Methods and compositions related to antibodies and antibody drug conjugates (ADCs) that bind to NECTIN-4 proteins

Abstract

The present invention describes antibody drug conjugates (ADCs) that bind to the NECTIN-4 protein and variants thereof. NECTIN-4 exhibits tissue-specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Thus, the ADC of the invention provides a therapeutic composition for the treatment of cancer.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 628,028, filed June 13, 2023, and U.S. Provisional Patent Application No. 63 / 575,054, filed April 5, 2024, the contents of which are incorporated herein by reference in their entirety.

[0003] Submission of the sequence list XML file (“Sequence List XML”) The following submission is incorporated herein by reference in its entirety: the contents of the computer-readable form (CRF) of the sequence list as an XML file (file name: 9300-20000.40 - SEQ LIST - XML ​​- 07-June-2024, entry date: June 6, 2024, size: 136 KB).

[0004] Claims of rights for inventions made under federally sponsored research not applicable. Technical Field

[0005] The present invention described herein relates to antibodies that bind to the NECTIN-4 protein, their antigen-binding fragments, and antibody-drug conjugates (ADCs). The invention further relates to prognostic, preventative, and therapeutic methods and compositions suitable for treating cancer and other immune and neurological disorders. Background Technology

[0006] Cancer is the second leading cause of death worldwide, after coronary disease.

[0007] Despite advancements in cancer therapies and improved survival rates over the past decades, the heterogeneity of cancer necessitates novel treatment strategies utilizing multiple therapeutic modalities. This is particularly relevant for solid tumors located at key anatomical sites (e.g., gliomas, head and neck squamous cell carcinomas, and lung adenocarcinomas), which are sometimes limited to standard radiotherapy and / or chemotherapy. However, these therapies have adverse effects such as chemotherapy and radioresistance, which exacerbate local regional recurrence, distant metastases, and secondary primary tumors, as well as severe side effects that reduce patients' quality of life.

[0008] The therapeutic efficacy of monoclonal antibodies (mAbs) in combating cancer and other medical conditions has been recognized (Georges Köhler and César Milstein, Nature 256:495-497 (1975)). Generally, antibodies function through many mechanisms, most of which are involved in other parts of the immune system.

[0009] Antibody-drug conjugates (ADCs) are a newly emerging class of targeted therapeutic agents with a therapeutic index superior to traditional chemotherapy. Besides (monoclonal) antibodies (mAbs) and target selection, the drug and the linker group have been the focus of ADC development. However, recently, the importance of conjugate homogeneity has been explored. It has been reported that the pharmacological profile of ADCs can be improved by applying site-specific binding techniques that utilize surface-exposed cysteine ​​residues in antibodies engineered to subsequently bind to the linker drug, thereby producing site-specifically bound ADCs with a defined drug-antibody ratio (DAR).

[0010] Several methods for obtaining ADCs are disclosed in the prior art. See, for example, WO2006 / 034488 (Genentech), SUTHERLAND et al., Blood 122(8):1455-1463 (2013), WO2014 / 124316 (Novartis), US2017 / 0080103 (Synthon Biopharmaceuticals), US11,559,582 (Agensys, Inc.), and WO2019 / 183438 (Seattle Genetics, Inc.), etc.

[0011] All existing techniques disclosed to date focus on site-binding of drug linkage groups at surface / solvent-exposed locations, at locations exhibiting high thiol reactivity, and at locations within specific constant regions of monoclonal antibodies, with the aim of improving homogeneity and pharmacokinetic properties.

[0012] While the conventional lysine and cysteine ​​binding methods described above have yielded FDA-approved antibody-drug conjugates and are used to construct a large portion of the numerous ADCs currently in preclinical and clinical trials, there is still a need for novel binding strategies aimed at (further) improving the physicochemical, pharmacokinetic, pharmacological, and / or toxicological properties of ADCs to obtain ADCs with acceptable antigen-binding properties, in vivo efficacy, therapeutic index, and / or stability.

[0013] Based on the foregoing, those skilled in the art will readily recognize the need for novel therapeutic paradigms to treat cancer and immune diseases. A new class of antibodies can be achieved through the use of modern antibody engineering techniques and novel binding methods, aiming to provide more effective treatment, reduce side effects, and lower production costs.

[0014] In view of the current deficiencies known in the art, one objective of the present invention is to provide novel and improved antibodies and binding ligands, and methods for treating cancer, immune disorders and other diseases using antibodies and ADCs. Summary of the Invention

[0015] This invention provides antibodies, antigen-binding fragments, antibody-drug conjugates (ADCs), antibody-immunomodulatory conjugates, antibody fusion proteins, and antibody fragment fusion proteins that bind to the NECTIN-4 protein and its polypeptide fragments. In some embodiments, this invention comprises a fully human antibody that binds to a therapeutic agent.

[0016] In some embodiments, the limitation is that the entire nucleic acid sequence of Table IV is not encoded and / or the entire amino acid sequence of Table V is not prepared. In some embodiments, the entire nucleic acid sequence of Table IV is encoded and / or the entire amino acid sequence of Table V is prepared, either in the form of the corresponding human unit dosage form.

[0017] The present invention further provides various immunogenic or therapeutic compositions, such as antibodies, antibody-drug conjugates, and strategies for treating cancers expressing NECTIN-4, such as those listed in Table I.

[0018] In another embodiment, the present invention teaches an antibody composition designated CL.Z.

[0019] In another embodiment, the present invention teaches an antibody composition labeled CL.X.

[0020] In another embodiment, the present invention teaches an antibody composition labeled CL.N.

[0021] In another embodiment, the present invention teaches an antibody composition labeled CL.I.

[0022] In another embodiment, the present invention teaches an antibody composition labeled CL.B.

[0023] In another embodiment, the present invention teaches an antibody composition labeled CL.D.

[0024] In another embodiment, the present invention teaches a method for synthesizing antibodies.

[0025] In another embodiment, the present invention teaches a method for synthesizing antibodies and for partially binding a drug to an antibody to form an ADC.

[0026] In another embodiment, the present invention teaches a method for treating cancer in humans.

[0027] In another embodiment, the present invention teaches a method for treating human immune or neurological disorders.

[0028] In another embodiment, the present invention teaches the use of one or more of the compositions described herein in the manufacture of a medicament for treating human cancer, immune and / or neurological disorders. Attached Figure Description

[0029] Figure 1 The Nectin-4 antibody exhibits specificity for antibody binding to multiple cell lines.

[0030] Figure 2 The Nectin-4 antibody binds to the T-47D breast cancer cell line. Figure 2 (A). Show Ab1. Figure 2 (B). Show Ab2. Figure 2 (C). Show Ab3. Figure 2 (D). Show Ab4. Figure 2 (E). Show Ab5. Figure 2 (F). Show Ab6.

[0031] Figure 3 The affinity of Nectin-4 antibody for the NCI-H292 lung cancer cell line compared to the corresponding ADC.

[0032] Figure 4 Nectin4 ADC demonstrated in vitro cytotoxicity across multiple cancer cell lines. Figure 4 (A). Display of the NCI-H322 lung cancer cell line. Figure 4 (B). PC3 prostate cancer cell lines expressing NECTIN-4 recombinant PC3-NECTIN-4. Figure 4 (C). Displaying the NECTIN-4 negative cell line PC3.

[0033] Figure 5 In vitro cytotoxicity of the Sum190PT breast cancer cell line using NECTIN-4 ADCs with multiple payloads. Figure 6 Bystander activity of ADC compared to enfortumab vedotin. Figure 6 (A). NECTIN-4 negative cell lines co-cultured with cell lines expressing NECTIN-4. Figure 6 (B). Demonstration of NECTIN-4 negative cell lines in monoclonal culture.

[0034] Figure 7 In vivo efficacy of NECTIN-4 ADC with multiple payloads in HT-1376 xenograft model.

[0035] Figure 8In vivo efficacy of NECTIN-4 ADC in a NECTIN-4 positive Sum190PT breast cancer xenograft model.

[0036] Figure 9 In vivo efficacy of NECTIN-4 ADC compared to Vitin-Enterotomab in a NECTIN-4 positive patient-derived head and neck cancer model.

[0037] Figure 10 Drug-linking group (DL) payload structure. Figure 10 (A). Show the structure marked DL-01. Figure 10 (B). Show the structure marked DL-02. Figure 10 (C). Show the structure marked DL-03. Figure 10 (D). The structure is shown as DL-04. Figure 10 (E). Show the structure marked DL-05. Figure 10 (F). Show the structure marked DL-06. Figure 10 (G). Show the structure marked DL-07. Figure 10 (H). Show the structure marked DL-08. Figure 10 (I). Show the structure marked DL-09. Figure 10 (J). The structure marked DL-10 is shown. Figure 10 (K). Show the structure marked DL-11. Figure 10 (L). The structure marked DL-12 is shown. Figure 10 (M). The structure marked DL-13 is shown. Figure 10 (N). Show the structure marked DL-14. Figure 10 (O). Show the structure marked DL-15. Figure 10 (P). Show the structure marked DL-16.

[0038] Figure 11 NECTIN-4 ADC demonstrates in vitro cytotoxicity across multiple primary cultures of normal human cells. Figure 11 (A). Displaying HCEpC cells. Figure 11 (B). Displaying HDFa cells. Figure 11 (C). Displaying HEKa cells.

[0039] Figure 12 Flow cytometry histograms of NECTIN-4, emfretuzumab, and Ab5 across multiple primary cultures of normal human cells. Figure 12 (A). Displaying HCEpC cells. Figure 12(B). Displaying HDFa cells. Figure 12 (C). Displaying HEKa cells.

[0040] Figure 13 Cell cycle analysis of Ab5-ADC2 in HT-1376 cells. Figure 13 (A). Showing the merged G2 and sub-G1 phases of HT-1376 cells after 72 hours of treatment with Ab5-ADC2. Figure 13 (B). A representative flow cytometry histogram showing the Ab5-ADC2-treated cells compared to untreated control cells.

[0041] Figure 14 Free payloads induced immunogenic cell death in NCI-H292 cancer cells compared to MMAE.

[0042] Figure 15 Complement-dependent cytotoxicity analysis of Ab5-ADC and Ab5 in HT-1376 cancer cells compared to emfretuzumab antibody.

[0043] Figure 16 Antibody-dependent cell-mediated cytotoxicity (ADCC) activities of Ab5-ADC2 and Ab5 in NCI-H292 cancer cells.

[0044] Figure 17 Ab5-ADC2 and Ab5 exhibit antibody-dependent cell-mediated phagocytosis (ADCP) activity across multiple cancer cell lines.

[0045] Figure 18 Pharmacokinetic (PK) profile of total IgG, ADC and free payload of Ab5-ADC2.

[0046] Figure 19 In vivo efficacy of NECTIN-4 ADC Ab5-ADC2 compared to Vitin-Enterotomab in the NECTIN-4 positive patient-derived cervical cancer model PDX36. Figure 19 (A) Demonstrating tumor growth dynamics. Figure 19 (B). Show the survival odds in the Kaplan-Meier plot.

[0047] Figure 20 In vivo efficacy of NECTIN-4 ADC Ab5-ADC2 in mouse clinical trials of six (6) cancer models. Figure 20 (A). Showing PDX10. Figure 20 (B). Showcase PDX12. Figure 20(C). Showing PDX13. Figure 20 (D). Showcase PDX16. Figure 20 (E). Showing PDX34. Figure 20 (F). Showing PDX36.

[0048] Figure 21 NECTIN-4 expression in a patient-derived cervical cancer xenograft model was determined via immunohistochemistry. Figure 21 (A), 21(C), 21(E), 21(G), 21(I) and 21(K) are presented in high resolution. Figure 21 (B), 21(D), 21(F), 21(H), 21(J) and 21(L) are displayed in low resolution. Figure 21 (A) and 21(B). Show PDX12. Figure 21 (C) and 21(D). Show PDX10. Figure 21 (E) and 21(F). Showing PDX16. Figure 21 (G) and 21(H). Show PDX13. Figure 21 (I) and 21(J). Showing PDX34. Figure 21 (K) and 21(L). Showing PDX36.

[0049] Figure 22 The concentration of free payload of Ab5-ADC2 or Vitin-Enterotomab in normal and tumor tissues. Figure 22 (A). Show the concentration of free payload in normal tissue. Figure 22 (B). Demonstrating the effective payload concentrations of Ab5-ADC2 and Vitin-Entertorumab.

[0050] Figure 23 Stability overview of Ab5-ADC2. Figure 23 (A). Showing the percentage of Ab5-ADC2 binding stability and initial drug-antibody ratio (DAR) compared to Vitin-Enterotomab. Figure 23 (B). Deconvolutional MS profiles of the heavy and light chains of affinity-purified Ab5-ADC2 are presented. Detailed Implementation

[0051] Chapter Overview I.) Definition II.) Antibodies III.) Antibody-drug conjugates IV.) Linking group unit V.) Extended group unit VI.) Amino acid units VII.) Spacer unit VIII.) Drug Unit IX.) Drug load X.) Methods for determining the cytotoxic effects of ADCs XI.) Treatment of cancers expressing NECTIN-4 XII.) NECTIN-4 ADC mixture XIII.) Combination Therapy XIV.) Reagent Kit / Product I.) Definition: Unless otherwise defined or explicitly indicated by the context, all technical terms, designations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention relates. In some cases, terms having their commonly understood meanings are defined herein for clarity and / or convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the general understanding in the art. Where necessary, unless otherwise indicated, procedures relating to the use of commercially available kits and reagents are generally performed according to the manufacturer's defined protocols and / or parameters.

[0052] Unless the context otherwise indicates, when a trademark is used herein, reference to the trademark also refers to the product formulation, generic medicine, and active pharmaceutical ingredient of the trademarked product.

[0053] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has extended through the associated tissue capsule and include stage C disease in the American Urological Association (AUA) system, stage C1-C2 disease in the Whitmore-Jewett system, and stage T3-T4 and N+ disease in the TNM (tumor, nodule, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease who have significantly worse outcomes compared to patients with clinically localized (organ-limiting) cancer.

[0054] The term "substituted" means that the specified group or portion carries one or more substituents. The term "unsubstituted" means that the specified group does not carry any substituents. The term "optionally substituted" means that the specified group is either unsubstituted or substituted by one or more substituents. When the term "substituted" is used to describe a structural system, substitution means occurring at any position on the system where the valence is allowed.

[0055] The term "analyte" refers to a molecule that is structurally similar to or shares similar or corresponding properties with another molecule (e.g., a NECTIN-4-related protein). For example, NECTIN-4 protein analogs can specifically bind to NECTIN-4 antibodies or T cells.

[0056] Unless otherwise explicitly indicated, the term "antibody" is used in its broadest sense. Thus, an "antibody" can be naturally occurring or synthetic, such as a monoclonal antibody produced by conventional fusion tumor or transgenic mouse technology. NECTIN-4 antibodies comprise monoclonal and polyclonal antibodies and fragments containing antigen-binding domains and / or one or more complementarity-determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds to NECTIN-4 and / or exhibits the desired biological activity, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they specifically bind to NECTIN-4 and / or exhibit the desired biological activity. Any specific antibody may be used in the methods and compositions provided herein. Thus, in one embodiment, the term "antibody" encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule, the variable regions combining to form a specific binding site against a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. The antibodies suitable for use in the methods and compositions of this invention can be generated in cell cultures, in bacteriophages, in yeast, or in various animals, including but not limited to cattle, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment, the antibody of this invention is a mammalian antibody. Phage technology can be used to isolate initial antibodies or to generate variants with altered specificity or affinity characteristics. Such techniques are conventional and well known in the art. In one embodiment, the antibody is generated by recombinant means known in the art. For example, recombinant antibodies can be generated by transfecting host cells with a vector containing a DNA sequence encoding the antibody. One or more vectors can be used to transfect DNA sequences expressing at least one VL and at least one VH region in the host cell.Exemplary descriptions of recombinant methods for antibody generation and production include Delves, *Antibody Production: Essential Techniques* (Wiley, 1997); Shepard et al., *Monoclonal Antibodies* (Oxford University Press, 2000); Goding, *Monoclonal Antibodies: Principles and Practice* (Academic Press, 1993); and *Current Protocols in Immunology* (John Wiley & Sons, latest edition). The antibodies of this invention can be modified by recombinant methods to enhance the efficacy of antibody-mediated desired functions. Therefore, within the scope of this invention, antibodies can be modified by substitution using recombinant methods. Typically, the substitution will be a conservative substitution. For example, at least one amino acid in the constant region of an antibody can be substituted with a different residue. See, for example, U.S. Patent Nos. 5,624,821, 6,194,551, and WO 9958572; and ANGAL et al., Molecular Immunology 30: 105-08 (1993). Modifications in amino acids include the deletion, addition, and substitution of amino acids. In some cases, such changes are made to reduce undesirable activities, such as complement-dependent cytotoxicity. Typically, antibodies are labeled by covalently or non-covalently binding to substances that provide a detectable signal. A wide variety of labeling and binding techniques are known and widely reported in scientific and patent literature. These antibodies can be screened for binding to normal or deficient NECTIN-4. See, for example, Antibody Engineering: A Practical Approach (Oxford University Press, 1996). Suitable antibodies with the desired biological activity can be identified using the following in vitro analyses, including but not limited to those for proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and the following in vivo analyses, such as inhibition of tumor growth. The antibodies described herein are also suitable for diagnostic applications. As capture antibodies or non-neutralizing antibodies, they can be screened to bind to specific antigens without inhibiting receptor binding or biological activity. As neutralizing antibodies, antibodies can be used for competitive binding assays.It can also be used to quantify NECTIN-4 and / or its receptors.

[0057] As used herein, the term "antigen-binding fragment" or "antibody fragment" (or simply "antibody portion") of an antibody refers to one or more fragments of a NECTIN-4 antibody that retain the ability to specifically bind to an antigen (e.g., NECTIN-4 and / or its variants). Antigen-binding function of an antibody has been shown to be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, a V... L V H C L and C H1 (ii) A monovalent segment composed of structural domains; (iii) A divalent segment consisting of two Fab segments linked by disulfide bridges at the hinge region; (iv) A Fd segment consisting of V H and C H1 (iv) Fv fragment, consisting of the V domain of the antibody's single arm. L and V H Structural domain composition; (v) dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of V H Structural domain composition; and (vi) separated complementary determinant regions (CDRs). Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by an independent gene, but which can be synthesized using recombinant methods by joining synthetic linker groups that enable it to be prepared as a single protein chain, wherein V L and V H The regions pair to form monovalent molecules (called single-chain Fv (scFv); see, for example, BIRD et al., (1988) Science 242:423-426; and HUSTON et al. (1988) Proceedings of the National Academy of Sciences of the United States of America 85:5879-5883). The single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are targeted for efficacy screening in the same manner as intact antibodies.

[0058] As used herein, the term "Fc" refers to the region containing the hinge region, CH2 and / or CH3 structural domains.

[0059] As used herein, any form of "antigen" can be used to generate antibodies specific to the NECTIN-4 of the present invention. Thus, the initiating antigen can be a single epitope, multiple epitopes, or the entire protein, or in combination with one or more immunogenic enhancers known in the art. The initiating antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunized with only the extracellular domain portion of the protein). The antigen can be generated in genetically modified cells. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term "portion" in the case of an antigen refers to the minimum number of amino acids or nucleic acids required to constitute an immunogenic epitope of the antigen of interest. Any gene vector suitable for transforming the cells of interest can be used, including but not limited to adenoviral vectors, plasmids, and nonviral vectors such as cationic lipids. In one embodiment, the antibody of the methods and compositions described herein specifically binds to at least a portion of the extracellular domain of the target.

