequence described in SEQ ID NO: 38 or SEQ ID NO: 60 Includes, The antibody or antigen-binding fragment thereof according to claim 1, wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions with respect to the sequence from which it is derived.

3. (1) VH or a variant thereof having the sequence described in SEQ ID NO: 37 and / or VL or a variant thereof having the sequence described in SEQ ID NO: 38; or (6) VH or a variant thereof having the sequence described in Sequence ID No. 59 and / or VL or a variant thereof having the sequence described in Sequence ID No. 60 Includes, The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived.

4. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a mouse antibody, a chimeric antibody, or a humanized antibody.

5. Further comprising a human immunoglobulin heavy chain constant region or a variant thereof and a light chain constant region or a variant thereof, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain constant region has an IgG heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, or IgG4 heavy chain constant regions, and the light chain constant region is a κ or λ light chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain constant region is the IgG1 heavy chain constant region.

8. The heavy chain steady region has the sequence described in SEQ ID NO: 61, and the light chain steady region is a κ-light chain steady region; and / or the light chain steady region has the sequence described in SEQ ID NO:

62. The antibody or antigen-binding fragment thereof according to claim 5.

9. Including heavy chains and light chains, (c) The heavy chain comprises a VH having the sequence described in SEQ ID NO: 37 and a heavy chain constant region having the sequence described in SEQ ID NO: 61, and the light chain comprises a VL having the sequence described in SEQ ID NO: 38 and a light chain constant region having the sequence described in SEQ ID NO: 62; or (f) The heavy chain comprises a VH having the sequence described in SEQ ID NO: 59 and a heavy chain constant region having the sequence described in SEQ ID NO: 61, and the light chain comprises a VL having the sequence described in SEQ ID NO: 60 and a light chain constant region having the sequence described in SEQ ID NO:

62. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

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

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

12. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, conjugated with at least one label.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the label is selected from the group consisting of enzymes, fluorescent dyes, radioisotopes, biotin, and colloidal gold.

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

15. An immunoeffector cell expressing the CAR described in claim 14 on its surface.

16. The immune effector cell according to claim 15, wherein the immune effector cell is selected from the group consisting of cytotoxic T cells, natural killer cells, and natural killer T cells.

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

18. An expression vector comprising the nucleic acid molecule described in claim 17.

19. A host cell comprising the nucleic acid molecule described in claim 17 or the expression vector described in claim 18.

20. A multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and at least one other antibody or antigen-binding fragment thereof or antibody analog, wherein the multispecific antibody binds to nectin-4 and at least one other antigen.

21. The multispecific antibody according to claim 20, wherein the multispecific antibody is a bispecific, triplicate, or quadruplicate antibody.

22. A fusion protein comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, and an additional biologically active polypeptide.

23. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, an immunoeffector cell according to claim 15, a multispecific antibody according to claim 20, or a fusion protein according to claim 22, and Pharmacopoecitable carriers and / or excipients, A pharmaceutical composition containing the following:

24. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, an immunoeffector cell according to claim 15, a multispecific antibody according to claim 20, a fusion protein according to claim 22, or a pharmaceutical composition according to claim 23, for use in the treatment of cancer.

25. An antibody or antigen-binding fragment thereof, immunoeffector cells, multispecific antibody, fusion protein, or pharmaceutical composition for use according to claim 24, wherein the cancer is selected from the group consisting of gastric cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, urethral cancer, renal pelvis cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, ductal carcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, and esophageal cancer.

26. An antibody or antigen-binding fragment thereof, an immunoeffector cell, a multispecific antibody, a fusion protein, or a pharmaceutical composition for use according to claim 24, to be used in combination with one or more therapeutic agents selected from the group consisting of antineoplastic agents, chemotherapeutic agents, radioisotopes, and immune checkpoint inhibitors.

27. An antibody or antigen-binding fragment thereof, immunoeffector cells, multispecific antibody, fusion protein, or pharmaceutical composition for use according to claim 24, to be used in combination with one or more treatment regimens selected from the group consisting of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative care.

28. A method for detecting the presence of nectin-4 in a sample or determining the expression level of nectin-4, (a) The step of contacting a sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, (b) The step of detecting the presence of nectin-4 in the sample or determining the expression level of nectin-4 by detecting the formation of an immune complex between an antibody or antigen-binding fragment in the sample and nectin-4, or by determining the amount thereof. Methods that include...

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