Methods and compositions relating to antibodies and antibody-drug conjugates (ADCs) that bind to the nectin-4 protein

Abstract

Antibody-drug conjugates (ADCs) that bind to nectin-4 protein(s) and its variants are described herein. Nectin-4 exhibits tissue-specific expression in normal adult tissues and is abnormally expressed in cancers listed in Table I. As a result, the ADCs of the present invention provide therapeutic compositions for the treatment of cancer. The present invention provides antibodies or antigen-binding fragments thereof that include a heavy chain variable region containing a complementarity-determining region (CDR) having sequences shown, for example, in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.

Description

[Technical Field]

[0001] Cross-references to related applications 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 thus fully incorporated herein by reference.

[0002] Submission of the sequence listing XML file ("SEQUENCE LISTING XML") The contents of the following submission are thus fully incorporated herein by reference: the contents of a sequence listing in a computer-readable form (CRF) XML file (filename: 9300-20000.40-SEQ LIST-XML-07-June-2024, date recorded: June 6, 2024, size: 136KB).

[0003] Statement of rights regarding inventions made under government-funded research Not applicable.

[0004] Field of Invention The inventions described herein relate to antibodies that bind to the nectin-4 protein, antigen-binding fragments thereof, and antibody-drug conjugates (ADCs). The inventions further relate to prognostic, prophylactic, and therapeutic methods, as well as compositions useful in the treatment of cancer and other immunological and neurological disorders. [Background technology]

[0005] Background of the Invention Cancer is the second leading cause of death worldwide, after coronary artery disease.

[0006] While cancer treatment has improved over the past few decades and survival rates have increased, the heterogeneity of cancer still demands new therapeutic strategies utilizing multiple treatment modalities. This is especially true when treating solid tumors in critical anatomical sites (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma) which are sometimes limited to standard radiotherapy and / or chemotherapy. Nevertheless, the adverse effects of these treatments include chemotherapy and radioresistance that promote localized recurrence, distant metastasis, and secondary primary tumors, in addition to severe side effects that reduce the patient's quality of life. The therapeutic utility of monoclonal antibodies (mAbs) in the fight against cancer and other medical conditions (G. KOHLER and C. MILSTEIN, Nature 256:495-497 (1975)) has been realized. Generally, antibodies act through several mechanisms, most of which involve other arms of the immune system.

[0007] Antibody-drug conjugates (ADCs) represent a newly emerging class of targeted therapeutics with improved therapeutic indices beyond conventional chemotherapy. Drugs and linkers, along with (monoclonal) antibodies (mAbs) and target selection, have been the focus of ADC development. However, the importance of conjugate homogeneity has recently been explored. It has been reported that the pharmacological profile of ADCs can be improved by applying site-specific conjugation techniques using manipulated surface-exposed cysteine ​​residues in the antibody conjugated to the linker drug, resulting in site-specifically conjugated ADCs with defined drug-to-antibody ratios (DARs).

[0008] Prior art discloses several approaches to obtaining ADCs. 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.).

[0009] All prior art methods disclosed to date have focused on the conjugation sites of the linker drug, specifically at surface / solvent-exposed locations, at highly thiol-reactive locations, and at locations specifically within the constant region of the monoclonal antibody, with the aim of improving homogeneity and pharmacokinetic properties.

[0010] While the conventional lysine and cysteine ​​conjugation methods described above have resulted in FDA-approved antibody-drug conjugates, which are currently used to construct most of the numerous ADCs in preclinical and clinical trials, there is still a need for novel conjugation strategies aimed at (further) improving the physicochemical, pharmacokinetic, pharmacological, and / or toxicological properties of ADCs to obtain ADCs with acceptable antigen-binding characteristics, in vivo efficacy, therapeutic index, and / or stability. From the above, it is readily apparent to those skilled in the art that a new treatment paradigm is needed in the treatment of cancer and immunological diseases. By using modern antibody manipulation techniques and novel conjugation methodologies, a new class of antibodies can be achieved with the overall objectives of more effective treatment, reduced side effects, and lower production costs. Considering the existing shortcomings known in the art, the object of the present invention is to provide novel and improved antibodies and binding ligands, as well as methods for treating cancer(s), immunological disorders and other diseases using antibodies and ADCs. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2006 / 034488 [Patent Document 2] International Publication No. 2014 / 124316 [Patent Document 3] U.S. Patent Application Publication No. 2017 / 0080103 [Patent Document 4] International Publication No. 2019 / 183438 [Non-patent literature]

[0012] [Non-Patent Document 1] G. KOHLER and C. MILSTEIN, Nature 256:495-497 (1975) [Non-Patent Document 2] SUTHERLAND, et. al., Blood 122(8):1455-1463 (2013) [Overview of the project] [Means for solving the problem]

[0013] Summary of the Invention The present invention provides antibodies that bind to nectin-4 protein and polypeptide fragments of nectin-4 protein, antigen-binding fragments, antibody-drug conjugates (ADCs), antibody-immunomodulatory conjugates, antibody fusion proteins, and antibody-fragment fusion proteins. In some embodiments, the present invention includes fully human antibodies conjugated to therapeutic agents.