[0060] The antibodies or antigen-binding fragments thereof provided herein may constitute or be part of a “bioactive agent.” As used herein, the term “bioactive agent” means any synthetic or naturally occurring compound that binds to an antigen and / or enhances or modulates a desired biological effect to enhance cell-killing toxins. In one embodiment, the binding fragment suitable for use in the present invention is a biologically active fragment. As used herein, the term “biologically active” means an antibody or antibody fragment capable of binding to a desired antigenic epitope and exerting a biological effect directly or indirectly. Direct effects include (but are not limited to) regulation, stimulation, and / or inhibition of growth signaling; regulation, stimulation, and / or inhibition of anti-apoptotic signaling; regulation, stimulation, and / or inhibition of apoptosis or necrosis signaling; regulation, stimulation, and / or inhibition of ADCC cascades; and regulation, stimulation, and / or inhibition of CDC cascades and / or Fc silencing.

[0061] As used herein in relation to antigen binding, the term "specifically binding" means that an antigen-binding protein binds to the target and discrete domains or discrete amino acid sequences within the target, without binding or significantly binding to other (e.g., unrelated) proteins. However, this term does not preclude the fact that antibodies or their binding fragments may also cross-react with closely related molecules. The antibodies and their fragments described herein, as well as antibody-drug conjugates containing them, can specifically bind to NECTIN-4 disclosed herein with an affinity at least 2, 5, 10, 50, 100, or 1000 times greater than that bound to closely related molecules.

[0062] Bispecific antibodies can also be used in the methods and compositions of this invention. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, that has binding specificity to at least two different antigenic epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be generated recombinantly using the co-expression of two immunoglobulin heavy / light chain pairs. See, for example, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical bonding. See, for example, Brennan et al., Science 229:81 (1985). Bispecific antibodies comprise bispecific antibody fragments. See, for example, Hollinger et al., Proceedings of the National Academy of Sciences 90:6444-48 (1993); Gruber et al., Journal of Immunology 152:5368 (1994).

[0063] The monoclonal antibodies described herein specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided that they specifically bind to the target antigen and / or exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences 81: 6851-6855 (1984)).

[0064] As used herein, the terms “cancer,” “tumor,” and “necrotic tumor” are used interchangeably and in the singular or plural form, referring to cells that have undergone malignant transformation to become pathological for the host organism. Primary cancer cells (i.e., cells derived from the vicinity of the malignant transformation site) can be readily distinguished from non-cancerous cells by recognized techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells and in vitro cultures and cell lines derived from cancer cells. When referring to cancer types that typically express as solid tumors, a “clinically detectable” tumor is one that can be detected based on a tumor mass; for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound, or palpation, and / or by the expression of one or more cancer-specific antigens available in a sample from the patient. A tumor can be a hematopoietic tumor, such as a tumor of blood cells, or a similar tumor, meaning a liquid tumor. Specific examples of clinical conditions based on these types of tumors include leukemia, such as chronic myeloid leukemia or acute myeloid leukemia; myeloma, such as multiple myeloma; lymphoma and similar tumors.

[0065] The term "therapeutic agent" refers to all agents that provide therapeutic benefit and / or are therapeutically effective as defined herein. Therapeutic agents may, for example, reverse, improve, alleviate, inhibit, or limit the progression of a disease, symptom, or condition, or reduce the severity of a disease, symptom, or condition, or affect, improve, or enhance one or more symptoms of a disease, such as cancer. Such agents may be cytotoxic agents or cell growth inhibitors. The term includes, but is not limited to, chemotherapeutic agents, anti-hypertrophic agents, and "pharmaceutical units" as defined herein.

[0066] The term "anti-hyperplastic agent" refers to all drugs that provide therapeutic benefit and / or are therapeutically effective (as defined herein) for the treatment of hyperplasia or cancer.

[0067] The term "chemotherapy agent" refers to all chemical compounds that effectively inhibit tumor growth. Non-limiting examples of chemotherapy agents include alkylating agents, such as nitrogen mustard, ethyleneimine compounds, and alkyl sulfonates; antimetabolites, such as folic acid, purine, or pyrimidine antagonists; mitotic inhibitors, such as anti-microtubule agents, such as vinca alkaloids, auristatin, and derivatives of podophyllotoxin; cytotoxic antibiotics; compounds that impair or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. Additionally, chemotherapy agents include cytotoxic agents (as defined herein), antibodies, biomolecules, and small molecules.

[0068] The terms “complementarity-determining region” and “CDR” are known in the field and refer to the discontinuous sequence of amino acids that confer antigen specificity and binding affinity within the variable region of an antibody. Generally, each heavy chain variable region contains three (3) CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region contains three (3) CDRs (CDR-L1, CDR-L2, CDR-L3).

[0069] The exact amino acid sequence boundaries of the CDR can be readily determined using any of many well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB273, 927-948 (“Chothia” numbering scheme); Maccalum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography”, J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains”, Developmental and Comparative Immunology (DevComp Immunol). ,January 2003; 27(1):55-77 (“IMGT” numbering scheme); and Honegger A. and Plicthun A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool”, Journal of Molecular Biology , June 8, 2001; 309(3):657-70, (AHo numbering scheme).

[0070] The boundaries of the CDRs can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody domain sequence, with some antibodies exhibiting insertions regulated by insert letters, such as "30a," and deletions. Both schemes place certain insertions and deletions ("insertions and deletions") in various positions, thus producing differential numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in several ways.

[0071] Therefore, unless otherwise specified, the terms “CDR” and “complementarity-determining region” or regions thereof for an antibody, such as the variable region, and individual CDRs (e.g., CDR-H1, CDR-H2) or regions thereof, should be understood to encompass complementarity-determining regions as defined by any of the known schemes described above herein. In some cases, a scheme is specified for identifying one or more particular CDRs, such as CDRs defined by the Kabat, Chothia, or Contact methods.

[0072] As used herein, the term “conservative substitution” refers to a substitution of an amino acid and / or amino acid sequence known to those skilled in the art, and which can generally be done without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., *Molecular Biology of the Gene*, The Benjamin / Cummings Pub. Co., p. 224 (4th ed., 1987)). Such exemplary substitutions are preferably made according to those set forth in Tables II and III. For example, such variations include replacing any other amino acid in these hydrophobic amino acids with any of isoleucine (I), valine (V), and leucine (L); replacing glutamic acid (E) with aspartic acid (D) and vice versa; replacing asparagine (N) with glutamine (Q) and vice versa; and replacing threonine (T) with serine (S) and vice versa. Depending on the specific amino acid's environment and its role in the protein's three-dimensional structure, other substitutions can also be considered conserved. For example, glycine (G) and alanine (A) are often interchangeable, just as alanine (A) and valine (V) are interchangeable. The relatively hydrophobic methionine (M) is often interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the amino acid residue's charge is a significant characteristic and the pK difference between the two residues is not significant. Other alterations may still be considered “conservative” in specific contexts (see, for example, Table III in this paper; pp. 13–15, “Biochemistry”, 2nd ed., Lubert Stryer (Stanford University); HENIKOFF et al., PNAS 1992, Vol. 89, 10915–10919; LEI et al., JBiol Chem, May 19, 1995; 270(20):11882–6). Other substitutions may also be permissible and can be determined empirically or based on known conserved substitutions.

[0073] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity, function, and / or causes cell damage. The term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auromycin, chlortetracycline, camptothecin (a topoisomerase 1 inhibitor), maytansine, ricin, ricin A chain, combrestatin, duocarmycins, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, CC1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin (PE) A. Pseudomonas exotoxin (PE) A), PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, soapwort ( Sapaonaria officinalis Inhibitors, and glucocorticoids and other chemotherapeutic agents, as well as radioactive isotopes such as At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 or 213 P 32 And radioactive isotopes of Lu, including Lu 177 .

[0074] Antibodies, including those of the present invention, may also bind to any of the aforementioned cytotoxic agents and to anticancer precursor activating enzymes capable of converting precursor drugs into their active forms.

[0075] As used herein, the term "bifunctional antibody" refers to a small antibody fragment having two antigen-binding sites, said fragment containing a polypeptide chain (V) linked to the same antigen-binding site. H -V L The light chain variable structural domain (V) in ) L The heavy chain variable structural domain (V) H By using a linker group that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain on another chain, resulting in two antigen-binding sites. Bifunctional antibodies are more fully described, for example, in EP404,097; WO 93 / 11161; and Hollinger et al., Proceedings of the National Academy of Sciences 90:6444-48 (1993).

[0076] The term "homology" refers to a molecule that exhibits homology with another molecule, for example, by having the same or similar sequence of chemical residues at the corresponding positions.

[0077] The term “consistent” or “sequence consistency” indicates the degree of consistency between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

[0078] Taking into account the number of vacancies and the length of each vacancy required for optimal alignment of two sequences, the "percentage consistency" between two sequences is a function of the number of consistent positions shared by the sequences (i.e., consistency % = number of consistent positions / total number of positions multiplied by 100). Sequence comparison and the determination of percentage consistency between two sequences can be performed using mathematical algorithms, as described below. Percentage consistency between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWS gap dna CMP matrix and vacancy weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Percentage consistency between two nucleotide or amino acid sequences can also be determined using the algorithm in Meyers et al., Comput. Appi. Biosci., 4:11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage consistency between two amino acid sequences can be determined using the algorithm of NEEDLEMAN et al., Journal of Molecular Biology 48:444-453 (1970), which is incorporated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0079] For example, a polynucleotide sequence may be identical to a reference polynucleotide sequence, i.e., 100% identical to the reference sequence, or it may include up to an integer number of nucleotide changes compared to the reference sequence, such as at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical. Such changes are selected from at least one nucleotide deletion, substitution (including transfer and transversion), or insertion, and said changes may occur at the 5' or 3' end position of the reference nucleotide sequence or at any position between those end positions, individually scattered between nucleotides in the reference sequence or within one or more adjacent groups in the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in the reference polynucleotide sequence as described herein by a numerical percentage of the corresponding percentage of identical nucleotides (divided by 100) and subtracting said product from the total number of nucleotides in the reference polynucleotide sequence, or: n n ≤x n -(x ny ), where n n x represents the number of nucleotide changes. n Let y be the total number of nucleotides in the reference polynucleotide sequence as described herein (see the nucleic acid sequences in the "Sequence Listing" for exemplary reference polynucleotide sequences), and let y be 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, and let x be the sign of the multiplication operator, where x is the number of nucleotides in the reference polynucleotide sequence as described herein (see the nucleic acid sequences in the "Sequence Listing" for exemplary reference polynucleotide sequences). n Any non-integer product of x and y is rounded down to the nearest integer, and then taken from x. n Subtract from the reference sequence. Similarly, a polypeptide sequence may be identical to a polypeptide reference sequence as described herein, i.e., 100% identical, or may include up to an integer number of amino acid changes compared to the reference sequence, such that the identity percentage is less than 100%, for example, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical. Such changes are selected from the group consisting of at least one amino acid deletion, substitution (including conserved and non-conserved substitutions), or insertion, and said changes may occur at the amino-terminal or carboxyl-terminal position of the reference polypeptide sequence or at any position between those terminal positions, individually scattered among amino acids in the reference sequence or in one or more adjacent groups within the reference sequence. The number of amino acid changes for a given identity percentage is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by the polypeptide reference sequence by a numerical percentage of the corresponding identity percentage (divided by 100) and then subtracting said product from the total number of amino acids in the polypeptide reference sequence as described herein, or: n a ≤xa -(x ay ), where n a x represents the number of amino acid changes. a Let y be the total number of amino acids in the reference polypeptide sequence as described herein, and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.75 for 75%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.98 for 98%, 0.99 for 99%, or 1.00 for 100%, where x is the sign of the multiplication operator. a Any non-integer product of x and y is rounded down to the nearest integer, and then taken from x. a Subtract from the middle. The percentage of consistency can be determined across the entire sequence length. As defined herein, the term "more than 75% consistency" includes more than 75%, 80%, 85%, 95%, and 99% consistency, as well as all discrete values ​​and discrete subranges within this range.

[0080] In one embodiment, the antibody presented herein is a “human antibody.” As used herein, the term “human antibody” refers to an antibody whose entire light and heavy chain sequence, including the complementarity-determining region (CDR), is derived from a human gene. In one embodiment, the human monoclonal antibody is prepared using trioma technology, human B cell technology (see, for example, Kozbor et al., *Immunol. Today* 4: 72 (1983)), EBV transgenic technology (see, for example, Cole et al., *Monoclonal Antibodies and Cancer Therapy* 77-96 (1985)), or using yeast or phage display (see, for example, Marks et al., *Molecular Biology Journal* 222:581 (1991)). In a particular embodiment, the human antibody is generated in transgenic mice. Techniques for preparing such partial to whole-human antibodies are known in the art and any such techniques may be used. According to a particularly preferred embodiment, a fully human antibody sequence is prepared in a transgenic mouse engineered to express human heavy and light chain antibody genes. Exemplary descriptions of the preparation of transgenic mice producing human antibodies are found in application WO02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix) and its sublines. Subsequently, B cells from the transgenic mice producing the desired antibodies can be fused to prepare fusionoma cell lines for continuous antibody production. See, for example, U.S. Patents 5,569,825; 5,625,126; 5,633,425; 5,661,016 and 5,545,806; and JAKOBovitz, Adv. Drug Del. Rev. 31:33-42 (1998); Green et al., J. Exp. Med. 188:483-95 (1998).

[0081] As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from non-human (e.g., mouse) antibodies as well as human antibodies. These antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain at least one, and typically two, variable domains, substantially all of which are variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin and all or substantially all of the FR regions are FR regions of the human immunoglobulin sequence. Optionally, a humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. See, for example, Cabilly, U.S. Patent No. 4,816,567; Queen et al., (1989) Proceedings of the National Academy of Sciences 86:10029-10033; and Antibody Engineering: A Practical Guide (Oxford University Press, 1996).

[0082] As used in this article, the term “inhibition” or “inhibition of…” means reducing a measurable amount or preventing it entirely.

[0083] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cattle, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0084] The terms “metastatic cancer” and “metastatic disease” mean cancer that has spread to local lymph nodes or distant sites and is intended to be included in stage D disease under the AUA system and stage T×N×M+ disease under the TNM system.

[0085] As used herein, the term "modified" refers to a change in a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such changes or modifications can be obtained through post-synthetic modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or through co-translation, or through post-translational modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide.

[0086] "Molecular recognition" refers to a chemical event in which a host molecule can form a complex with a second molecule (i.e., a guest molecule). This process occurs via non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a largely homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies exhibit high specificity against a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically comprise multiple antibodies targeting (or specific to) different epitopes. In one embodiment, a polyclonal antibody contains multiple monoclonal antibodies with different epitope specificities, affinities, or affinity for a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a largely homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention may be prepared by a fusion tumor method first described by Keller et al., Nature 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described in Clarkson et al., Nature 352: 624-628 (1991) and Max et al., Journal of Molecular Biology 222: 581-597 (1991). These monoclonal antibodies are typically bound to Kd at at least about 1 μM, more usually at least about 300 nM, usually at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM, or even more preferably, and are typically determined by ELISA.

[0088] "Non-natural amino acid" or "nnAA" refers to an amino acid that is not one of the twenty (20) common amino acids, or pyrolysine, or selenocysteine. Other terms that may be used synonymously with "nnAA" include "non-natural encoded amino acid," "unnatural amino acid," and "non-natural occurring amino acid." In addition, the term nnAA includes, but is not limited to, amino acids that are not naturally occurring and can be obtained synthetically or by modifying non-natural amino acids.

[0089] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, wetting agents, preservatives and the like.

[0090] "Pharmaceutical acceptable" means a non-toxic, inert, and / or composition that is physiologically compatible with humans or other mammals.

[0091] The term "polypeptide" means a polymer having at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, amino acids are represented by a standard three-letter name (see Table II) or a single-letter name. In the relevant field, this term is often used interchangeably with "peptide" or "protein."

[0092] As used herein, the terms "single-chain Fv" or "scFv" or "single-chain" antibody refer to a single-chain antibody containing a V-type antibody. H and V L Antibody fragments containing domains, wherein these domains are present within a single polypeptide chain. Generally, Fv polypeptides further contain V... H With V L The polypeptide linker groups between the domains enable sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0093] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the selective binding of an antibody to an epitope of a target antigen. Binding specificity of an antibody can be tested by comparing binding to an appropriate antigen with binding to an unrelated antigen or a mixture of antigens under a given set of conditions. An antibody is considered specific if it binds to an appropriate antigen at least 2, 5, 7, and preferably 10 times more to an unrelated antigen or a mixture of antigens. In one embodiment, a specific antibody is an antibody that binds only to the NECTIN-4 antigen but not to any other unrelated antigens. In another embodiment, a specific antibody is an antibody that binds to a human NECTIN-4 antigen but not to a non-human NECTIN-4 antigen having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid homology to the NECTIN-4 antigen. In another embodiment, the specific antibody is an antibody that binds to human NECTIN-4 antigen but not to non-human NECTIN-4 antigens having a concordance percentage of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher with respect to the amino acid sequence of the NECTIN-4 antigen. In another embodiment, the specific antibody is an antibody that binds to both human and murine NECTIN-4 antigens, but with a higher degree of binding to the human antigen. In another embodiment, the specific antibody is an antibody that binds to both human and primate NECTIN-4 antigens, but with a higher degree of binding to the human antigen. In yet another embodiment, the specific antibody binds to both human and any non-human NECTIN-4 antigen, but with a higher degree of binding to the human antigen or any combination thereof.

[0094] As used herein, the terms “to treat” or “therapeutic” and grammatically related terms refer to any improvement in disease outcomes, such as long-term survival, lower morbidity, and / or reduced side effects, as a byproduct of alternative treatment modalities; as is readily understood in the art, complete eradication of disease is preferred, but not a requirement for therapeutic actions.

[0095] The term "variant" refers to a molecule that exhibits variation from the described type or standard, such as a protein having one or more different amino acid residues at the corresponding position in a particular described protein (e.g., the NECTIN-4 protein shown in Table IV). Analogs are one example of variant proteins. Splice isotypes and single nucleotide polymorphisms (SNPs) are other examples of variants.

[0096] The phrase "isolated" or "biopure" refers to a material that is substantially or essentially free of components typically associated with the material as found in its native state. Therefore, the isolated peptides according to the invention preferably do not contain materials typically associated with peptides in their in-situ environment. For example, a polynucleotide is called "isolated" when it is isolated from contaminant polynucleotides that correspond to or complement genes other than the NECTIN-4 gene or encode polypeptides other than the NECTIN-4 gene product or fragments thereof. Those skilled in the art can readily obtain isolated NECTIN-4 polynucleotides using nucleic acid isolation procedures. For example, a protein is called "isolated" when NECTIN-4 protein is removed from cellular components typically associated with proteins using physical, mechanical, or chemical methods. Those skilled in the art can readily obtain isolated NECTIN-4 protein using standard purification methods. Alternatively, isolated proteins can be prepared by chemical means.

[0097] Suitable “labels” include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Additionally, the antibodies presented herein can be used as antigen-binding components of fluorinated antibodies. (See, for example, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003)).

[0098] The “NECTIN-4 protein” and / or “NECTIN-4 related protein” of this invention include proteins specifically identified herein (see Table IV), as well as allelic variants, conserved substitution variants, analogs, and homologs that can be isolated / generated and characterized without excessive experimentation by following methods outlined herein or readily available in the art. It also includes fusion proteins combining portions of different NECTIN-4 proteins or fragments thereof, and fusion proteins of NECTIN-4 protein and heterologous polypeptides. These NECTIN-4 proteins are collectively referred to as NECTIN-4 related proteins, proteins of this invention, or NECTIN-4. The term "NECTIN-4 related protein" refers to a polypeptide fragment or NECTIN-4 protein sequence having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 1 35, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 515, 516, 517, 518, 519 or more amino acids.

[0099] II.) Antibodies Another aspect of the invention provides an antibody that binds to NECTIN-4 as disclosed herein. In one embodiment, the antibody binds to NECTIN-4 (Table IV) and other NECTIN-4-related proteins.

[0100] As known in the art, the NECTIN-4 antibodies of the present invention are particularly suitable for cancers (see, for example, Table I) for prognostic analysis, imaging, diagnosis, and treatment methods. In one embodiment, NECTIN-4 binding assays for detecting cancer, for example, in an immunoassay, are disclosed herein. Similarly, such NECTIN-4 antibodies are suitable (e.g., when combined with a therapeutic agent, such as in an ADC) for the treatment and / or prognosis of cancers (e.g., those described in Table I) in which NECTIN-4 is also expressed or overexpressed. Furthermore, intracellularly expressed antibodies (e.g., single-chain antibodies) are therapeutically suitable for treating cancers involving the expression of NECTIN-4 and other targets.

[0101] Various methods for preparing antibodies, especially monoclonal antibodies, are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host with NECTIN-4-related proteins, peptides, or fragments in isolated or immunobinding forms (Antibodies: A Laboratory Manual, CSH Press, eds. Harlow and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Laboratory Press, New York State (1989)). Alternatively, fusion proteins of NECTIN-4, such as the NECTIN-4 GST fusion protein, can be used. In one particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of NECTIN-4 is generated and subsequently used as an immunogen to generate appropriate antibodies. In another embodiment, a NECTIN-4-related protein is synthesized and used as an immunogen.

[0102] In addition, naked DNA immunization techniques known in the field (with or without purified NECTIN-4-related proteins or NECTIN-4-expressing cells) were used to generate an immune response against the encoded immunogen (for the review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0103] Preferred methods for generating NECTIN-4 antibodies are further illustrated with the examples provided herein. Methods for preparing proteins or peptides to be used as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers, such as BSA, KLH, or another carrier protein, are also well known in the art. In some cases, direct binding is used, for example, with carbodiimide reagents; in others, conjugating reagents, such as those supplied by Pierce Chemical Co., Rockford, Illinois, are effective. As understood in the art, the administration of the NECTIN-4 immunogen is typically carried out by injection at an appropriate time and with the use of a suitable adjuvant. During the immunization schedule, the antibody titer can be used to determine the suitability for antibody formation.

[0104] NECTIN-4 monoclonal antibodies can be produced through various methods well-known in the field. For example, immortalized cell lines secreting the desired monoclonal antibody can be prepared using the standard fusion tumor technique of Köhler and Milstein, or by modifying antibody-producing B cells to immortalize them, as is commonly known. Immortalized cell lines secreting the desired antibody are screened by immunoassay, where the antigen is a NECTIN-4-related protein. When a suitable immortalized cell culture is identified, the cells can expand, and the antibody is produced either from the in vitro culture or from ascites.

[0105] The antibodies or fragments of the present invention can also be generated by recombinant means. Regions that specifically bind to the desired region of the NECTIN-4 protein can also be generated in the case of chimeric or complementarity-determining region (CDR) transplanted antibodies from multiple species. Humanized or human NECTIN-4 antibodies can also be generated and are preferably used in therapeutic settings. Methods for humanizing murine and other non-human antibodies by replacing one or more of the corresponding human antibody sequences in a non-human antibody CDR are well known (see, for example, Jones et al., 1986, Nature 321: 522-525; RIECHMANN et al., 1988, Nature 332: 323-327; VERHOEYEN et al., 1988, Science 239: 1534-1536). See also Carter et al., 1993, Proceedings of the National Academy of Sciences 89: 4285 and Sims et al., 1993, Journal of Immunology 151: 2296.

[0106] In one embodiment, the human monoclonal antibody of the present invention can be prepared using VelocImmune mice, wherein the genomic sequence of an endogenous mouse variable segment carrying the immunoglobulin heavy chain (VH, DH, and JH fragments) and / or κ light chain (VK and JK) locus has been wholly or partially replaced by a human genomic sequence carrying the unrearranged germline variable segment of the human immunoglobulin heavy chain (VH, DH, and JH) and / or κ light chain (VK and JK) locus (Regeneron, Tarrytown, NY). See, for example, U.S. Patents 6,586,251, 6,596,541, 7,105,348, 6,528,313, 6,638,768, and 6,528,314.

[0107] In addition, the human antibodies of the present invention can be generated using human immunoglobulin gene mini-loci containing sequences encoding unrearranged human heavy chain (mu and γ) and κ light chain immunoglobulin sequences and targeted mutants of HuMAb mice (Medarex, Inc.) that inactivate endogenous mu and κ chain loci (see, for example, LONBERG et al., (1994) Nature 368(6474): 856-859).

[0108] In another embodiment, the all-human antibody of the present invention can be produced using mice carrying human immunoglobulin sequences on transgenic and transchromosomal transfections, such as mice carrying human heavy chain transgenic and human light chain transchromosomal transfections. These mice are referred to herein as "KM mice," and are described in Tomizuka et al., (2000) Proceedings of the National Academy of Sciences 97:722-727 and PCT Publication WO 02 / 43478 granted to Tomizuka et al.

[0109] The human monoclonal antibodies of the present invention can also be prepared using phage or yeast display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies have been established in the art. See, for example: U.S. Patent Nos. 5,223,409 and 5,403,484; and U.S. Patent No. 5,571,698 to Ladener et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793, 6,521,404, 6,544,731, and 6,555,313 to Griffiths et al.; and U.S. Patent Nos. 6,582,915 and 6,593,081 to Griffiths et al.

[0110] The human monoclonal antibodies of the present invention can also be prepared using SCID mice in which human immune cells have been reconstituted to produce a human antibody response upon immunization. Such mice are described, for example, in U.S. Patents Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0111] Alternatively, the human antibodies of the present invention can be prepared using transgenic mice that are inactivated against antibody production and are engineered with human heavy and light chain loci, and are referred to as transgenic mice (Amgen Fremont, Inc., formerly Abgenix, Inc.). An exemplary description of the preparation of transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103. See also U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Mendez et al., Nature Genetics, 15: 146-156 (1998); Kellerman SA and Green LL, Curr. Opin. Biotechnol 13,593-597 (2002).

[0112] Any of the above methods yields an antibody with a certain ability to bind to NECTIN-4, or a homolog or fragment or polypeptide sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9%, 98%, or 99% sequence identity with NECTIN-4.

[0113] Binding affinity (KbA1c) of antibodies against NECTIN-4, their binding fragments, and antibody-drug conjugates containing them. D ) can be 1 mM or lower, 100 nM or lower, 10 nM or lower, 2 nM or lower, or 1 nM or lower. Or, K D It can be between 5 and 10 nM; or between 1 and 2 nM. K D It can be between 1 micromolar and 500 micromolars or between 500 micromolars and 1 nM.

[0114] The binding affinity of an antigen-binding protein is determined by the association constant (Ka) and dissociation constant (Kd) (KD = Kd / Ka). Binding affinity can be measured via BIACORE, for example, by capturing the test antibody on a protein A-coated sensor surface and allowing NECTIN-4 to flow across this surface. Alternatively, binding affinity can be measured via FORTEBIO, for example, by capturing the test antibody receptor on a protein A-coated needle and allowing NECTIN-4 to flow across this surface. Those skilled in the art can identify other suitable analytical methods known in the art for measuring binding affinity.

[0115] The engineered antibody of the present invention includes V H and / or V L Antibodies whose framework residues have been modified (e.g., to improve antibody properties). Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to "reverse mutate" one or more framework residues to their corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody was derived. These residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody was derived. To restore the germline configuration of the framework region sequence, somatic mutations can be "reverse mutated" to germline sequences (e.g., leucine "reverse mutated" to methionine) through, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverse mutated" antibodies are also intended to be included in this invention.

[0116] V can also be used H and / or V L Engineering modifications can be made to alter the binding affinity to the antigen. For example, this invention also intends to cover altering residues within the framework and / or CDR region to increase affinity or decrease affinity for Nectin-4.

[0117] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization" and is described in further detail in U.S. Patent Publication No. 2003 / 0153043 by Carl et al.

[0118] In addition to or alternatively to modifications made within the framework or CDR region, the antibodies of the present invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the NECTIN-4 antibody of the present invention may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or modified to alter its glycosylation, similarly altering one or more functional properties of the antibody. These embodiments are further described in detail below.

[0119] In one embodiment, the hinge region of CH1 is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing the number. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Changing the number of cysteine ​​residues in the CH1 hinge region can, for example, promote light and heavy chain assembly or improve or decrease the stability of the NECTIN-4 antibody.

[0120] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the NECTIN-4 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment to weaken the binding of the antibody to Staphylococcyl protein A (SpA) relative to the binding to the native Fc hinge domain SpA. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0121] In another embodiment, the NECTIN-4 antibody is modified to prolong its biological half-life. Various methods can be employed. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to prolong the biological half-life, the antibody can be modified within the CH1 or CL region to contain a rescue receptor-binding epitope consisting of two loops derived from the CH2 domain of the Fc region of IgG, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 to Presta et al.

[0122] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(A).

[0123] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(B).

[0124] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(C).

[0125] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(D).

[0126] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(E).

[0127] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain sequence described in Table VI(F).

[0128] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(G).

[0129] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(H).

[0130] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(I).

[0131] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(J).

[0132] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(K).

[0133] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain sequence described in Table VI(L).

[0134] In another embodiment, the NECTIN-4 antibody comprises the antibody heavy chain variable region sequence as described in Table VIII.

[0135] In another embodiment, the NECTIN-4 antibody comprises the antibody light chain variable region sequence as described in Table IX.

[0136] In another embodiment, the NECTIN-4 antibody comprises the antibody CDR sequence described in Table X.

[0137] In another embodiment, the NECTIN-4 antibody binds to the therapeutic agent.

[0138] The reactivity of NECTIN-4 antibodies can be established using a variety of well-known methods, including Western blotting, immunoprecipitation, ELISA, and FACS analysis, using NECTIN-4-related proteins, NECTIN-4-expressing cells, or extracts thereof as needed. NECTIN-4 antibodies or fragments thereof can be labeled with detectable markers or bound to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes.

[0139] III.) Antibody-drug conjugates In another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody (preferably, the NECTIN-4 antibody disclosed herein) bound to a therapeutic agent. The therapeutic agent may be a cytotoxic agent, a cell growth inhibitor, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzyme-active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioactive conjugate). In another aspect, the present invention further provides a method of using the ADC. In one aspect, the ADC comprises any one of the above-mentioned NECTIN-4 antibodies covalently linked or linked via an oxime bond to a cytotoxic agent or a detectable drug.

[0140] In another embodiment, the ADC comprises a NECTIN-4 antibody bound to a therapeutic agent using autologous hydrolyzed maleimide for cysteine ​​modification (see WO 2013 / 173337).

[0141] In another embodiment, the ADC comprises a NECTIN-4 antibody that binds to a therapeutic agent using cysteine ​​modification and further comprises reducing cysteine ​​residues to form a sulfhydryl moiety.

[0142] In another embodiment, the ADC comprises a NECTIN-4 antibody that binds to a therapeutic agent using both a polypeptide portion and a self-degrading portion.

[0143] In another embodiment, the ADC comprises a NECTIN-4 antibody that binds to a therapeutic agent, wherein the ADC has a high drug-antibody ratio (DAR).

[0144] Using existing technologies, the use of antibody-drug conjugates in the treatment of cancer for the local delivery of cytotoxic agents or cell growth inhibitors (Syrigos and Epinetos (1999) Anticancer Research 19:605-614; NICULESCU-DUVAZ and SPRINGER (1997) Advanced Drug Delivery Review 26:151-172; U.S. Patent No. 4,975,278) allows for the partial targeted delivery of drugs to tumors and the formation of intracellular accumulation therein, where systemic administration of these unbound drug agents can produce unacceptable levels of toxicity to normal cells as well as tumor cells that attempt to be eliminated (BALDWIN et al. (1986) The Lancet, pp. (March 15, 1986):603-05; Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506. The aim is to achieve maximum efficacy with minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be suitable for these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these approaches include daunomycin, doxorubicin, methotrexate, and vindesin (Rowland et al., (1986), see above).Toxins used in antibody-toxin conjugates include: bacterial toxins, such as diphtheria toxin; plant toxins, such as ricin; small molecule toxins, such as geldanamycin (MANDLER et al., (2000) Journal of the National Cancer Institute 92(19):1573-1581; MANDLER et al., (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; MANDLER et al., (2002) Bioconjugate Chem. 13:786-791); and maytansine (EP 1391213; LIU et al., (1996)). Proceedings of the National Academy of Sciences of the United States of America 93:8618-8623; and kazimidox (LODE et al., (1998) Cancer Res. 58:2928; HINMAN et al., (1993) Cancer Res. 53:3336-3342). Toxins can affect their cytotoxic and cell growth-inhibiting effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or weakly active when they bind to large antibody or protein receptor ligands.

[0145] To date, the FDA has approved twelve (12) ADCs, including gemtuzumabozogamicin (MYLOTARG, Wyeth Pharmaceuticals), which was the first ADC approved by the FDA in 2000. (See, for example, Drago et al., Nature Reviews, 2021, 18, 327-344; Mckertish et al., Biomedicines, 2021, 9, 872; Khongorzui et al., Molecular Cancer Res., 2020, 18:3-19; Bross et al., Clin. Cancer Res., 2001, 7, 1490-1496; Hamann et al., Bioconjugation Chemistry, 2002, 13, 47-58; Lamb, Drugs, 2017, 77, 1603-1610).

[0146] Other examples of commercially available antibody drug conjugates include ADCETRIS (brentuximab vedotin, Seattle Genentech), ZEVALIN® (ibritumomabtiuxetan, Biogen / Idec), KADCYLA® (ado-trastuzumab emtansine, Genentech), BESPONSA® (inotuzumabozogamicin, Pfizer / Wyeth), POLIVY (polatuzumab vedotin, Genentech / Roche), Cantuzumab mertansine (Immunogen, Inc.), MLN-2704 (Millennium Pharm., BZL Biotech). Biologics, Immunogen Inc. and PADCEV (enfortumab vedotin-ejfv, Seattle Gene / Astellas, Inc., Santa Monica, California).

[0147] Furthermore, this document describes therapeutic agents, including but not limited to, those suitable for generating ADCs. Usable enzymatically active toxins and their fragments include the diphtheria A chain, the non-bound active fragment of diphtheria toxin, and the exotoxin A chain (from Pseudomonas aeruginosa). Pseudomonas aeruginosa ), ricin A chain, absinin A chain, modizoxin A chain, α-coriacin, guarcinia ( Aleurites fordii Protein, carnation protein, pokeweed protein Phytolaca americana Proteins (PAPI, PAPII, and PAP-S), bitter melon ( momordica charantia Inhibitors, jatropha toxin, croton toxin, soapwort inhibitors, leucopicrin, mitogens, aspergillin, phenolmycin, enoxacin, and trichothecene. See, for example, WO 93 / 21232, published October 28, 1993. Various radionuclides can be used to generate radioactively bound antibodies. Examples include... 177 Lu、 89 Zr、 212 Bi、 131 I, 131 In、 90 Y and 186Re. Antibody-cytotoxic agent conjugates can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithiool)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imide esters (e.g., dimethyl adipamide hydrochloride), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazidides (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazinon derivatives (e.g., bis(p-diazinonbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the binding of radioactive nucleotides to antibodies (WO94 / 11026).

[0148] Other antitumor agents that can bind to the antibodies of the present invention include BCNU; streptozoicin; vincristine and 5-fluorouracil; the family of agents collectively referred to as the LL-E33288 complex as described in U.S. Patent Nos. 5,053,394 and 5,770,710; and esperamicins (U.S. Patent No. 5,877,296).

[0149] Usable enzymatically active toxins and their fragments include diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modizol A chain, α-broomrin, sclerotin, carnation protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin, crotonin, soapwort inhibitor, leucopicrin, mitogen, localized aspergillin, phenolmycin, enoxacin, and trichothecene. For example, ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science, 238:1098. See, for example, WO 93 / 21232 (published October 28, 1993).

[0150] The present invention further covers ADCs formed between antibodies and compounds with nuclear degradation activity (e.g., ribonucleases or DNA endonucleases, such as deoxyribonucleases; DNA hydrolases).

[0151] To selectively destroy tumors, antibodies can contain highly radioactive atoms. Various radioactive isotopes can be used to generate radioactively binding antibodies. Examples include At... 211 I 131 I 125 Y 90 Re186 Re 88 、Sm 53 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu. When conjugates are used for detection, they may contain radioactive atoms, such as tc, used for scintillation scanning studies. 99m Or I 123 Spin labeling, or spin labeling used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as the same iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0152] Radioactive or other labeling can be incorporated into conjugates in known ways. For example, peptides can be biosynthesized or synthesized via chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 replacing hydrogen. For example, tc 99m Or I 123 Re 186 Re 188 and In 111 The label can be linked via cysteine ​​residues in the peptide. Yttrium-90 can be linked via lysine residues. The IODOGEN method (FRAKER et al., (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (CHATAL, CRC Press, 1989).

[0153] This invention particularly provides antibody-drug conjugate compounds for targeted delivery of therapeutic agents. The inventors have discovered that antibody-drug conjugate compounds possess potent cytotoxic and / or cell growth-inhibiting activity against cells expressing NECTIN-4 and its variants.

[0154] The antibody-drug conjugate compound comprises an antibody unit covalently linked to at least one drug unit. The drug unit may be directly covalently linked to the antibody unit or linked to the antibody unit via a linker unit (-LU-).

[0155] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0156] Or its pharmaceutically acceptable salts or solvates; wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; • (LU-D) represents the linker unit-drug unit portion, where: • LU- is the linking group unit, and • -D represents a drug unit that exhibits cell growth inhibition or cytotoxic activity against target cells; and • p is in the range of 1 to 20 or alternatively 1 to 50.

[0157] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0158] Or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of this invention; and • -A a -W w -Y y - is the linking unit (LU), where: • -A- is the extension group unit. • a can be 0, 1, 2, or 3. • Each -W- is an independent amino acid unit. • w is an integer in the range of 0 to 12. • -Y- represents a self-decomposing spacer unit. • y is 0, 1, or 2; • -D represents a drug unit that exhibits cell growth inhibition or cytotoxic activity against target cells; and • p is an integer from 1 to 20, or alternatively from 1 to 50.