[0014] In certain embodiments, 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 certain embodiments, the entire nucleic acid sequence of Table IV is encoded, and / or the entire amino acid sequence of Table V is prepared, and either of them is in their respective human unit dose forms.

[0015] The present invention further provides various immunogenic or therapeutic compositions, such as antibodies, antibody-drug conjugates, and strategies for treating cancers that express nectin-4, such as those listed in Table I.

[0016] In another embodiment, the present disclosure teaches an antibody composition represented by CL.Z.

[0017] In another embodiment, the present disclosure teaches an antibody composition represented by CL.X.

[0018] In another embodiment, the present disclosure teaches an antibody composition represented by CL.N.

[0019] In another embodiment, the present disclosure teaches an antibody composition shown in CL.I.

[0020] In another embodiment, the present disclosure teaches an antibody composition represented by CL.B.

[0021] In another embodiment, the present disclosure teaches an antibody composition shown in CL.D.

[0022] In another embodiment, the present disclosure teaches a method for synthesizing antibodies.

[0023] In another embodiment, the Disclosure teaches a method for synthesizing an antibody and conjugating a drug moiety to the antibody to form an ADC.

[0024] In another embodiment, the disclosure teaches a method for treating cancer(s) in humans.

[0025] In another embodiment, the disclosure teaches a method for treating immunological or neurological disorders in humans.

[0026] In another embodiment, the Disclosure teaches the use of one or more compositions of this Specified in the manufacture of pharmaceuticals for treating cancer(s), immunological and / or neurological disorders(s) in humans. [Brief explanation of the drawing]