[0159] In some embodiments, the antibody-drug conjugate compound has the following formula: ADC Solution (III) or ADC Solution (IV) Or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; • Each R is independently selected from N, CH, or C; • R' is either C or CH; • W is selected from:

[0160] In some embodiments, the antibody-drug conjugate compound has the following formula: ADC solution (V) ADC Solution (VI) • Ab is an antibody unit, such as the anti-NECTIN-4 antibody of the present invention; • Each R is independently selected from N, CH, or C; • W is selected from:

[0161] • X b The spacer group is selected from the group consisting of: alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide; • b is 0, 1, or 2; • Y b A polypeptide moiety comprising about 1 to about 6 amino acids that are natural and / or non-natural amino acids; • Z b For self-decomposing components, including but not limited to:

[0162] • D represents a drug unit that has cell growth inhibitory or cytotoxic activity against the target cells; • p is an integer from 1 to 20, or alternatively from 1 to 50.

[0163] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0164] ADC scheme (VII) Or a pharmaceutically acceptable salt or solvate thereof, wherein: • Ab is an antibody unit, such as the NECTIN-4 antibody of the present invention; • R 1 for , where R 2 It can be an unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteralkyl group; • R x and R y Each of them is independently selected from R and LR. z The premise is that when R x and R y One of them is NR z At that time, the other is R; • R 5 It is H or CR'3, where each R' is independently H or F; • R 6 It is H or CH2CN; • LU is a linking unit; and • R is H or a C1-C3 alkyl group; and • i It is an integer in the range of 1 to 20.

[0165] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0166] ADC Solution (VIII) Or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab represents antibody units, such as the NECTIN-4 antibody of this invention; R 1 for , where R 2 It can be an unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteralkyl group; R x and R y Each of them is independently selected from R and LR. z The premise is that when R x and R y One of them is NR z At that time, the other is R; R 5 It is H or CR'3, where each R' is independently H or F; R 6 It is H or CH2CN; LU is a linking group unit; and R is H or a C1-C3 alkyl group; and j It is an integer in the range of 1 to 20.

[0167] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0168] ADC Solution (IX) Or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab represents antibody units, such as the NECTIN-4 antibody of this invention; R 1 for , where R 2 It can be an unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteralkyl group; R xand R y Each of them is independently selected from R and LR. z The premise is that when R x and R y One of them is NR z At that time, the other is R; R 5 It is H or CR'3, where each R' is independently H or F; R 6 It is H or CH2CN; LU is a linking group unit; and R is H or a C1-C3 alkyl group; and k It is an integer in the range of 1 to 20.

[0169] In some embodiments, the antibody-drug conjugate compound has the following formula:

[0170] • R 1 for , where R 2 It can be an unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteralkyl group; • R 3 It is H or C1-C3 alkyl; • R 5 It is H or CR'3, where each R' is independently H or F; • R 6 It is H or CH2CN; • Ab is an antibody unit, such as the anti-NECTIN-4 antibody of the present invention; • Each R is independently selected from N, CH, or C; • J represents the joined part; • X b The spacer group is selected from the group consisting of: alkyl, heteroalkyl, polyethylene glycol (PEG), and peptide; • b is 0, 1, or 2; • Y b A polypeptide moiety comprising about 1 to about 6 amino acids that are natural and / or non-natural amino acids; • Z b For self-decomposing components, including but not limited to:

[0171] For compositions comprising multiple antibodies, the drug load is represented by the average number of drug molecules, p, per antibody. The drug load can range from 1 to 24 drug molecules (D) per antibody. The average number of drug molecules per antibody in the preparation of the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA analysis, and HPLC. The quantitative distribution of the antibody-drug conjugate can also be determined based on p. In some cases, when p is a specific value from antibody-drug conjugates with other drug loads, the homogeneous antibody-drug conjugates can be separated, purified, and characterized by methods such as reverse-phase HPLC or electrophoresis. In exemplary embodiments, p is 2 to 8. In some embodiments, p is 2 to 24.

[0172] The formation of antibody-drug conjugates can be achieved using techniques known to those skilled in the art. In simple terms, antibody-drug conjugates contain NECTIN-4 antibodies.

[0173] In one embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(A).

[0174] In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(B).

[0175] In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(C).

[0176] In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(D).

[0177] In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(E).

[0178] In another embodiment, the NECTIN-4 ADC comprises the antibody heavy chain sequence described in Table VI(F).

[0179] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(G).

[0180] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(H).

[0181] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(I).

[0182] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(J).

[0183] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(K).

[0184] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain sequence described in Table VI(L).

[0185] In another embodiment, the NECTIN-4 ADC includes the antibody heavy chain variable region sequence as described in Table VIII.

[0186] In another embodiment, the NECTIN-4 ADC comprises the antibody light chain variable region sequence as described in Table IX.

[0187] In another embodiment, the NECTIN-4 ADC comprises the antibody CDR sequence described in Table X.

[0188] In another embodiment, the aforementioned NECTIN-4 antibody binds to the therapeutic agent.

[0189] In one embodiment, the therapeutic agent is Figure 10 The drug-linking group (DL) payload described in the paper.

[0190] In one embodiment, the DL payload is described as follows: Figure 10 (A) contains the following chemical structures:

[0191] Or in its pharmaceutically acceptable salt or solvate form.

[0192] In one embodiment, the DL payload is described as follows: Figure 10 (B) contains the following chemical structures:

[0193] Or in its pharmaceutically acceptable salt or solvate form.

[0194] In one embodiment, the DL payload is described as follows: Figure 10 (C) contains the following chemical structures:

[0195] Or in its pharmaceutically acceptable salt or solvate form.

[0196] In one embodiment, the DL payload is described as follows: Figure 10 (D) contains the following chemical structures:

[0197] Or in its pharmaceutically acceptable salt or solvate form.

[0198] In one embodiment, the DL payload is described as follows: Figure 10 (E) and has the following chemical structure:

[0199] Or in its pharmaceutically acceptable salt or solvate form.

[0200] In one embodiment, the DL payload is described as follows: Figure 10 (F) contains the following chemical structures:

[0201] Or in its pharmaceutically acceptable salt or solvate form.

[0202] In one embodiment, the DL payload is described as follows: Figure 10 (G) contains the following chemical structures:

[0203] Or in its pharmaceutically acceptable salt or solvate form.

[0204] In one embodiment, the DL payload is described as follows: Figure 10 (H) and has the following chemical structure:

[0205] Or in its pharmaceutically acceptable salt or solvate form.

[0206] In one embodiment, the DL payload is described as follows: Figure 10 (I) and has the following chemical structure:

[0207] Or in its pharmaceutically acceptable salt or solvate form.

[0208] In one embodiment, the DL payload is described as follows: Figure 10 (J) contains the following chemical structures:

[0209] Or in its pharmaceutically acceptable salt or solvate form.

[0210] In one embodiment, the DL payload is described as follows: Figure 10 (K) and has the following chemical structure:

[0211] Or in its pharmaceutically acceptable salt or solvate form.

[0212] In one embodiment, the DL payload is described as follows: Figure 10 (L) and has the following chemical structure:

[0213] Or in its pharmaceutically acceptable salt or solvate form.

[0214] In one embodiment, the DL payload is described as follows: Figure 10 (M) contains the following chemical structures:

[0215] Or in its pharmaceutically acceptable salt or solvate form.

[0216] In one embodiment, the DL payload is described as follows: Figure 10 (N) and has the following chemical structure:

[0217] Or in its pharmaceutically acceptable salt or solvate form.

[0218] In one embodiment, the DL payload is described as follows: Figure 10 (O) and has the following chemical structure:

[0219] Or in its pharmaceutically acceptable salt or solvate form.

[0220] In one embodiment, the DL payload is described as follows: Figure 10 (P) contains the following chemical structures:

[0221] Or in its pharmaceutically acceptable salt or solvate form.

[0222] A variety of reactions can be used for the covalent linking of drugs and / or linking groups to conjugates. This is typically achieved through reactions involving amino acid residues of the conjugate, such as the amino group of lysine, the free carboxyl groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and multiple moieties of aromatic amino acids. One of the most common nonspecific methods of covalent linking is the carbodiimide reaction, which links the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody.

[0223] Additionally, bifunctional agents such as dialdehydes or imide esters have been used to link the amino group of a compound to the amino group of an antibody molecule. Schiff base reactions can also be used to link drugs to conjugates. This method involves the oxidation of the periodate of a drug containing a diol or hydroxyl group, thereby forming an aldehyde that subsequently reacts with the conjugate. The linking occurs via the formation of a Schiff base with the amino group of the conjugate. Isothiocyanates can also be used as coupling agents for the covalent linking of drugs to conjugates. Other techniques are known to those skilled in the art and are within the scope of this invention.

[0224] In some embodiments, an intermediate of the precursor linking group reacts with the drug under appropriate conditions. In some embodiments, a reactive group is used on the drug and / or the intermediate. The reaction product between the drug and the intermediate, or a derivative of the drug, is subsequently reacted with the NECTIN-4 antibody under appropriate conditions.

[0225] IV.) Linking group unit Typically, antibody-drug conjugate compounds include a linker unit between a drug unit and an antibody unit. In some embodiments, the linker unit is cleavable under intracellular conditions, and this cleavage releases the drug unit from the antibody in the intracellular environment. In other embodiments, the linker unit is not cleavable, and the drug is released, for example, through antibody degradation.

[0226] In a preferred embodiment, the linker group is bound to the NECTIN-4 antibody described herein.

[0227] In some embodiments, the linker group can be cleaved by a lysing agent present in the intracellular environment (e.g., within a lysosome, nucleosome, or caveole). The linker group can be, for example, a peptidyl linker group, cleaved by intracellular peptidases or proteases, including (but not limited to) lysosomal or endosomal proteases. The linker group can also be cleaved by a lysing agent present in the extracellular environment (e.g., near the cell membrane or tissue space). The linker group can be, for example, a peptidyl linker group, cleaved by extracellular peptidases or proteases, including (but not limited to) cathepsin family enzymes or matrix metalloproteinases.

[0228] In other embodiments, the cleavable linker is pH-sensitive, meaning it is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., oximes, hydrazones, hemicarbazones, thiohemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, or analogs thereof) can be used. (See, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; Dubochick and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biochemistry 264:14653-14661.) In other embodiments, the linker group may be cleaved under reducing conditions known in the art. (See, for example, Thorp et al., 1987, Cancer Research 47:5924-5931; Wavzynčak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford University Press, 1987). See also U.S. Patent No. 4,880,935). The linker group may also be cleaved under reducing conditions found intracellularly (or extracellularly). For example, in a preferred embodiment, the specific linker group NO bond may be formally reduced and broken, resulting in linker group cleavage.

[0229] In other specific embodiments, the linking group is a malonate linking group (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl linking group (Lau et al., 1995, Bioorganic and Pharmaceutical Chemistry 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorganic and Pharmaceutical Chemistry 3(10):1305-12).

[0230] In other embodiments, the linker unit is non-cleavable, and the drug is released via antibody degradation. (See PCT Publication WO2012 / 166560 (Ambrx, Inc.), which is incorporated herein by reference in its entirety for all purposes.)

[0231] Typically, linker groups are substantially insensitive to the extracellular environment. As used herein, in the case of linker groups, "substantially insensitive to the extracellular environment" means that when the antibody-drug conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker group in the antibody-drug conjugate sample is cleaved. Whether a linker group is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and subsequently quantifying the amount of free drug present in the plasma.

[0232] In other non-mutually exclusive embodiments, as is known in the art, the linker group promotes cell internalization.

[0233] A variety of exemplary linking groups that can be used with the compositions and methods of the present invention are described in WO 2004 / 010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649 and U.S. Publication No. 2006 / 0024317 (all of which are incorporated herein by reference in their entirety for all purposes).

[0234] For the purposes of this disclosure, a "linking unit" (LU) is a bifunctional compound that can be used to link a drug unit and an antibody unit to form an antibody-drug conjugate compound. In some embodiments, the linking unit has the following formula: -A a -W w -Y y - ○ Where: -A- is the extension group unit, ○ a is 0 or 1, ○ Each -W- is an independent amino acid unit. ○ w is an integer in the range of 0 to 12. ○ -Y- is a self-decomposing spacer unit, and ○ y is 0, 1 or 2.

[0235] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

[0236] V.) Extended group unit The extension group unit (A), when present, can link antibody units to amino acid units (-W-) (if present), to spacer group units (-Y-) (if present), or to pharmaceutical units (-D). Suitable functional groups that may be naturally present or chemically manipulated on the NECTIN-4 antibody include, but are not limited to, ketone, aldehyde, thioglycolic acid, amino, hydroxyl, terminal hydroxyl groups of sugars, and carboxyl groups. Suitable functional groups are ketone, aldehyde, thioglycolic acid, and amino. In one example, the ketone group is on a non-natural amino acid (nnAA) incorporated into the antibody of the present invention. In another example, the aldehyde group is on an nnAA incorporated into the antibody of the present invention. In another example, the thioglycolic acid group can be generated by reducing the intramolecular disulfide bond of the NECTIN-4 antibody. In another embodiment, the thioglycolic acid group can be generated by reacting the amino group of the lysine moiety of the NECTIN-4 antibody with 2-iminothiacyclopentane (Traut's reagent) or other thioglycolic acid generating reagents. In some embodiments, the NECTIN-4 antibody is a recombinant antibody and engineered to carry one or more lysine residues. In some other embodiments, the recombinant NECTIN-4 antibody is engineered to carry an additional sulfhydryl group, such as an additional cysteine ​​residue.

[0237] In one embodiment, the extension group unit forms a bond with the sulfur atom of the antibody unit. The sulfur atom may be derived from the thiodiol group of the antibody. In some embodiments, the extension group unit is connected to the antibody unit via a disulfide bond between the sulfur atom of the antibody unit and the sulfur atom of the extension group unit. In other embodiments, the extension group contains a reactive site that can form a bond with the primary or secondary amino group of the antibody. Examples of such reactive sites include, but are not limited to, activated esters such as succinimidyl ester, 4-nitrobenzene ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0238] In some embodiments, the extension group contains a reactive site that is reactive to the (-CHO) group of the modified sugar that may be present on the antibody. For example, the sugar can be lightly oxidized using a reagent such as sodium periodate, and the resulting (-CHO) unit of the oxidized sugar can be condensed with an extension group containing, for example, an acylhydrazine, oxime, primary or secondary amine, hydrazine, thiohexahydrazone, hydrazine carboxylate, and aryl acylhydrazine (e.g., those aryl acylhydrazines described in Kaneko et al., 1991, Bioconjugation Chemistry 2:133-41).

[0239] VI.) Amino acid units When an amino acid unit (-W-) is present: if a spacer unit is present, then an extension unit is connected to the spacer unit; if a spacer unit is not present, then an extension unit is connected to the drug moiety; and if neither the extension unit nor the spacer unit is present, then an antibody unit is connected to the drug moiety.

[0240] In some embodiments, the amino acid unit may comprise natural amino acids. In other embodiments, the amino acid unit may comprise non-natural amino acids.

[0241] In some embodiments, the amino acid unit may be enzymatically cleaved by one or more enzymes, including cancer or tumor-associated proteases, to release the drug unit (-D). In one embodiment, the drug unit is protonated in vivo upon release to deliver the drug (D).

[0242] In one aspect, the amino acid unit is valine-citrulline (vc or Val-Cit). In another aspect, the amino acid unit is phenylalanine-lysine. In yet another aspect, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinoline carboxylate-lysine, cyclohexylalanine-lysine, isoperidinic acid-lysine, β-alanine-lysine, glycine-serine-valine-glutamine, and isoperidinic acid.

[0243] VII.) Spacer unit When an amino acid unit is present, a spacer group unit (-Y-) connects the amino acid unit to the drug unit. Alternatively, when an amino acid unit is absent, the spacer group unit connects the extension group unit to the drug unit. When neither the amino acid unit nor the extension group unit is present, the spacer group unit also connects the drug unit to the antibody unit. The spacer group unit has two general types: non-disintegrating or disintegrating. Examples of possible spacer groups of the present invention are known in the art. See TOKI et al., 2002, J. Org. Chem. 67:1866-1872 and Nature Biotechnology 21(7):778-784.

[0244] Other examples of self-decomposing spacer groups include, but are not limited to, aromatic compounds that are electronically similar to PAB groups, such as 2-aminoimidazol-5-methanol derivatives (HAY et al., 1999, Bioorganic & Medicinal Chemistry Letters 9:2237) and o-aminobenzyl acetaldehyde or p-aminobenzyl acetaldehyde.

[0245] Spacer groups that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic amides (Amsbury et al., 1990, Journal of Organic Chemistry 55:5867). Elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-decomposing spacer group.

[0246] VIII.) Drug Unit The pharmaceutical moiety (D) can be any cytotoxic, cell growth inhibitory, or immunomodulatory (e.g., immunosuppressive) drug. D is a pharmaceutical unit (mole) having atoms that can form bonds with spacer units, amino acid units, extended unit units, or antibody units. In some embodiments, the pharmaceutical unit D has a nitrogen atom that can form a bond with a spacer unit. As used herein, the terms "pharmaceutical unit" and "pharmaceutical moiety" are synonymous and used interchangeably.

[0247] Applicable categories of cytotoxic agents, cell growth inhibitors, or immunomodulators include, for example, anti-microtubule agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0248] In some embodiments, the drug is olistatin, such as olistatin E (also known in the art as a derivative of saccharin-10) or a derivative thereof. Olistatin may be, for example, an ester formed between olistatin E and a keto acid. For example, olistatin E may react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEB, respectively.

[0249] In some embodiments, the pharmaceutical unit is chalcogenide, camptothecin, maytansine, or anthracycline. In some embodiments, the pharmaceutical unit is taxane, topoisomerase inhibitor, or vinca alkaloid.

[0250] In some typical embodiments, suitable cytotoxic agents include, for example, DNA groove binders (e.g., enediyne and lexitropsins, a CBI compound; see also U.S. Patent No. 6,130,237), pyroxine, taxanes (e.g., paclitaxel and docetaxel), puromycins, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyano-N-morpholino-doxorubicin, echinomycin, compretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansine, discormolide, eleutherobin, and mitoxantrone.

[0251] In some embodiments, the drug is an anti-tubulin agent. Examples of anti-tubulin agents include olistatin, taxanes (e.g., Taxol®, Taxotere®), T67, and vinorelbine alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vinorelbine). Other anti-tubulin agents include, for example, bacardine derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colchicine, estradiol, cryptophycins, cimadolicine, maytansine, compressoritine, spongiformin, and ileoxetine.

[0252] In some embodiments, the cytotoxic agent is a maytansine-like substance (another group of anti-microtubule agents). For example, in certain embodiments, the maytansine-like substance is maytansine or DM-1 (Immunogen Corporation; see also Chari et al., 1992, Cancer Research 52:127-131).

[0253] In some embodiments, the cytotoxic agent or cell growth inhibitor is sulphurin. In some embodiments, the cytotoxic agent or cell growth inhibitor is an olistatin, such as sulphurin-10, olistatin E, or olistatin PHE.

[0254] In some embodiments, the pharmaceutical unit (D) is an olritamine analog having the following structural formula:

[0255] Or its pharmaceutically acceptable salt. in R 1 for , where R 2 It can be an unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteralkyl group; R a R b and R c The elements in the text are selected from H and NR. x R y The premise is R a R b and R c Only one of them is NR x R y And each of the others is H; R x and R y Each of them is independently selected from R, R r and LR z The premise is that when R x and R y One of them is LR z Or R r At that time, the other is R; R 5 It is H or CR'3, where each R' is independently H or F; R 6 It is H or CH2CN; L is a linking group; R r It is (C=O)-O-(CH2) p -R v Or (C=O)-(CH2) q -R v ; R v It can be R, OR, NHR, NR2, aryl, or amino acid; p is 0, 1, 2, 3, 4, 5 or 6; q can be 0, 1, 2, 3, 4, 5, or 6; R z It contains functional or reactive groups; and R is H or C1-C3 alkyl.

[0256] IX.) Drug load Drug load is represented by p and is the average number of drug moieties in each antibody in the molecule. Drug load can range from 1 to 24 drug moieties (D) per antibody. The ADC of the present invention comprises a collection of antibodies conjugated to drug moieties ranging from 1 to 24. The average number of drug moieties of each antibody in the preparation of the ADC by the conjugation reaction can be characterized by conventional methods such as mass spectrometry and ELISA analysis. The quantitative distribution of the ADC can also be determined according to p. In some cases, when p is a specific value from an ADC with other drug loads, the separation, purification, and characterization of the homogeneous ADC can be achieved by means such as electrophoresis.

[0257] For some antibody-drug conjugates, p may be limited by the number of linking sites on the antibody. For example, in the case of cysteine ​​thiol linkage, as in the exemplary embodiments described above, the antibody may have only one or more cysteine ​​thiol groups, or may have only one or more fully reactive thiol groups that can be linked to the linking group. In some embodiments, higher drug loading, such as p>5, can cause aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates. In some embodiments, the drug loading of the ADC of the present invention is in the following ranges: 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In fact, it has been shown that for some ADCs, the most preferred ratio of the drug portion per antibody may be less than 8 and may be about 2 to about 5. See U.S. Patent No. 7,498,298 (which is incorporated herein by reference in its entirety).

[0258] In some embodiments, less than the theoretical maximum amount of drug moiety is conjugated to the antibody during the binding reaction. The antibody may contain, for example, lysine residues that do not react with drug-linking intermediates or linking reagents, as discussed below. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can be linked to the drug moiety. In fact, most cysteine ​​thiols in antibodies are present as disulfide bridges. In some embodiments, the antibody may be reduced under partially or completely reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiols. In some embodiments, the antibody undergoes denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0259] The loading (drug / antibody ratio) of an ADC can be controlled, for example, by: (i) limiting the molar excess of the drug-linking intermediate or linking agent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) using partial or limiting reduction conditions for cysteine ​​thiol modification; (iv) engineering the amino acid sequence of the antibody through recombinant technology such that the number and position of cysteine ​​residues are modified to control the number and / or position of the linking group-drug linkage (e.g., thioMab or thioFab prepared as disclosed herein and in WO2006 / 034488 (incorporated herein by reference in its entirety)).

[0260] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, followed by a reaction with a drug moiety reagent, the resulting product is a mixture of the ADC compound and a distribution of one or more drug moieties linked to the antibody. The average number of drugs per antibody can be calculated from the mixture using a dual ELISA antibody assay that is specific to both the antibody and the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example, Hamblett KJ et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley SC et al., "Controlling the location of drug attachment in antibody-drug conjugates", Abstract No. 627). American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004 (AACR Conference Proceedings, Volume 45, March 2004). In some embodiments, homogeneous ADCs with a single loading value can be separated from the binding mixture by electrophoresis or chromatography.

[0261] X.) Methods for determining the cytotoxic effects of ADCs Methods for determining whether a drug or antibody-drug conjugate exerts cell growth inhibitory and / or cytotoxic effects on cells are known. Generally, the cytotoxic or cell growth inhibitory activity of an ADC can be measured by: exposing mammalian cells expressing the target protein of the antibody-drug conjugate to cell culture medium; culturing the cells for a period of approximately 6 hours to approximately 5 days; and measuring cell viability. Cell-based in vitro analyses can be used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (cysteine ​​activity) of the antibody-drug conjugate.

[0262] To determine whether an ADC exerts a cell growth inhibitory effect, thymidine incorporation analysis can be used. For example, cancer cells expressing the target antigen can be cultured at a density of 5,000 cells / well in a 96-well plate for a 72-hour period, and exposed to 0.5 μCi of thymidine during the last 8 hours of this 72-hour period. 3 H-thymidine. Measured in the presence and absence of ADC. 3 Incorporation of H-thymidine into the cells of the culture.

[0263] To determine cytotoxicity, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased plasma membrane permeability; cell swelling and membrane rupture. Apoptosis is typically characterized by membrane bubbling, cytoplasmic condensation, and activation of endogenous endonucleases. Measurement of any of these effects on cancer cells suggests that ADCs are suitable for cancer treatment.

[0264] Cell viability can be measured by determining the uptake of dyes such as Neutral Red, Trypanosome Blue, or Alamar™ Blue by cells (see, for example, Page et al., 1993, International Journal of Oncology 3:473-476). In this type of analysis, cells are cultured in a dye-containing medium, washed, and the remaining dye, reflecting the cells' uptake of the dye, is measured spectrophotometrically. Cytotoxicity can also be measured using the protein-bound dye sulforhodamine B (SRB) (SKEHAN et al., 1990, Journal of the National Cancer Institute 82:1107-12).

[0265] Alternatively, tetrazolium salts, such as MTT or CellTiter-Glo®, can be used to quantify mammalian cell survival and proliferation by detecting live cells rather than dead cells (see, for example, Mosmann, 1983, J. Immunol. Methods 65:55-63).

[0266] Apoptosis can be quantified by measuring, for example, DNA fragmentation. Commercially available optical measurement methods are available for the quantitative in vitro determination of DNA fragmentation. Examples of such analyses, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based analyses, are described in Biochemica, 1999, Vol. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0267] Apoptosis can also be determined by measuring morphological changes in cells. For example, similar to necrosis, the loss of plasma membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells have been described by Duke and Cohen, Current Laboratory Protocols in Immunology (Coligan et al., 1992, pp. 3.17.1–3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and chromatin condensation and margination along the inner nuclear membrane can be observed. Other morphological changes that can be measured to indicate apoptosis include, for example, cytoplasmic condensation, increased membrane vesicle formation, and cell contraction.

[0268] The presence of apoptotic cells can be measured in both the attached and “floating” compartments of the culture. For example, both compartments can be collected by: removing the supernatant, digesting the conjugated cells with protein trypsin, combining the preparations after a centrifugation and washing step (e.g., at 2000 rpm for 10 minutes), and detecting apoptosis (e.g., by measuring DNA fragmentation). (See, for example, Piazza et al., 1995, Cancer Research 55:3110-16).

[0269] In vivo, the effects of the NECTIN-4 antibody therapeutic composition can be evaluated in suitable animal models. For example, xenogeneic cancer models can be used, in which cancer explants or subsequent xenograft tissues are introduced into immunocompromised animals, such as nude mice or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT patent application WO98 / 16628 and U.S. Patent No. 6,107,540 describe various xenograft models of human prostate cancer that can reproduce the development of the primary tumor, micrometastases, and the formation of osteoblastic bone metastases characterizing advanced disease. Efficacy can be predicted using analyses measuring inhibition of tumor formation, tumor regression or metastasis, and similar parameters.

[0270] In vivo analyses assessing apoptosis promotion are suitable for evaluating therapeutic compositions. In one embodiment, the presence of apoptotic foci in xenografts from tumor-bearing mice treated with the therapeutic composition can be examined and compared with untreated control mice carrying xenografts. The extent to which apoptotic foci are found in the tumors of treated mice provides an indication of the therapeutic efficacy of the composition.

[0271] The therapeutic composition used to perform the methods described above can be formulated into a pharmaceutical composition comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that retains the antitumor function of the therapeutic composition when combined with it and is generally unresponsive to the patient's immune system. Examples include, but are not limited to, any of a variety of standard pharmaceutical carriers, such as sterile phosphate-buffered saline solutions, antibacterial water, and the like (see generally Remington's Pharmaceutical Sciences, 16th edition, ed. A. Osal, 1980).

[0272] Therapeutic formulations can be dissolved and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorganic, in situ, and similar routes. Preferred formulations for intravenous injection comprise the therapeutic composition in a preservative-sterile aqueous solution, sterile unpreserved aqueous solution, and / or diluted in a polyvinyl chloride or polyethylene bag containing 0.9% sterile sodium chloride for injection, USP. Therapeutic protein formulations may be lyophilized and stored as sterile powders, preferably under vacuum, and subsequently reconstituted in preservative-sterile aqueous solution (containing, for example, a benzyl alcohol preservative) or sterile aqueous solution prior to injection.

[0273] The dosage and administration regimen for treating cancer using the aforementioned methods will vary depending on the method and the target cancer, and will generally depend on a number of other factors known in the field.

[0274] In one embodiment, due to the modification of the NECTIN-4 antibody, the pharmaceutical composition of the present invention may comprise more than one of the ADC species of the present invention. For example, the present invention includes a pharmaceutical composition comprising the ADC of the present invention, wherein the NECTIN-4 antibody is an antibody with partial or complete removal of the C-terminal lysine, an antibody having an N-terminal post-translational modification, an antibody lacking a heavy chain C-terminal lysine but having an N-terminal post-translational modification, and / or an antibody having a heavy chain C-terminal lysine but not having an N-terminal post-translational modification.

[0275] In a preferred embodiment, the NECTIN-4 antibody is described in Tables VI and VII.

[0276] XI.) Treatment of cancers expressing NECTIN-4 Identifying NECTIN-4 as a protein that is typically expressed in a limited group of tissues or cells but is also expressed in cancers such as those listed in Table I opens up a variety of treatment options for these types of cancers.

[0277] It is worth noting that targeted anti-tumor therapy is applicable even when the target protein is expressed on normal tissues or cells, or even on vital normal organs. Vital organs are those essential for maintaining life, such as the heart or colon. Non-vital organs are those that can be removed without the individual surviving. Examples of non-vital organs include the ovary, breast, and prostate.

[0278] Expression of the target protein in normal tissues, or even vital normal tissues, does not impair the efficacy of the protein's target agent as a therapeutic agent for certain tumors in which the protein is also overexpressed. For example, expression in vital organs can be altered and is itself harmful. Additionally, organs considered non-essential, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some vital organs are immune to the effects of expression in normal organs due to immune exemption. Immune-exempt organs are protected from the effects of blood through the blood-organ barrier and are therefore ineligible for immunotherapy. Examples of immune-exempt organs include the brain and testes.

[0279] Therefore, treatments that inhibit the activity of the NECTIN-4 protein are suitable for patients with cancers expressing NECTIN-4 (such as those described in Table I). These treatments generally fall into three categories. The first category modulates the function of NECTIN-4 when it is associated with tumor cell growth, causing inhibition or arrest of tumor cell growth or inducing its killing. The second category includes various methods for inhibiting the NECTIN-4 protein from its binding complexes or from binding to or associating with other proteins. The third category includes various methods for inhibiting the transcription of the NECTIN-4 gene or the translation of NECTIN-4 mRNA.

[0280] Therefore, the presence and level of NECTIN-4 expression in cancer patients can be assessed, preferably using immunohistochemical evaluation of tumor tissue, quantitative NECTIN-4 imaging, or other techniques that reliably indicate the presence and extent of NECTIN-4 expression. If applicable, immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0281] XII.) NECTIN-4 ADC mixture The therapeutic methods of this invention encompass the administration of a single NECTIN-4 ADC and compositions or mixtures of different antibodies (i.e., NECTIN-4 antibodies or antibodies binding to another protein). Such antibody mixtures may have certain advantages because they contain antibodies targeting different epitopes, utilizing different effector mechanisms, or directly combining cytotoxic antibodies with antibodies whose functionality depends on immune effectors. Such antibody combinations can exhibit synergistic therapeutic efficacy. Furthermore, NECTIN-4 antibodies can be administered simultaneously with other therapeutic modalities, including but not limited to various chemotherapeutic agents and biologics, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF, PD1, PD-L1), surgery, or radiation.

[0282] In a preferred embodiment, the NECTIN-4 antibody is administered in a conjugated form.

[0283] In another preferred embodiment, the NECTIN-4 antibody is described in Tables VI and VII.

[0284] NECTIN-4 ADC formulations can be administered via any route capable of delivering antibodies to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and similar routes. Treatment generally involves repeated administration of the NECTIN-4 ADC formulation via an acceptable route of administration, such as intravenous (IV) injection, typically at doses including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight. Generally, doses in the range of 10–1000 mg MAb weekly are effective and well-tolerated.

[0285] Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg / kg patient body weight IV, followed by weekly doses of the MAb formulation of approximately 2 mg / kg IV, represents an acceptable dosing regimen. Preferably, the initial loading dose is administered via infusion over 90 minutes or longer. The maintenance dose is administered via infusion over 30 minutes or longer, subject to the initial dose being well tolerated. As those skilled in the art will appreciate, many factors can influence the ideal dosing regimen under specific circumstances. These factors include, for example, the binding affinity and half-life of the antibody used, the level of NECTIN-4 expression in the patient, the amount of NECTIN-4 antigen expended in circulation, the required steady-state antibody concentration level, the frequency of treatment, and the effects of chemotherapeutic agents or other agents used in combination with the treatment method of the present invention, as well as the specific patient's health condition.

[0286] Optionally, NECTIN-4 levels in a given patient sample (e.g., levels of circulating NECTIN-4 antigen and / or NECTIN-4-expressing cells) should be assessed to help determine the most effective dosing regimen, etc. Such assessments are also used to monitor objectives throughout the therapy and are suitable for use in combination with assessments of other parameters (e.g., urinary cytology and / or immune cell levels in bladder cancer therapy, or similarly, serum PSA levels in prostate cancer therapy) to measure treatment success.

[0287] One objective of this invention is to provide a NECTIN-4 ADC that inhibits or delays the growth of tumor cells expressing NECTIN-4. Another objective of this invention is to provide a method for using such NECTIN-4 ADCs, and particularly for using such NECTIN-4 ADCs in combination with other drugs or immunotherapies, to inhibit angiogenesis and other biological functions in mammals, preferably humans, and thereby reduce tumor growth.

[0288] XIII.) Combination Therapy In one embodiment, a synergistic effect exists when a tumor, including a human tumor, is treated with a NECTIN-4 ADC in combination with a chemotherapy agent or radiation, or a combination thereof. In other words, when combined with a chemotherapy agent or radiation, or a combination thereof, the inhibition of tumor growth by the NECTIN-4 ADC is enhanced beyond expectation. The synergistic effect can be demonstrated, for example, by a greater inhibition of tumor growth by the combination therapy compared to the expected effect of treatment with the NECTIN-4 ADC alone, or the additive effect of treatment with the NECTIN-4 ADC and chemotherapy or radiation. Preferably, the synergistic effect is demonstrated by cancer remission, wherein treatment with the NECTIN-4 ADC or the additive combination of the NECTIN-4 ADC with chemotherapy or radiation or immunotherapy, such as CAR-T or NK cell therapy, does not result in remission.

[0289] Methods of inhibiting tumor cell growth using a combination of NECTIN-4 ADC and chemotherapy or radiation, or both, include administering the NECTIN-4 ADC before, during, or after the initiation of chemotherapy or radiation therapy, and any combination thereof (i.e., before and during, before and after, during and after, or before, during and after). For example, the NECTIN-4 ADC is typically administered between 1 and 60 days, preferably between 3 and 40 days, and more preferably between 5 and 12 days, before the initiation of radiation therapy and / or chemotherapy. However, depending on the treatment regimen and the specific needs of the patient, the method is performed in a manner that will provide the most effective treatment and ultimately prolong the patient's life.

[0290] Chemotherapy agents can be administered in various ways, including systemic administration via parenteral and enteral routes. In one embodiment, the NECTIN-4 ADC and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy therapies include cisplatin, dacarbazine (DTIC), dactinomycin D, dichloromethyldiethylamine (nitrogen mustard), streptozotocin, cyclophosphamide, carmustine (BCNU), and lomustine. (CCNU), Doxorubicin (adriamycin), Zorobacterium, Procarbazine, Mitomycin, Cytarabine, Etoposide, Methotrexate, 5-Fluorouracil, Vincristine, Bleomycin, Paclitaxel, Docetaxel, Aldesleukin, Asparaginase, Busulfan, Carboplatin, Cladribine, Dacarbazine, Fluuridine, Fludarabine, Hydroxyurea, Evoramide, Interferon Alpha, Leuproli (de), megestrol, melphalan, mecaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, streptozotocin, tamoxifen, teniposide, testrolide, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, paclitaxel, and combinations thereof.

[0291] The radiation source used in conjunction with the NECTIN-4 ADC can be external to or internal to the patient being treated. When the radiation source is external to the patient, the therapy is called external beam radiotherapy (EBRT). When the radiation source is internal to the patient, the therapy is called proximal therapy (BT). In one embodiment, the radiotherapy is boron neutron capture therapy. In one embodiment, the radiotherapy is proton-boron fusion therapy.

[0292] The treatment regimens described above can be further combined with other cancer therapies and / or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents suitable for treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies as described in WO / 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R and cytokines.

[0293] When mammals undergo additional chemotherapy, the chemotherapeutic agents described above can be used. Additionally, growth factor inhibitors, biological response modifiers, anti-hormonal therapies, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti-androgens can be used. For example, anti-hormonal agents such as Nolvadex (tamoxifen) or anti-androgens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionylaniline) can be used.

[0294] The above treatment methods can be combined with any of a wide variety of surgical, chemotherapy, or radiation therapy regimens. The treatment methods of this invention enable the use of reduced chemotherapy (or other therapies) doses and / or less frequent administration, which is an advantage for all patients, especially those who do not tolerate the toxicity of chemotherapy agents well.

[0295] XIV.) Reagent Kit / Product For laboratory, prognostic, preventative, diagnostic, and therapeutic applications described herein, the kits are within the scope of this invention. Such kits may comprise a carrier, a package, or a container segmented to receive one or more containers (e.g., vials, tubes, and the like), each containing one of the individual elements for the methods described, and a label or insert containing instructions for use, such as those for the purposes described herein. For example, the container may contain a NECTIN-4 antibody or several NECTIN-4 antibodies of this disclosure (see Tables VI and VII). The kit may comprise a container containing a pharmaceutical unit. The kit may include all or part of a NECTIN-4 ADC and / or diagnostic assays for the detection of cancer and / or other immune disorders.

[0296] The kit of the present invention will generally comprise the container described above and one or more other related containers, which contain materials that are commercially and user-desirable, including buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or tubes listing the contents, labels and / or instructions for use, and a pharmaceutical instruction leaflet with instructions for use.

[0297] Labels may be present on or with the container to indicate that the composition is intended for a specific therapeutic or non-therapeutic application, such as prognostic, preventative, diagnostic, or laboratory use, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and / or other information may also be included in inserts or labels included with or on the kit. Labels may be on or associated with the container. When the letters, numbers, or other characters forming the label are molded or etched into the container itself, the label may be located on the container; when the label is present within a reservoir or carrier that also contains the container, the label may be associated with the container, for example, in the form of a drug instruction leaflet. Markings may indicate that the composition is intended for the diagnosis, treatment, prevention, or prognosis of a condition, such as cancer or other immune disorders.

[0298] The terms “kit” and “product” can be used as synonyms.

[0299] In another embodiment of the invention, the article contains a composition, such as the NECTIN-4 ADC of this disclosure. The article typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, such as glass, metal, or plastic. The container can contain therapeutic doses of one or more NECTIN-4 ADCs and / or one or more NECTIN-4 ADCs.

[0300] The container may alternatively contain a composition effective for the treatment, diagnosis, prognosis, or prevention of a condition and may have a sterile dispensing port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). The active agent in the composition may be the NECTIN-4 antibody or ADC of the present invention.