[0027] [Figure 1] Antibody binding specificity of Nectin-4 Ab to multiple cell lines. [Figure 2-1] Antibody binding of Nectin-4 Ab to T-47D breast cancer cell lineage. Figure 2(A) shows Ab1. Figure 2(B) shows Ab2. Figure 2(C) shows Ab3. Figure 2(D) shows Ab4. Figure 2(E) shows Ab5. Figure 2(F) shows Ab6. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3] Antibody affinity of Nectin-4 Ab against NCI-H292 lung cancer cell lineage compared to each ADC. [Figure 4-1] In vitro cytotoxicity of nectin-4 ADCs across multiple cancer cell lines. Figure 4(A) shows the NCI-H322 lung adenocarcinoma cell line. Figure 4(B) shows the PC3-nectin-4 recombinant nectin-4-expressing PC3 prostate cancer cell line. Figure 4(C) shows the nectin-4-negative PC3 cell line. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] In vitro cytotoxicity of Sum190PT breast cancer cell line with nectin-4 ADC using multiple payloads. [Figure 6] Bystander activity of ADC compared to enfortumab vedotin. Figure 6(A) shows a nectin-4 negative cell line in a co-culture with a nectin-4 expressing cell line. Figure 6(B) shows a nectin-4 negative cell line in a single culture. [Figure 7] In vivo efficacy of multiple nectin-4 ADCs using multiple payloads in an HT-1376 xenograft model. [Figure 8] In vivo efficacy of nectin-4 ADC in a nectin-4-positive Sum190PT breast cancer xenograft model. [Figure 9] In vivo efficacy of nectin-4 ADC compared to enfortumab vedotin in a head and neck cancer model derived from nectin-4-positive patients. [Figure 10-1] Drug-linker (DL) payload structure. Figure 10(A) shows the structure indicated by DL-01. Figure 10(B) shows the structure indicated by DL-02. Figure 10(C) shows the structure indicated by DL-03. Figure 10(D) shows the structure indicated by DL-04. Figure 10(E) shows the structure indicated by DL-05. Figure 10(F) shows the structure indicated by DL-06. Figure 10(G) shows the structure indicated by DL-07. Figure 10(H) shows the structure indicated by DL-08. Figure 10(I) shows the structure indicated by DL-09. Figure 10(J) shows the structure indicated by DL-10. Figure 10(K) shows the structure indicated by DL-11. Figure 10(L) shows the structure indicated by DL-12. Figure 10(M) shows the structure indicated by DL-13. Figure 10(N) shows the structure indicated by DL-14. Figure 10(O) shows the structure indicated by DL-15. Figure 10(P) shows the structure indicated by DL-16. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. [Figure 10-7] Same as above. [Figure 10-8] Same as above. [Figure 11-1]In vitro cytotoxicity of nectin-4 ADCs across multiple primary cultures of normal human cells. Figure 11(A) shows HCEpC cells. Figure 11(B) shows HDFa cells. Figure 11(C) shows HEKa cells. [Figure 11-2] Same as above. [Figure 12-1] Flow cytometry histograms of nectin-4 mediated by enfortumab and Ab5 across multiple primary cultures of normal human cells. Figure 12(A) shows HCEpC cells. Figure 12(B) shows HDFa cells. Figure 12(C) shows HEKa cells. [Figure 12-2] Same as above. [Figure 13-1] Cell cycle analysis of Ab5-ADC2 in HT-1376 cells. Figure 13(A) shows the combined percentage of HT-1376 cells in the G2 and sub-G1 phases after 72 hours of treatment with Ab5-ADC2. Figure 13(B) shows representative flow cytometry histograms of Ab5-ADC2-treated cells compared to untreated control cells. [Figure 13-2] Same as above. [Figure 14] Induction of immunogenic cell death by free payload compared to MMAE in NCI-H292 cancer cells. [Figure 15] Complement-dependent cytotoxicity analysis of Ab5-ADC and Ab5 compared to enfortumab antibody in HT-1376 cancer cells. [Figure 16] Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of Ab5-ADC2 and Ab5 in NCI-H292 cancer cells. [Figure 17] Antibody-dependent cell-mediated phagocytosis (ADCP) activity of Ab5-ADC2 and Ab5 across multiple cancer cell lines. [Figure 18] Pharmacokinetic (PK) profiles of total IgG, ADC, and free payload of Ab5-ADC2. [Figure 19-1]In vivo efficacy of nectin-4 ADC Ab5-ADC2 compared to enfortumab vedotin in the PDX36 cervical cancer model derived from nectin-4-positive patients. Figure 19(A) shows tumor growth dynamics. Figure 19(B) shows survival probability in Kaplan-Meier plots. [Figure 19-2] Same as above. [Figure 20-1] In vivo efficacy of nectin-4 ADC Ab5-ADC2 in mouse clinical trials of six cancer models. Figure 20(A) shows PDX10. Figure 20(B) shows PDX12. Figure 20(C) shows PDX13. Figure 20(D) shows PDX16. Figure 20(E) shows PDX34. Figure 20(F) shows PDX36. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 21-1] Nectin-4 expression in patient-derived xenograft models of cervical cancer mediated by immunohistochemistry. Figures 21(A), 21(C), 21(E), 21(G), 21(I), and 21(K) show high-resolution expression. Figures 21(B), 21(D), 21(F), 21(H), 21(J), and 21(L) show low-resolution expression. Figures 21(A) and 21(B) show PDX12. Figures 21(C) and 21(D) show PDX10. Figures 21(E) and 21(F) show PDX16. Figures 21(G) and 21(H) show PDX13. Figures 21(I) and 21(J) show PDX34. Figures 21(K) and 21(L) show PDX36. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 22] Free payload concentrations of Ab5-ADC2 or enfortumab vedotin in normal and tumor tissues. Figure 22(A) shows the free payload concentration in normal tissue. Figure 22(B) shows the payload concentrations of Ab5-ADC2 and enfortumab vedotin. [Figure 23-1]Stability profile of Ab5-ADC2. Figure 23(A) shows the conjugation stability and initial drug-to-antibody ratio (DAR) percentage of Ab5-ADC2 compared to enfortumab vedotin. Figure 23(B) shows the deconvoluted MS profiles of the heavy and light chains of affinity-purified Ab5-ADC2. [Figure 23-2] Same as above. [Modes for carrying out the invention]

[0028] Detailed description of the invention Section Overview I.) Definition II.) Antibodies III.) Antibody-drug conjugates IV.) Linker Unit V.) Stretcher Unit VI.) Amino Acid Units VII.) Spacer Unit VIII.) Drug Unit IX.) Drug Loading X.) Method for determining the cytotoxic effect of ADCs XI.) Treatment of cancer(s) expressing nectin-4 XII.) Nectin-4 ADC Cocktail XIII.) Combination Therapy XIV.) Kits / Manufactured Products

[0029] I.) Definition: Unless otherwise defined, all terms, notations, and other scientific or technical terms used herein are intended to have meanings generally understood by those skilled in the art, unless the context clearly indicates otherwise. In some cases, terms having generally understood meanings are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be interpreted as indicating a substantial difference beyond the generally understood meanings in the art. Where necessary, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters specified by the manufacturer, unless otherwise indicated.