[0301] The product may further comprise a second container containing a pharmaceutically acceptable buffer solution, such as phosphate-buffered saline, Ringer's solution, and / or dextran solution. It may further include other materials deemed commercially and user-appropriate, including additional buffers, diluents, filters, stirrers, needles, syringes, and / or product information leaflets with indications and / or instructions for use.

[0302] Exemplary Examples 1) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.

[0303] 2) The antibody or its antigen-binding fragment according to technical solution 1 further comprises a light chain variable region, the light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

[0304] 3) The antibody or its antigen-binding fragment according to technical solution 1, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 28.

[0305] 4) The antibody or its antigen-binding fragment according to technical solution 2, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 34.

[0306] 5) The antibody or its antigen-binding fragment according to technical solution 1, wherein the heavy chain comprises the sequence set forth in SEQ ID NO:5.

[0307] 6) The antibody or its antigen-binding fragment according to technical solution 2, wherein the light chain comprises the sequence set forth in SEQ ID NO:17.

[0308] 7) The antibody or antigen-binding fragment thereof according to technical solution 1, comprising: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 28; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 34.

[0309] 8) The antibody or its antigen-binding fragment according to technical solution 1, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0310] 9) The antibody or its antigen-binding fragment according to technical solution 1, wherein the antibody is a whole human antibody.

[0311] 10) The antibody or antigen-binding fragment thereof according to technical solution 1, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0312] 11) The antibody or antigen-binding fragment thereof according to technical solution 1, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0313] 12) The antibody or its antigen-binding fragment according to technical solution 11, wherein the drug is an olistatin analogue.

[0314] 13) The antibody or its antigen-binding fragment according to technical solution 11 further comprises an extension group unit.

[0315] 14) The antibody or its antigen-binding fragment according to technical solution 11 further comprises a spacer unit.

[0316] 15) The antibody or its antigen-binding fragment according to technical solution 11 further comprises amino acid units.

[0317] 16) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

[0318] 17) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 16.

[0319] 18) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 16.

[0320] 19) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 17.

[0321] 20) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15.

[0322] 21) The method according to any one of the technical solutions 18 to 20, wherein the individual is a human individual.

[0323] 22) The method according to technical solution 21, wherein the cancer is described in Table I.

[0324] 23) The method according to technical solution 22, wherein the method further comprises administering a radiotherapy or chemotherapy agent.

[0325] 24) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.

[0326] 25) The antibody or antigen-binding fragment thereof according to technical solution 24 further comprises a light chain variable region, said light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75.

[0327] 26) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 29.

[0328] 27) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 35.

[0329] 28) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 6.

[0330] 29) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the light chain comprises the sequence set forth in SEQ ID NO: 18.

[0331] 30) The antibody or antigen-binding fragment thereof according to technical solution 24 comprises: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 29; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 35.

[0332] 31) The antibody or its antigen-binding fragment according to technical solution 24, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0333] 32) The antibody or its antigen-binding fragment according to technical solution 24, wherein the antibody is a whole human antibody.

[0334] 33) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0335] 34) The antibody or antigen-binding fragment thereof according to technical solution 24, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0336] 35) The antibody or its antigen-binding fragment according to technical solution 34, wherein the drug is an olistatin analogue.

[0337] 36) The antibody or its antigen-binding fragment according to technical solution 34 further comprises an extension group unit.

[0338] 37) The antibody or its antigen-binding fragment according to technical solution 34 further comprises a spacer unit.

[0339] 38) The antibody or its antigen-binding fragment according to technical solution 34 further comprises amino acid units.

[0340] 39) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 24 to 38 and a pharmaceutically acceptable excipient.

[0341] 40) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 24 to 39.

[0342] 41) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 39.

[0343] 42) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 40.

[0344] 43) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 24 to 39.

[0345] 44) The method according to any one of the technical solutions 41 to 43, wherein the individual is a human individual.

[0346] 45) The method according to technical solution 43, wherein the cancer is described in Table I.

[0347] 46) The method according to technical solution 43, wherein the method further comprises administering a radiotherapy or chemotherapy agent.

[0348] 47) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.

[0349] 48) The antibody or antigen-binding fragment thereof according to technical solution 47 further comprises a light chain variable region, said light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78.

[0350] 49) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 30.

[0351] 50) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 36.

[0352] 51) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 8.

[0353] 52) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the light chain comprises the sequence set forth in SEQ ID NO: 20.

[0354] 53) The antibody or antigen-binding fragment thereof according to technical solution 47 comprises: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 30; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 36.

[0355] 54) The antibody or its antigen-binding fragment according to technical solution 47, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0356] 55) The antibody or its antigen-binding fragment according to technical solution 47, wherein the antibody is a whole human antibody.

[0357] 56) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0358] 57) The antibody or antigen-binding fragment thereof according to technical solution 47, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0359] 58) The antibody or its antigen-binding fragment according to technical solution 47, wherein the drug is an olistatin analogue.

[0360] 59) The antibody or its antigen-binding fragment according to technical solution 47 further comprises an extension group unit.

[0361] 60) The antibody or its antigen-binding fragment according to technical solution 47 further comprises a spacer group unit.

[0362] 61) The antibody or its antigen-binding fragment according to technical solution 47 further comprises amino acid units.

[0363] 62) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 47 to 61 and a pharmaceutically acceptable excipient.

[0364] 63) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 47 to 62.

[0365] 64) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 62.

[0366] 65) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 63.

[0367] 66) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 47 to 65.

[0368] 67) The method according to any one of the technical solutions 64 to 66, wherein the individual is a human individual.

[0369] 68) The method according to technical solution 66, wherein the cancer is described in Table I.

[0370] 69) The method according to technical solution 66, wherein the method further comprises administering a radiotherapy or chemotherapy agent.

[0371] 70) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63.

[0372] 71) The antibody or antigen-binding fragment thereof according to technical solution 70 further comprises a light chain variable region, said light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81.

[0373] 72) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 31.

[0374] 73) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 37.

[0375] 74) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 10.

[0376] 75) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the light chain comprises the sequence set forth in SEQ ID NO: 22.

[0377] 76) The antibody or antigen-binding fragment thereof according to technical solution 70 comprises: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 31; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 37.

[0378] 77) The antibody or its antigen-binding fragment according to technical solution 70, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0379] 78) The antibody or its antigen-binding fragment according to technical solution 70, wherein the antibody is a whole human antibody.

[0380] 79) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0381] 80) The antibody or antigen-binding fragment thereof according to technical solution 70, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0382] 81) The antibody or its antigen-binding fragment according to technical solution 70, wherein the drug is an olistatin analogue.

[0383] 82) The antibody or its antigen-binding fragment according to technical solution 70 further comprises an extension group unit.

[0384] 83) The antibody or its antigen-binding fragment according to technical solution 70 further comprises a spacer group unit.

[0385] 84) The antibody or its antigen-binding fragment according to technical solution 70 further comprises amino acid units.

[0386] 85) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 70 to 84 and a pharmaceutically acceptable excipient.

[0387] 86) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 70 to 84.

[0388] 87) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 85.

[0389] 88) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 86.

[0390] 89) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 70 to 88.

[0391] 90) The method according to any one of the technical solutions 87 to 89, wherein the individual is a human individual.

[0392] 91) The method according to technical solution 89, wherein the cancer is described in Table I.

[0393] 92) The method according to technical solution 89, wherein the method further comprises administering a radiotherapy or chemotherapy agent.

[0394] 93) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

[0395] 94) The antibody or its antigen-binding fragment according to technical solution 93 further comprises a light chain variable region, said light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84.

[0396] 95) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 32.

[0397] 96) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 38.

[0398] 97) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 12.

[0399] 98) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the light chain comprises the sequence set forth in SEQ ID NO: 24.

[0400] 99) The antibody or antigen-binding fragment thereof according to technical solution 93 comprises: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 32; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 38.

[0401] 100) The antibody or its antigen-binding fragment according to technical solution 93, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0402] 101) The antibody or its antigen-binding fragment according to technical solution 93, wherein the antibody is a whole human antibody.

[0403] 102) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0404] 103) The antibody or antigen-binding fragment thereof according to technical solution 93, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0405] 104) The antibody or its antigen-binding fragment according to technical solution 93, wherein the drug is an olistatin analogue.

[0406] 105) The antibody or its antigen-binding fragment according to technical solution 93 further comprises an extension group unit.

[0407] 106) The antibody or its antigen-binding fragment according to technical solution 93 further comprises a spacer group unit.

[0408] 107) The antibody or its antigen-binding fragment according to technical solution 93 further comprises amino acid units.

[0409] 108) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 93 to 107 and a pharmaceutically acceptable excipient.

[0410] 109) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of technical solutions 93 to 108.

[0411] 110) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 108.

[0412] 111) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 109.

[0413] 112) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of technical solutions 93 to 107.

[0414] 113) The method according to any one of the technical solutions 110 to 112, wherein the individual is a human individual.

[0415] 114) The method according to technical solution 112, wherein the cancer is described in Table I.

[0416] 115) The method according to technical solution 112, wherein the method further comprises administering a radiotherapy or chemotherapy agent.

[0417] 116) An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69.

[0418] 117) The antibody or antigen-binding fragment thereof according to technical solution 116 further comprises a light chain variable region, said light chain variable region comprising a complementarity-determining region (CDR) having the sequences set forth in SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87.

[0419] 118) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 33.

[0420] 119) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 39.

[0421] 120) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the heavy chain comprises the sequence set forth in SEQ ID NO: 14.

[0422] 121) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the light chain comprises the sequence set forth in SEQ ID NO: 26.

[0423] 122) The antibody or antigen-binding fragment thereof according to technical solution 116 comprises: a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 33; and a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequence of the light chain variable region described in SEQ ID NO: 39.

[0424] 123) The antibody or its antigen-binding fragment according to technical solution 116, wherein the antigen-binding fragment is Fab, F(ab')2, Fv or scFv.

[0425] 124) The antibody or its antigen-binding fragment according to technical solution 116, wherein the antibody is a whole human antibody.

[0426] 125) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the antibody or antigen-binding fragment thereof is generated in a recombinant manner.

[0427] 126) The antibody or antigen-binding fragment thereof according to technical solution 116, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker group.

[0428] 127) The antibody or its antigen-binding fragment according to technical solution 116, wherein the drug is an olistatin analogue.

[0429] 128) The antibody or its antigen-binding fragment according to technical solution 116 further comprises an extension group unit.

[0430] 129) The antibody or its antigen-binding fragment according to technical solution 116 further comprises a spacer unit.

[0431] 130) The antibody or its antigen-binding fragment according to technical solution 116 further comprises amino acid units.

[0432] 131) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 116 to 130 and a pharmaceutically acceptable excipient.

[0433] 132) A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 116 to 131.

[0434] 133) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 131.

[0435] 134) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the kit according to claim 132.

[0436] 135) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 116 to 130.

[0437] 136) The method according to any one of the technical solutions 133 to 135, wherein the individual is a human individual.

[0438] 137) The method according to technical solution 135, wherein the cancer is described in Table I.

[0439] 138) The method according to technical solution 135, wherein the method further comprises administering a radiotherapy or chemotherapy agent or CAR-T therapy or NK cell therapy.

[0440] 139) An antibody-drug conjugate (ADC) comprising a NECTIN-4 antibody or an antigen-binding fragment thereof conjugated to a drug-linking group (DL) payload, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR region comprising the amino acid sequence set forth in any one of SEQ ID NO: 52 to SEQ ID NO: 69.

[0441] 140) The ADC according to technical solution 139 further comprises a NECTIN-4 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a light chain CDR region, the light chain CDR region comprising the amino acid sequence set forth in any one of SEQ ID NO: 70 to SEQ ID NO: 87.

[0442] 141) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0443] 142) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0444] 143) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0445] 144) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0446] 145) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0447] 146) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0448] 147) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0449] 148) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0450] 149) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0451] 150) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0452] 151) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0453] 152) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0454] 153) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0455] 154) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0456] 155) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0457] 156) The ADC according to technical solution 139 or 140, wherein the DL payload comprises the following chemical structure: .

[0458] 157) A pharmaceutical composition comprising an ADC according to any one of claims 139 to 156 and a pharmaceutically acceptable excipient.

[0459] 158) A kit comprising an ADC according to any one of claims 139 to 156.

[0460] 159) A kit comprising the pharmaceutical composition according to claim 157.

[0461] 160) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of an ADC according to any one of claims 139 to 156.

[0462] 161) A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to claim 157.

[0463] 162) The method according to technical solution 160, wherein the individual is a human being.

[0464] 163) The method according to technical solution 161, wherein the individual is a human being.

[0465] 164) The method according to technical solution 160, wherein the cancer is described in Table I.

[0466] 165) The method according to technical solution 161, wherein the cancer is described in Table I.

[0467] 166) The method according to technical solution 160, wherein the method further comprises administering a radiotherapy or chemotherapy agent or CAR-T therapy or NK cell therapy.

[0468] 167) The method according to technical solution 161, wherein the method further comprises administering a radiotherapy or chemotherapy agent or CAR-T therapy or NK cell therapy.

[0469] Example: Various aspects of the invention are further described and illustrated by means of the following examples, none of which are intended to limit the scope of the invention.

[0470] Example 1: Methods for generating antibodies.

[0471] NECTIN-4 antibodies were generated by rediscovering the activity of a human Fab antibody library using yeast. After several rounds of enrichment followed by clonal screening, clones specifically recognizing human NECTIN-4 expressed on cancer cells were identified. Subsequently, the variable heavy and light chains of the antibodies were sequenced from the DNA isolated from the yeast clones. To express the NECTIN-4 antibody recombinantly, the variable heavy and light chain sequences of the antibody were cloned upstream of the human heavy chain IgG1 and the human light chain Igκ constant region, respectively. Signal peptides were inserted upstream of the heavy and light chains to allow antibody secretion. The complete anti-nectin-4 antibody human heavy and light chain cassettes were cloned downstream of the CMV promoter / enhancer in a cloning vector. A polyadenylation site was included downstream of the MAb coding sequence. The expression constructs of the recombinant anti-nectin-4 antibody heavy and light chains were transfected into CHO cells.

[0472] Stably transfected Chinese hamster ovary (CHO) cells underwent a selection and recovery process to generate stable pools expressing recombinant antibodies and Fc variants. For antibody generation, a feed-batch production process with a typical culture duration of 8 to 15 days was used for the stable transfected pools prior to culture collection. Alternatively, transiently transfected cells were cultured for a typical duration of 3–15 days prior to culture collection. Subsequently, protein-A affinity purification was performed on the collected cell culture medium, and the purified material was buffer-exchanged for phosphate-buffered saline (PBS) or other preferred antibody formulation buffers. The quality of the recombinant antibodies was assessed by size exclusion chromatography, SDS-PAGE, and other methods known in the art.

[0473] The obtained NECTIN-4 antibodies are described in Tables VI and VII and include (SEQ ID NO: 4) to (SEQ ID NO: 27).

[0474] Example 2: Binding analysis of NECTIN-4 antibody.

[0475] The binding affinity of the NECTIN-4 antibody of the present invention was evaluated using the following protocol. In short, tumor cell lines were collected and resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated on ice for one (1) hour with the antibody (10 µg / mL). For cell binding specificity assays, 10 µg / mL of antibody was added. After incubation, cells were granulated by centrifugation at 200×g for 5 minutes, washed twice with FACS buffer, and counterstained on ice for one (1) hour in the dark with R-PE-labeled goat anti-human Fcγ fragment specific secondary antibody (Jackson Immuno Research; West Grove, PA)). The labeled cells were then washed twice with FACS buffer and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA) for flow cytometry analysis.

[0476] The results showed that the NECTIN-4 antibody specifically bound to NECTIN-4 on multiple cancer cell lines (T-47D, RT4, NCI-H1781, NCI-H322, PC-3, L-540, SU-DHL-1, and K562). (See also: [link to original text]) Figure 1 ).

[0477] Example 3: Binding analysis of NECTIN-4 antibody.

[0478] In another example, the binding affinity of the NECTIN-4 antibody of the present invention was assessed using the following protocol. In short, tumor cell lines were collected and resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated on ice for one (1) hour with the antibody (10 µg / mL). For cell binding specificity assays, a 3-fold dilution of the antibody (10 µg / mL) was added. After incubation, cells were granulated by centrifugation at 200×g for 5 min, washed twice with FACS buffer, and counterstained on ice for one (1) hour in the dark with an R-PE-labeled goat anti-human Fcγ fragment-specific secondary antibody (Jackson Immunological Research; Sigrove, PA). The labeled cells were then washed twice with FACS buffer and analyzed using flow cytometry on an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

[0479] The results showed that the NECTIN-4 antibody specifically bound to NECTIN-4 on the T-47D breast cancer cell line. (See also: Figure 2 (and Table XII).

[0480] Example 4: Binding analysis of NECTIN-4 antibody.

[0481] In another example, the binding affinity of the NECTIN-4 antibody of the present invention was assessed using the following protocol. In short, tumor cell lines were harvested and resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). Cells were seeded into 96-well round-bottom plates and incubated on ice for one (1) hour with the antibody (10 µg / mL). For cell binding affinity assays, a 3-fold dilution of the antibody (10 µg / mL) was added. After incubation, cells were granulated by centrifugation at 200×g for 5 min, washed twice with FACS buffer, and counterstained on ice for one (1) hour in the dark with an R-PE-labeled goat anti-human Fcγ fragment-specific secondary antibody (Jackson Immunological Research; Sigrove, PA). The labeled cells were then washed twice with FACS buffer and analyzed using flow cytometry on an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

[0482] The results showed that the NECTIN-4 antibody specifically bound to NECTIN-4 in the NCI-H292 lung cancer cell line and also bound to NCI-H292 cells in its corresponding ADC form. (See...) Figure 3 (and Table XIII).

[0483] Example 5: In vitro cytotoxicity of NECTIN-4 ADC.

[0484] The in vitro cytotoxicity of NECTIN-4 ADC was determined using the following protocol. In short, tumor cell lines were harvested, seeded into 384-well white flat-bottomed culture plates, and incubated at 37°C while allowing reattachment for 2–4 hours. Cells were then treated with the ADC or free payload assay via dose-adjustment (maximum 500 nM and 5-fold dilution). After 5 days of treatment, residual cell viability was determined using CellTiterGlo analysis based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized relative to untreated control cells, and dose-response curves were fitted using GraphPad Prism software (version 9; La Jolla, CA) with a 4-parameter logistic equation.

[0485] The results show that NECTIN-4 ADC is effective against multiple cancer cell lines, including NECTIN-4-positive lung adenocarcinoma NCI-H322 cells (see [link to results]). Figure 4 (A)), NECTIN-4 positive PC3-NECTIN-4 recombinant prostate cancer cells (see...) Figure 4 (B)) but not for NECTIN-4 negative cells (see Figure 4 (C)) exhibits in vitro cytotoxic effects. See also Table XIV.

[0486] In another set of experiments using the scheme described above, the same NECTIN-4 Ab conjugated with nine (9) different linker groups / payloads demonstrated in vitro cytotoxicity titers (IC50). 50 IC 50 (nM) is described in Table XV. These results further confirm that NECTIN-4 ADCs with multiple linker groups / loads have in vitro cytotoxic effects on the NECTIN-4 positive cell line PC3-NECTIN-4.

[0487] Example 6: In vitro cytotoxicity of NECTIN-4 ADC payload.

[0488] The in vitro cytotoxicity of the NECTIN-4 ADC payload was determined using the following protocol. In short, tumor cell lines were harvested, seeded into 384-well white flat-bottomed culture plates, and incubated at 37°C for 2–4 hours while allowing reattachment. Cells were then treated with either the ADC or the free payload test sample at dose-adjusted levels (maximum 500 nM and 5-fold dilution). After 5 days of treatment, residual cell viability was determined using CellTiter Glo analysis according to the manufacturer's instructions (Promega; Madison, Wisconsin). Data were normalized relative to untreated control cells, and dose-response curves were fitted using GraphPad Prism software (version 9; La Jolla, CA) with a 4-parameter logistic equation.

[0489] Results are presented in the Nectin4-positive breast cancer cell line Sum190PT, showing the in vitro cytotoxicity of three (3) different payload solids (filled symbols) of Nectin-4 ADC compared with the corresponding isotype control ADC (hollow symbols) and the corresponding free payload (dotted lines). (See also) Figure 5 ).

[0490] Example 7: Bystander activity of NECTIN-4 ADC compared to Vitin-Entorutumab.

[0491] The bystander activity of NECTIN-4 ADC compared to the commercially available Vitin-Enflutumab (PADCEV) was determined using the following protocol. In short, a co-culture model was performed in 24-well plates using PC3-Nectin4 recombinant cells expressing NECTIN-4 and SU-DHL-1 cells not expressing Nectin-4, labeled with Cell Trace Violet (Invitrogen; Waltham, MA). The target-positive cell to target-negative cell ratio was 1:2. After 96 hours of treatment with the ADC assay, SU-DHL-1 cells were harvested and collected into 96-well round-bottom plates, stained sequentially with a fixable viability dye (FVD) eFluor780 (Invitrogen) and APC-Annexin V (BioLegend; San Diego, CA), and then analyzed by flow cytometry. Single cultures of SU-DHL-1 cells treated with ADC were used as controls to demonstrate non-target-specific killing or its absence. Early apoptosis (single Annexin V positive), late apoptosis (single FVD eFluor780 positive), and necrosis (double Annexin V and FVD eFluor780 positive) cells were considered non-viable.

[0492] The results show bystander activity (solid squares) of the representative ADC compared to vitine-entaftuzumab (solid circles) and to no treatment (hollow triangles). Bystander activity was determined by plotting cell death in Nectin4-negative cell lines while co-cultured with Nectin4-expressing cell lines. Figure 6 (A)). Compared to vitine-emfretuzumab in target negative cell monoclonal culture, the nonspecific activity of the representative ADC was absent. Figure 6 (B)).

[0493] Example 8: In vivo work of NECTIN-4 ADC with multiple payloads in HT1-376 xenograft model effect.

[0494] The in vivo efficacy of NECTIN-4 ADC was assessed using the following protocol. In short, HT-1376 cell suspension was mixed with Matrigel at a 1:1 ratio. 5,000,000 live cells were subcutaneously injected into the posterior abdomen of female BALB / c nude mice. Tumors were evaluated when the average tumor size reached approximately 100 mm. 3 Mice were randomly divided into five (5) groups. The test sample was administered once at a dose of 5 mg / kg. The study was terminated on day 28.

[0495] The results showed that all NECTIN-4 ADCs inhibited tumor growth compared to the control group. (See...) Figure 7 ).

[0496] Example 9: In vivo efficacy of NECTIN-4 ADC in the Sum190PT breast cancer xenograft model.

[0497] In addition, the in vivo efficacy of NECTIN-4 ADC (Ab5-ADC2) was further investigated using the following protocol. In short, Sum190PT cell suspension was mixed with Cultrex ECM at a 1:1 ratio. 3,000,000 live cells were subcutaneously injected into the posterior abdomen of female NSG mice. When the average tumor size reached approximately 130 mm... 3 Mice were randomly divided into groups of five (5) mice each. The test product was administered twice a week at 10 mg / kg intervals. The study was terminated on day 28.

[0498] Results show the in vivo efficacy of Nectin-4 ADC (solid circles) compared to the corresponding isotype control ADC (hollow squares) and PBS group (hollow circles) in a Nectin4-positive Sum190PT breast cancer xenograft model. (See also: [link to original text]) Figure 8 ).

[0499] Example 10: In vivo efficacy of NECTIN-4 ADC compared to PADCEV in a patient-derived head and neck cancer model.

[0500] In addition, the in vivo efficacy of NECTIN-4 ADC (Ab5-ADC2) compared to the commercially available Vitin-Enterotolumab (PADCEV) was further investigated using the following protocol. In short, tumors derived from patients with head and neck squamous cell carcinoma were proliferated in vivo in immunocompromised mice as patient-derived xenografts (PDXs). Tumor fragments (2-3 mm in diameter) from stock mice were harvested and subcutaneously inoculated into female NOD / SCID mice. When the average tumor size reached approximately 150 mm... 3 Mice were randomly divided into groups of five (5) mice each. The test product was administered as a single dose or twice at two (2) week intervals at 5 mg / kg or 10 mg / kg.

[0501] Results showed that in a Nectin4-positive patient-derived head and neck cancer model, the Nectin-4 ADC (round) demonstrated better efficacy and complete tumor eradication compared to Vitin-Enflutumab (square). (See also...) Figure 9 ).

[0502] Example 11: Use of chimeric antigen receptor (CAR) T-cell therapy in cancers expressing NECTIN-4.

[0503] Generally, T cells help detect and fight infections and diseases, such as cancer in the body. Many cancers can evade T cells, so cancer can grow in the body when T cells cannot "see" it. A promising form of cancer immunotherapy is called CAR-T therapy.

[0504] In CAR-T therapy, chimeric antigen receptors (CARs) are engineered to recognize specific markers expressed in cancer (such as NECTIN-4). Studies have shown that when CARs are linked to specific antigens, they induce immune responses and T cells to recognize cancer and inhibit cancer growth.

[0505] Using a non-limiting example, blood was collected from patients with cancer expressing NECTIN-4. T cells from the patient's blood were isolated and genetically engineered to generate CAR-T cells. The CAR-T cells were cultured and expanded using techniques known in the art. Finally, the CAR-T cells were perfused into the patient's bloodstream. See JIN et al., Cancer Cell International, 21:83 (2021).

[0506] The trial initially demonstrated safety and subsequently confirmed efficacy with repeated doses. The trial was open-label, comparing standard chemotherapy with standard chemotherapy plus NECTIN-4 CAR-T cells. As will be understood, a non-limiting criterion for use in conjunction with patient registration is the NECTIN-4 concentration in the tumor as determined by standards known in the field.

[0507] Example 12: Use of natural killer (NK) cell therapy in cancers expressing NECTIN-4.

[0508] Similar to CAR-T therapy, natural killer (NK) cell therapy is a form of immunotherapy that has shown promise in treating cancers, such as those expressing NECTIN-4. Unlike T cells, NK cells are not suited to specific antigens. However, while NK cells can recognize and invade cancer cells, they cannot survive long enough or proliferate rapidly enough to fully combat cancer cells. However, studies have shown that NK cells can be enhanced by treating them with immune system proteins called cytokines. Studies have shown that enhancing NK cells with cytokines leads to a more stable immune response. One advantage of NK cell therapy is the lack of side effects compared to CAR-T therapy. In some cases, NK cells are also enhanced with CARs to make them more suitable for fighting cancer. See LU et al., Frontiers in Oncology, Vol. 11, Paper 720501 (August 2021).

[0509] Using non-limiting examples, several strategies can be employed to enhance the efficacy of NK cell therapy. First, NK cells are generated from peripheral blood (PB), umbilical cord blood (UCB), induced pluripotent stem cells (iPSCs), and the NK92 cell line. After isolation from these sources, NK cells are stimulated with cytokines such as IL-2, IL-15, and / or IL-18. Furthermore, NK cells can be modified in vitro to express CARs, allowing them to recognize specific tumor-associated antigens, such as NECTIN-4. Finally, the NK cells are perfused into the patient's bloodstream. See MEHTA et al., Int. J. of Hematology, 107:262-270 (2018).

[0510] The trial initially demonstrated safety and subsequently confirmed efficacy with repeated doses. The trial was open-label, comparing standard chemotherapy with standard chemotherapy plus NECTIN-4 NK cells. As will be understood, a non-limiting criterion for use in conjunction with patient registration is the NECTIN-4 concentration in the tumor as determined by standards known in the field.

[0511] Example 13: Characterization analysis of NECTIN-4 antibody and NECTIN-4 ADC.

[0512] The Nectin-4 antibody and NECTIN-4 ADC composition of the present invention were further characterized using analyses known in the art.

[0513] Figure 11 and Figure 12 The results presented confirm a better safety profile and stronger efficacy, resulting in an improved therapeutic window compared to other known NECTIN-4 antibodies and NECTIN-4 ADCs in the field.

[0514] Example 14: In vitro safety assessment of NECTIN-4 ADC across multiple first-generation cultures of normal human cells.

[0515] The in vitro cytotoxicity of NECTIN-4 ADC was determined using the following protocol. In short, normal human primary cells, including corneal epithelial cells, adult dermal fibroblasts, and adult epidermal keratinocytes, were collected, plated into 384-well white flat-bottomed culture plates, and incubated at 37°C for 2–4 hours while allowing reattachment. Cells were then treated with the ADC or free payload assay via dose-adjustment (maximum 1000 nM and 5-fold dilution). Cell viability was then determined using CellTiter Glo analysis based on the manufacturer's instructions (Promega; Madison, Wisconsin) after 5 or 6 days of treatment. Data were normalized relative to untreated control cells, and dose-response curves were fitted using GraphPad Prism software (version 9; La Jolla, CA) with a 4-parameter logistic equation.

[0516] The results show that, for example, in human corneal epithelial cells (see...) Figure 11 (A)), adult dermal fibroblasts (see also...) Figure 11 (B)) and adult epidermal keratinocytes (see Figure 11 On various normal primary cell lines (C), NECTIN-4 ADC Ab5-ADC2 exhibited significantly weaker in vitro cytotoxicity than vitine-emfretuzumab. See also Table XVI, which shows the toxicokinetic parameters of ADC and total IgG at repeated doses of up to 18 mg / kg, and thus provides evidence of increased HNSTD in NHP as a result of the described improved in vitro safety profile.

[0517] In another set of experiments using the protocol described above, histograms of flow cytometry analysis of the aforementioned cells are shown. Human corneal epithelial cells (see...) Figure 12 (A)); Adult dermal fibroblasts (see also...) Figure 12 (B)) shows no NECTIN-4 expression and indicates a toxic tendency due to non-targeted ADC uptake; and adult epidermal keratinocytes (see Figure 12 (C)) expresses NECTIN-4 and indicates the mid-target toxicity tendency of NECTIN-4 ADC.

[0518] Example 15: Cell cycle analysis of Ab5-ADC2.

[0519] In this experiment, the following protocol was used to determine cell cycle analysis for Ab5-ADC2. In short, HT-1376 tumor cells were harvested, seeded into 6-well plates, and cultured overnight at 37°C. The following day, cells were treated with 5 nM Ab5-ADC2 for 72 hours, followed by propidium iodide staining with FxCycle PI / RNase staining solution (Engineers, Waltham, MA, USA). DNA content was measured by flow cytometry, and cell cycle data were analyzed using the Watson Pragmatic algorithm and FlowJo software (version 10; BD Biosciences, Franklin Lakes, NJ, USA) for modeling.

[0520] The results present a cell cycle analysis of the percentage (%) of G2 and G1 phase confluence in HT-1376 cells after 72 hours of Ab5-ADC2 treatment. (See also: [link to results]) Figure 13 (A)). Figure 13(B) A representative flow cytometry histogram of Ab5-ADC2-treated cells shows that, compared to untreated control cells, Ab5-ADC2-treated cells exhibited increased cell populations in both sub-G1 and G2 phases of the cell cycle, as expected for this payload class.

[0521] Example 16: Free payload compared to MMAE Immunogenic cell death (ICD) analyze 。

[0522] In this experiment, the following protocol was used to determine the ICD analysis of the free payload compared to MMAE. In short, NCI-H292 tumor cells were harvested and seeded into 100 mm culture dishes and incubated overnight at 37°C. The following day, cells were treated with 10 nM of free payload for 48 hours, followed by live cell staining with the following immunogenic cell death (ICD) markers: anti-calreticulin, anti-HSP70, and anti-HMGB1. All marker antibodies were labeled with Alexa Fluor 488 (Novus Biologicals, Centennial, CO, USA). Cells were analyzed using an Attune NxT flow cytometer, and ICD marker positivity was determined relative to isotype control antibody staining.

[0523] The results showed that, as expected for this payload category, the increase in immunogenic cell death markers was similar for both payloads. (See [link to previous section]) Figure 14 ).

[0524] Example 17: Complement-dependent cytotoxicity (CDC) analysis of Ab5-ADC2 and Ab5 。

[0525] In this experiment, the following protocol was used to determine the CDC assay for Ab5-ADC2. In short, HT-1376 tumor cells were collected and plated in 96-well white flat-bottomed culture plates and incubated on ice for one (1) hour with the specified serially diluted test sample to allow the antibody or ADC to bind to the cells. Subsequently, the cells were opsonized with rabbit complement at 37°C for 1 hour, and the remaining cell viability after cell lysis due to CDC activity was determined by CellTiter-Glo 2.0 assay (Promega).

[0526] The results showed that Ab5-ADC2 and entaftozumab lacked any significant CDC activity. (See also...) Figure 15 ).

[0527] Example 18:Ab5-ADC2 and Ab5 Antibody Cell-dependent cell-mediated cytotoxicity (ADCC) analysis 。

[0528] In this experiment, the ADCC analysis of Ab5-ADC2 was determined using the following protocol. In short, NCI-H292 tumor cells were harvested and seeded into 96-well white flat-bottomed culture plates and incubated overnight at 37°C. The following day, the tumor cells were co-incubated for 6 hours with engineered effector cells (effector cell to target cell ratio of 6:1) in the presence of the specified antibody or ADC assay, and ADCC activity was measured using a commercial ADCC Reporter Bioassay V Variant Kit (Promega, Madison, Wisconsin, USA).

[0529] The results showed that although the positive control (Her2 Ab) exhibited strong ADCC activity, no significant ADCC activity was observed for Ab5-ADC2. (See also...) Figure 16 ).

[0530] Example 19: Ab5-ADC2 and Ab5 Antibody Cell-dependent phagocytosis (ADCP) analysis 。

[0531] In this experiment, the ADCP assay for Ab5-ADC2 was determined using the following protocol. In short, tumor cells were harvested and seeded in 384-well white flat-bottomed culture plates and incubated overnight at 37°C. The following day, the tumor cells were co-incubated for 6 hours with engineered effector cells (effector cell to target cell ratio of 6:1 to 7.5:1) in the presence of the specified antibody or ADC assay sample, and ADCP activity was measured using a commercially available FcγRI ADCP bioassay kit (Promega).

[0532] The results showed that weak ADCP activity for Ab5-ADC2 was observed in the SUM190PT and HT-1376 cancer cell lines expressing NECTIN-4, compared to the analytical control reagent (isoCTRLAb in RAMOS cells expressing the target isoCTRLAb). (See also...) Figure 17 ).

[0533] Overall, the lack of findings from CDC, ADCC, and ADCP excludes any significant contribution of FcR-mediated effector activity to the mechanism of action of Ab5-ADC2.

[0534] Example 20: Pharmacokinetics of Ab5-ADC2's ADC and free payload in stone crab macaques.

[0535] In this experiment, the pharmacokinetic profile of Ab5-ADC2 and its corresponding free payload was determined using the following protocol. In short, the ADC test sample Ab5-ADC2 was administered to rhesus macaques at two different dose levels. Blood was collected at different time points and processed into serum for total antibody and ADC quantification and plasma for free payload analysis. Enzyme-linked immunosorbent assay (ELISA) was used to quantify total antibody and ADC concentrations by capture with soluble recombinant NECTIN-4 and detection with biotin-labeled anti-human IgG or biotin-labeled anti-payload, respectively, followed by binding to streptavidin-HRP. TMB was used as the colorimetric substrate, and the color reaction was stopped with H2SO4 solution. Absorbance was measured at 450 nm–630 nm on a disc reader. Calibration curves were generated by plotting the reactions of serially diluted test samples against calibration sample concentrations (average absorbance of each calibration sample minus the average absorbance of the blank) and fitting them with a four-parameter logistic model. All data were analyzed using SoftMax Pro 7.0 software.

[0536] For quantification of free payload, the payload was extracted from 20 μL of K2EDTA-containing plasma from stone crab macaques via protein precipitation, followed by AB SCIEX TRIPLE QUAD. TM The 6500+ mass spectrometer uses internal standards for detection and quantification. TK parameters are summarized in Table XVI.

[0537] The results showed a linear pharmacokinetic profile for total IgG, ADC, and free payload without significant accumulation. Notably, the total IgG and ADC curves overlapped at all dose levels and throughout the 21-day dosing cycle, with a half-life of approximately 5 days (Table XVI), achieved via a highly stable binding technique for generating Ab5-ADC2. Importantly, repeated dosings of 18 mg / kg Ab5-ADC2 were well tolerated in non-human primates, achieved through increased conjugation stability and superior tumor-targeting payload accumulation while preserving normal tissue (see [link to table]). Figure 18 ).

[0538] Example 21: NECTIN-4 ADC compared to Vitin-Enterotoxiprob in the patient-derived cervical cancer model PDX36 Its internal effects.

[0539] Additionally, the in vivo efficacy of NECTIN-4 ADC (Ab5-ADC2) compared to the commercially available vitin-enterotoxin was investigated using the following protocol. In short, PDX derived from cervical cancer patients (in this case, PDX36) was replicated in vivo in immunocompromised mice. Tumor fragments (2-3 mm in diameter) from native mice were harvested and subcutaneously inoculated into female NOD / SCID mice. When the average tumor size reached approximately 150 mm... 3Mice were randomly divided into groups of 5. The test product was administered at doses of 2.5, 5, or 10 mg / kg.

[0540] Results showed that in a patient-derived cervical cancer model, Ab5-ADC2 (black circle) demonstrated better efficacy and tumor suppression compared to vitine-enfretuzumab (dark triangle), which translated into longer study survival in tumor-bearing mice treated with Ab5-ADC2. (See also...) Figure 19 (A) and 19(B)).

[0541] Example 22: NECTIN-4 ADC in multiple patient-derived cervical cancer models (PDX10, PDX12, PDX13, ... In vivo efficacy of PDX16, PDX34 and PDX36.

[0542] In addition, the in vivo efficacy of NECTIN-4 ADC (Ab5-ADC2) was investigated in several cervical cancer models using the following protocol. In short, PDX derived from cervical cancer patients (PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36 in this case) were propagated in vivo in immunocompromised mice. Tumor fragments (2-3 mm in diameter) from propagating mice were collected and subcutaneously inoculated into female NOD / SCID mice. When the average tumor size reached approximately 150 mm... 3 Mice were randomly divided into groups of 5. The test product was administered at doses of 2.5, 5, or 10 mg / kg.

[0543] Results showed that in multiple patient-derived cervical cancer models, Ab5-ADC2 (black circle) exhibited significant efficacy and tumor inhibition compared to the mediator control. (See also...) Figure 20 (A) to 20(F)).

[0544] Example 23: NECTIN-4 expression in a patient-derived cervical cancer model via immunohistochemistry.