[0030] Where a trade name is used herein, references to that trade name also refer to the product formulation, generic drugs, and active medicinal ingredients of the product of that trade name, unless otherwise indicated by the context.

[0031] The terms “advanced cancer,” “locally advanced cancer,” “progressive disease,” and “locally advanced disease” refer to cancer that has spread through the associated tissue capsule, and include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, nodule, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, as these patients have substantially less favorable outcomes compared to patients with clinically localized (organ-limited) cancer.

[0032] The term "substituted" means that the identified group or part has one or more substituents. The term "unsubstituted" means that the identified group does not have substituents. The term "substituted as necessary" means that the identified group is either unsubstituted or substituted with one or more substituents. When the term "substituted" is used to describe a structural system, the substitution is intended to be at any valence-possible position on that system.

[0033] The term "analog" refers to a molecule that is structurally similar to another molecule (e.g., a nectin-4 related protein) or that shares similar or corresponding properties with another molecule. For example, an analog of the nectin-4 protein may be an antibody or T cell that specifically binds to nectin-4.

[0034] The term “antibody” is used in its broadest sense unless otherwise clearly indicated. Therefore, “antibody” can be naturally occurring or synthetic, such as monoclonal antibodies produced by conventional hybridoma or transgenic mouse technologies. Nectin-4 antibodies include monoclonal and polyclonal antibodies, as well as fragments containing the antigen-binding domain 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, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they specifically bind to nectin-4 and / or exhibit desired biological activity. Any specific antibody may be used in the methods and compositions provided herein. Therefore, in one embodiment, the term “antibody” encompasses molecules comprising at least one variable region derived from a light chain immunoglobulin molecule and at least one variable region derived from a heavy chain molecule, which jointly form a specific binding site to a target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. Antibodies useful in the methods and compositions of the present invention can be produced in cell cultures, in phages, in yeast, or in a variety of 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 the present invention is a mammalian antibody. Phage techniques can be used to isolate initial antibodies or to generate variants having altered specificity or binding affinity characteristics. Such techniques are conventional and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector containing a DNA sequence encoding the antibody.One or more vectors may be used to transfect host cells with DNA sequences expressing at least one VL and at least one VH region. Exemplary descriptions of recombinant means 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, most recent edition). The antibodies of the present invention may be modified by recombinant means to increase the effectiveness of the antibody in mediating a desired function. Therefore, the modification of antibodies by substitution using recombinant means falls within the scope of the present invention. Typically, the substitutions are conservative substitutions. For example, at least one amino acid in the constant region of the antibody may be replaced with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, application number WO9958572; and ANGAL, et al., Mol. Immunol. 30: 105-08 (1993). Amino acid modifications include deletions, additions, and substitutions. In some cases, such changes are made to reduce undesirable activity, such as complement-dependent cytotoxicity. Frequently, antibodies are labeled by conjugating a substance that provides a detectable signal, either covalently or noncovalently. A wide variety of labeling and conjugation techniques are known and extensively reported in both 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 having the desired biological activity can be identified using the following in vitro assays, and the following in vivo assays, including but not limited to proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, and signal transduction, for example, inhibition of tumor growth. The antibodies provided herein may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind specifically to an antigen without inhibiting either antigen receptor binding or biological activity. As neutralizing antibodies, the antibodies may be useful in competitive binding assays. They can also be used to quantify nectin-4 and / or its receptor.

[0035] 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 variant). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment which is a monovalent fragment consisting of a V L domain, a V H domain, a C L domain and a C H1 domain; (ii) an F(ab’)2 fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of a V H domain and a C H1 domain; (iv) an Fv fragment consisting of a V L domain and a V H domain of a single arm of an antibody; (v) a dAb fragment consisting of a V H domain (WARD et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs). Further, the two domains V L and V HThese are encoded by separate genes, but these can be combined using recombination. L Region and V H These single-chain antibodies can be linked by synthetic linkers, which allow them to be constructed as single protein chains (also known as single-chain Fv (scFv); see, for example, BIRD et. al. (1988) Science 242:423-426; and HUSTON et. al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) in which the regions pair up to form a monovalent molecule. Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0036] As used herein, the term "Fc" refers to the region containing the hinge region, the CH2 domain, and / or the CH3 domain.