[0545] The following protocol was used to evaluate the expression characteristics of NECTIN-4 in a patient-derived cervical cancer xenograft model. In short, tissue samples were fixed in 10% buffered neutral formalin, processed, and embedded in paraffin, then prepared into 4 µm tissue sections. After dewaxing and rehydration, the sections underwent antigen retrieval. The antigen-retrieval-treated tissue sections were then incubated with either mouse anti-nectin-4 primary antibody or IgG (antibody control). The tissue sections bound to the primary antibody were washed and detected with enzyme-labeled secondary antibodies. The bound secondary antibodies were stained using a Leica Bond Refine Polymer Detection system. The stained tissue sections were then scanned and imaged using an Aperio ScanScope CS (Aperio; Vista, CA) and evaluated using optical microscopy. All stained sections were scanned at 40× magnification using a NanoZoomer-HT 2.0 Image system to generate images.

[0546] The results showed that NECTIN-4 expression was confirmed by IHC using an anti-Nectin-4 antibody. PDX12 (21(A) and 21(B)), PDX10 (21(C) and 21(D)), PDX16 (21(E) and 21(F)), PDX13 (21(G) and 21(H)), PDX34 (21(I) and 21(J)), and PDX36 (21(K) and 21(L)) are shown in the corresponding figures, with images shown at high resolution (21(A), (C), (E), (G), (I), and (K)) and low resolution (21(B), (D), (F), (H), (J), and (L)). (See also...) Figure 21 These PDX models exhibit a range of NECTIN-4 expression patterns, including PDX models with highly heterogeneous NECTIN-4 expression. Nevertheless, Ab5-ADC2 was able to achieve significant tumor growth inhibition in all models.

[0547] Example 24: Analysis of free payload concentration of Ab5-ADC2 or Vitin-Enterotomab via LC-MS / MS.

[0548] The following protocol was used to evaluate the free payload concentrations of Ab5-ADC2 or Vitin-Entaftumab in multiple tissues of NECTIN-4 positive breast cancer-bearing mice. In short, Ab5-ADC2 or Vitin-Entaftumab was administered to a Sum190PT inflammatory breast cancer xenograft mouse model. Tumor, normal tissue, and plasma were collected, and payload concentrations were analyzed by LCMS / MS.

[0549] Figure 22 The results in (A) show the free payload concentration relative to MMAE. Figure 22 The results in (B) show the payload concentration of Ab5-ADC2 relative to vitine-emfretuzumab. Notably, Ab5-ADC2 was able to deliver more payload to NECTIN-4-expressing tumors while reducing payload exposure in normal tissues, which underpins its better safety profile than vitine-emfretuzumab.

[0550] Example 25: Stability and DAR retention analysis of Ab5-ADC2.

[0551] Stability assessment and DAR retention analysis were performed using the following protocol. In short, Sprague Dawley rats, arriving at 10–12 weeks of age, were acclimatized for one week and subsequently given a single intravenous dose of either Ab5-ADC2 or Vittin-Enfretuzumab. Blood samples were collected at different time points following administration: 1 hour, 4 hours, day 3, day 7, day 14, and day 21. Blood collected in EDTA-coated tubes was processed into plasma by centrifugation at 10,000 g for 10 minutes at 4°C. The plasma was aliquoted and frozen at -80°C until analysis. Plasma samples were diluted in TBS, then combined with biotin-labeled anti-human antibody-coated beads, followed by mixing with streptavidin-coated DynaBeads and incubating for approximately 2 hours. The affinity-captured ADC on the beads was subjected to a magnet to collect the complex, followed by washing of the ADC. The affinity-purified ADC was operated on an Agilent QTOF 6550 B under MassHunter B.07.00 or equivalent. Peaks were integrated, extracted, and spectrally deconvolved using the maximum entropy algorithm. The deconvolved spectra were output as CSV files and imported into the DAR calculator. The DAR of each sample was measured and plotted over time.

[0552] Figure 23 The results in (A) show the stability of Ab5-ADC2 over time in terms of conjugation and drug-antibody ratio (DAR) compared to Vitin-Enterotomab after intravenous injection. Figure 23 The results in (B) present deconvolutiond MS profiles of the heavy and light chains of affinity-purified Ab5-ADC2 from plasma 1 hour and 21 days after injection into Sporgodory rats. Taken together, these results indicate that Ab5-ADC2 is highly stable in circulation, allowing for increased payload delivery to tumors compared to normal tissues, such as… Figure 22 This is what is shown. Importantly, this makes the HNSTD (maximum non-serious toxicity dose) dose in non-human primates 18 mg / kg, such as... Figure 18 As shown in the image.

[0553] Example 26: Human clinical trials using NECTIN-4 antibody and NECTIN-4 ADC to treat human cancers.

[0554] According to the present invention, NECTIN-4 antibodies and NECTIN-4 ADCs are synthesized, which specifically accumulate in tumor cells and are used to treat certain tumors and other immune disorders and / or other diseases (see Table I). Two clinical approaches have been successfully implemented for each of these indications.

[0555] I.) adjunctive therapy In adjuvant therapy, patients are treated with NECTIN-4 antibodies and NECTIN-4 ADCs, along with chemotherapeutic agents, pharmaceuticals, or biologics, or combinations thereof. The primary cancer target is treated under standard regimens by adding NECTIN-4 antibodies and NECTIN-4 ADCs. The regimen design addresses efficacy assessed through examples such as, but not limited to, reduction in tumor mass at primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce the common doses of standard chemotherapy and other biologics. These dose reductions allow for additional and / or longer-term therapy by reducing dose-related toxicities of chemotherapeutic agents or biologics.

[0556] II.) Monotherapy This involves combining NECTIN-4 antibody and NECTIN-4 ADC in monotherapy for cancer, administering the NECTIN-4 antibody and NECTIN-4 ADC to patients in the absence of chemotherapy, pharmaceuticals, or biologics. In one embodiment, monotherapy is administered clinically to patients with advanced cancer suffering from extensive metastatic disease. The regimen design addresses efficacy assessed through examples including, but not limited to, reduction in tumor mass of primary or metastatic lesions, increased progression-free survival, overall survival, improvement in patient health, disease stabilization, and the ability to reduce common doses of standard chemotherapy and other biologics.

[0557] dose Dosing regimens can be adjusted to provide the optimal desired response. For example, a single NECTIN-4 antibody and NECTIN-4 ADC can be administered via injection, administered in fractions over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. As used herein, “Dosage Unit Form” refers to a physically discontinuous unit suitable as a unit dose for use in a mammalian individual to be treated; each unit contains a predetermined amount of active compound, calculated to produce the desired therapeutic effect, associated with the desired drug delivery system. The specifications of the Dosage Unit Form of the present invention are determined by and directly dependent on the following factors: (a) the unique characteristics of the NECTIN-4 antibody and NECTIN-4 ADC, the individual mechanisms of the radiation mechanism (reactor), and the specific therapeutic or preventative effect to be achieved, and (b) the inherent limitations of mixing such compounds in the art for treating individual sensitivities.

[0558] Clinical Development Program (CDP) CDP adheres to and develops treatments for cancer and / or immune disorders using the NECTIN-4 antibody and NECTIN-4 ADC disclosed herein (see Table I). Trials initially demonstrated safety and subsequently confirmed efficacy with repeated doses. Trials were open-label, comparing standard chemotherapy with standard therapy plus NECTIN-4 antibody and NECTIN-4 ADC. As will be understood, a non-limiting criterion for use in conjunction with patient registration is the concentration of NECTIN-4 antibody and NECTIN-4 ADC in the tumor, as determined by standard assays known in the art.

[0559] The scope of this invention is not limited to the embodiments disclosed herein, which are intended as a single illustration of individual aspects of the invention, and any functionally equivalent embodiments are within the scope of this invention. Those skilled in the art will readily recognize, from the foregoing description and teachings, various modifications to the model, method, and lifecycle methodology of this invention, other than those described herein, and similarly intend to fall within the scope of this invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of this invention.

[0560] Table I. Representative list of cancers awaiting treatment

[0561] Table II. amino acid abbreviation

[0562]

[0563] Table III. amino acid substitution matrix Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the greater the likelihood of finding substitutions in the relevant natural protein.

[0564]

[0565] Table IV. Nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human adhesion molecule-4 (NECTIN-4)

[0566] Table V. Amino acid sequence of human adhesion molecule-4 (NECTIN-4) (SEQ ID NO: 3). Signal peptide underlined .

[0567]

[0568] Tables VI(A) to VI(F). Sequences of the antibody heavy chain. Table VI(A). cDNA sequence (SEQ ID NO: 4) and amino acid sequence (SEQ ID NO: 5) of the CL.Z heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0569]

[0570] Table VI(B). cDNA sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7) of the CL.X heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0571]

[0572] Table VI(C). cDNA sequence (SEQ ID NO: 8) and amino acid sequence (SEQ ID NO: 9) of the CL.N heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0573]

[0574] Table VI(D). cDNA sequence (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 11) of the CL.I heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0575]

[0576] Table VI(E). cDNA sequence (SEQ ID NO: 12) and amino acid sequence (SEQ ID NO: 13) of the CL.B heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0577]

[0578] Table VI(F). cDNA (SEQ ID NO: 14) and amino acid sequence (SEQ ID NO: 15) of the CL.D heavy chain. Nucleotide sequence encoding the variable region. underlined .

[0579]

[0580] Tables VI(G) to VI(L). Sequences of the antibody light chain Table VI(G). cDNA sequence (SEQ ID NO: 16) and amino acid sequence (SEQ ID NO: 17) of the CL.Z light chain. Nucleotide sequence encoding the variable region. underlined .

[0581]

[0582] Table VI(H). cDNA sequence (SEQ ID NO: 18) and amino acid sequence (SEQ ID NO: 19) of the CL.X light chain. Nucleotide sequence encoding the variable region. underlined .

[0583]

[0584] Table VI(I). cDNA sequence (SEQ ID NO: 20) and amino acid sequence (SEQ ID NO: 21) of the CL.N light chain. Nucleotide sequence encoding the variable region. underlined .

[0585]

[0586] Table VI(J). cDNA sequence (SEQ ID NO: 22) and amino acid sequence (SEQ ID NO: 23) of the CL.I light chain. Nucleotide sequence encoding the variable region. underlined .

[0587]

[0588] Table VI(K). cDNA sequence (SEQ ID NO: 24) and amino acid sequence (SEQ ID NO: 25) of the CL.B light chain. Nucleotide sequence encoding the variable region. underlined .

[0589]

[0590] Table VI(L). cDNA sequence (SEQ ID NO: 26) and amino acid sequence (SEQ ID NO: 27) of the CL.D light chain. Nucleotide sequence encoding the variable region. underlined .

[0591]

[0592] Tables VII(A) to VII(F). Amino acid sequences of the antibody heavy chain. Table VII(A). Amino acid sequence of the CL.Z heavy chain (SEQ ID NO: 28). Variable region underlined Furthermore, the KabatCDR area is framed.

[0593]

[0594] Table VII(B). Amino acid sequence of the CL.X heavy chain (SEQ ID NO: 29). Variable region underlined Furthermore, the KabatCDR area is framed.

[0595]

[0596] Table VII(C). Amino acid sequence of the CL.N heavy chain (SEQ ID NO: 30). Variable region underlined Furthermore, the KabatCDR area is framed.

[0597]

[0598] Table VII(D). Amino acid sequence of the CL.I heavy chain (SEQ ID NO: 31). Variable region underlined Furthermore, the KabatCDR area is framed.

[0599]

[0600] Table VII(E). Amino acid sequence of the CL.B heavy chain (SEQ ID NO: 32). Variable region underlined Furthermore, the KabatCDR area is framed.

[0601]

[0602] Table VII(F). Amino acid sequence of the CL.D heavy chain (SEQ ID NO: 33). Variable region. underlined Furthermore, the KabatCDR area is framed.

[0603]

[0604] Tables VII(G) to VII(L). Amino acid sequences of the antibody light chain. Table VII(G). Amino acid sequence of the CL.Z light chain (SEQ ID NO: 34). Variable region underlined Furthermore, the KabatCDR area is framed.

[0605]

[0606] Table VII(H). Amino acid sequence of the CL.X light chain (SEQ ID NO: 35). Variable region underlined Furthermore, the KabatCDR area is framed.

[0607]

[0608] Table VII(I). Amino acid sequence of the CL.N light chain (SEQ ID NO: 36). Variable region underlined Furthermore, the KabatCDR area is framed.

[0609]

[0610] Table VII(J). Amino acid sequence of the CL.I light chain (SEQ ID NO: 37). Variable region underlined Furthermore, the KabatCDR area is framed.

[0611]

[0612] Table VII(K). Amino acid sequence of the CL.B light chain (SEQ ID NO: 38). Variable region underlined Furthermore, the KabatCDR area is framed.

[0613]

[0614] Table VII(L). Amino acid sequence of the CL.D light chain (SEQ ID NO: 39). Variable region underlined Furthermore, the KabatCDR area is framed.

[0615]

[0616] Table VIII. Amino acid sequence of the variable region of the antibody heavy chain

[0617] Table IX. Amino acid sequence of the variable region of the antibody light chain

[0618] Table X. Amino acid sequence of antibody CDR

[0619]

[0620] Tables XI(A) to XI(F). Alignment of amino acid variable heavy chain regions with corresponding germline sequences. Table XI(A). Alignment of the amino acid sequence of the CL.Z heavy chain variable region (SEQ ID NO: 88) with the corresponding top V (SEQ ID NO: 89), D (SEQ ID NO: 90), and J (SEQ ID NO: 91) germline sequences. Kabat CDR region is boxed.

[0621]

[0622] Table XI(B). Alignment of the amino acid sequence of the CL.X heavy chain variable region (SEQ ID NO: 92) with the corresponding top V (SEQ ID NO: 93), D (SEQ ID NO: 94), and J (SEQ ID NO: 95) germline sequences. Kabat CDR region is boxed.

[0623]

[0624] Table XI(C). Alignment of the amino acid sequence (SEQ ID NO: 96) of the variable region of the CL.N heavy chain with the corresponding top V (SEQ ID NO: 97), D (SEQ ID NO: 98), and J (SEQ ID NO: 99) germline sequences. Kabat CDR regions are boxed.

[0625]

[0626] Table XI(D). Alignment of the amino acid sequence of the CL.I heavy chain variable region (SEQ ID NO: 100) with the corresponding top V (SEQ ID NO: 101), D (SEQ ID NO: 102), and J (SEQ ID NO: 103) germline sequences. Kabat CDR regions are boxed.

[0627]

[0628] Table XI(E). Alignment of the amino acid sequence of the CL.B heavy chain variable region (SEQ ID NO: 104) with the corresponding top V (SEQ ID NO: 105), D (SEQ ID NO: 106), and J (SEQ ID NO: 107) germline sequences. Kabat CDR region is boxed.

[0629]

[0630] Table XI(F). Alignment of the amino acid sequence (SEQ ID NO: 108) of the variable region of the CL.D heavy chain with the corresponding top V (SEQ ID NO: 109), D (SEQ ID NO: 110), and J (SEQ ID NO: 111) germline sequences. Kabat CDR region is boxed.

[0631]

[0632] Tables XI(G) to XI(L). Alignment of amino acid light chain variable regions with corresponding germline sequences. Table XI(G). Alignment of the amino acid sequence of the CL.Z light chain variable region (SEQ ID NO: 112) with the corresponding top V (SEQ ID NO: 113) and J (SEQ ID NO: 114) germline sequences. Kabat CDR region is boxed.

[0633]

[0634] Table XI(H). Alignment of the amino acid sequence of the CL.X light chain variable region (SEQ ID NO: 115) with the corresponding top V (SEQ ID NO: 116) and J (SEQ ID NO: 117) germline sequences. Kabat CDR region is boxed.

[0635]

[0636] Table XI(I). Alignment of the amino acid sequence (SEQ ID NO: 118) of the variable region of the CL.N light chain with the corresponding top V (SEQ ID NO: 119) and J (SEQ ID NO: 120) germline sequences. Kabat CDR region is boxed.

[0637]

[0638] Table XI(J). Alignment of the amino acid sequence of the CL.I light chain variable region (SEQ ID NO: 121) with the corresponding top V (SEQ ID NO: 122) and J (SEQ ID NO: 123) germline sequences. Kabat CDR region is boxed.

[0639]

[0640] Table XI(K). Alignment of the amino acid sequence (SEQ ID NO: 124) of the variable region of the CL.B light chain with the corresponding top V (SEQ ID NO: 125) and J (SEQ ID NO: 126) germline sequences. Kabat CDR region is boxed.

[0641]

[0642] Table XI(L). Alignment of the amino acid sequence of the variable region of the CL.D light chain (SEQ ID NO: 127) with the corresponding top V (SEQ ID NO: 128) and J (SEQ ID NO: 129) germline sequences. Kabat CDR region is boxed.

[0643]

[0644] Table XII. Summary of mean fluorescence intensity ratio (MFIR) values ​​for the binding of various Nectin-4 antibodies to the human breast cancer cell line T-47D.

[0645] Table XIII. Summary of binding parameters Bmax, Kmax of various Nectin-4 antibodies and derived ADCs with the human lung cancer cell line NCI-H292. D and the fitted curve R2

[0646] Table XIV. Summary of in vitro cytotoxic titers (IC50) in Nectin4-positive cell lines, NCI-H322 and PC3-Nectin4, and the Nectin4-negative cell line PC3. 50 )

[0647]

[0648] Table XV. Summary of in vitro cytotoxic titers (IC50) of Nectin-4 ADCs containing nine (9) different linker-loads in the Nectin-4 positive cell line PC3-Nectin4. 50 )

[0649] Table XVI. Summary of toxicological parameters of ADC and total IgG antibodies following intravenous administration of Ab5-ADC2 at doses of 6, 12, or 18 mg / kg in stone crab macaques.

Claims

1. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO:

54.

2. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO:

57.

3. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO:

60.

4. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO:

63.

5. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO:

66.

6. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) having the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO:

69.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof is conjugated to a drug via a linker group.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the drug is an auristatin analog.

9. An antibody drug conjugate (ADC) comprising a NECTIN-4 antibody or antigen-binding fragment thereof conjugated to a drug-linker (DL) payload, wherein the antibody or antigen-binding fragment thereof comprises an antibody according to any one of claims 1-6.

10. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 11. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 12. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 13. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 14. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 15. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 16. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 17. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 18. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 19. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 20. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 21. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 22. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 23. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 24. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 25. The ADC of claim 9, wherein the DL payload comprises the following chemical structure: 。 26. A pharmaceutical composition comprising the ADC of any one of claims 9-25 and a pharmaceutically acceptable excipient.

27. A kit comprising the ADC of any one of claims 9-25.

28. A kit comprising the pharmaceutical composition of claim 26.

29. A method of treating cancer in an individual comprising administering to the individual a therapeutically effective amount of the ADC of any one of claims 9-25.

30. A method of treating cancer in an individual comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition of claim 26.

31. The method of claim 29, wherein the individual is a human.

32. The method of claim 30, wherein the individual is a human.

33. The method of claim 29, wherein the cancer is listed in Table I.

34. The method of claim 30, wherein the cancer is listed in Table I.

35. The method of claim 29, wherein the method further comprises administering a radiation or chemical therapeutic agent or a CAR-T therapy or an NK cell therapy.

36. The method of claim 30, wherein the method further comprises administering a radiation or chemical therapeutic agent or a CAR-T therapy or an NK cell therapy.

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