[0037] As used herein, any form of “antigen” may be used to produce antibodies specific to Nectin-4 of the present invention. Thus, the eliciting antigen may be a single epitope, multiple epitopes, or an entire protein, either alone or in combination with one or more immunogenicity enhancers known in the art. The eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing at least a portion of the antigen in transfected cells), or a soluble protein (e.g., immunizing only the extracellular domain portion of the protein). The antigen may be produced in genetically modified cells. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term “portion” means, as necessary, the minimum number of amino acids or nucleic acids that constitute the immunogenic epitope of the antigen of interest. Any gene vector suitable for the transformation of the cells of interest may be used, including but not limited to adenovirus vectors, plasmids, and nonviral vectors, such as cationic lipids. In one embodiment, the antibody of the method and composition described herein specifically binds to at least a portion of the extracellular domain of the target of interest.

[0038] The antibodies or antigen-binding fragments provided herein may constitute or be part of a “bioactive agent.” As used herein, the term “bioactive agent” refers to any synthetic or naturally occurring compound that enhances a cell-killing toxin, binds to an antigen, and / or enhances or mediates a desired biological effect. In one embodiment, the binding fragment useful in the present invention is a biologically active fragment. As used herein, the term “bioactive” refers to an antibody or antibody fragment that is capable of binding to a desired antigenic epitope and exerting a biological effect directly or indirectly. Direct effects include, but are not limited to, modulation, stimulation, and / or inhibition of growth signals; modulation, stimulation, and / or inhibition of anti-apoptotic signals; modulation, stimulation, and / or inhibition of apoptosis or necrosis signals; modulation, stimulation, and / or inhibition of the ADCC cascade; and modulation, stimulation, and / or inhibition of the CDC cascade and / or Fc silencing.

[0039] The term "specifically binds," as used herein, means that, with respect to antigen-binding proteins, the antigen-binding protein binds to a target and separate domains or separate amino acid sequences within the target with no or insignificant binding to other (e.g., unrelated) proteins. However, this term does not preclude the fact that antibodies or their binding fragments may also be cross-reactive with closely related molecules. Antibodies and their fragments, and antibody-drug conjugates containing them as described herein, may bind specifically to nectin-4 as disclosed herein with an affinity at least 2, 5, 10, 50, 100, or 1000 times higher than when they bind to closely related molecules.

[0040] "Bispecific" antibodies are also useful in the methods and compositions of the present invention. As used herein, the term "bispecific antibody" refers to an antibody having binding specificity to at least two different antigenic epitopes, typically a monoclonal antibody. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitopes are derived from two different antigens. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced 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 linkage. See, for example, BRENNAN, et. al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. For example, see HOLLINGER, et. al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) and GRUBER, et. al., J. Immunol. 152:5368 (1994).

[0041] The monoclonal antibodies described herein specifically include “chimeric” antibodies, insofar as they specifically bind to a target antigen and / or exhibit the desired biological activity, in which portions of the heavy chain and / or light chain are identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (U.S. Patent No. 4,816,567; and MORRISON et. al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

[0042] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are interchangeable and refer to malignantly transformed cells that make them pathological to the host organism, either singular or plural. Primary cancer cells (i.e., cells obtained near the site of malignant transformation) can be readily identified from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells, as used herein, includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a “clinically detectable” tumor is one that is detectable based on tumor volume; for example, one that is detectable by procedures such as CAT scans, MR imaging, X-rays, ultrasound or palpation, and / or one that is detectable due to the expression of one or more cancer-specific antigens in a sample obtainable from a patient. A tumor can be a hematopoietic tumor, meaning a liquid tumor, such as a tumor of blood cells. Specific examples of clinical conditions based on such tumors include leukemia, such as chronic myeloid leukemia or acute myeloid leukemia; myeloma, such as multiple myeloma; and lymphoma.

[0043] The term “therapeutic agent,” as defined herein, refers to all agents that provide a therapeutic benefit and / or are therapeutically effective. A therapeutic agent may, for example, reverse, ameliorate, alleviate, inhibit, or limit the progression of a disease, disorder, or condition, or reduce the severity of a disease, disorder, or condition; it may affect, improve, or improve one or more symptoms of a disease, such as cancer. Such agents may be cytotoxic or inhibitory. The term includes, but is not limited to, chemotherapeutic agents, antineoplastic agents, and “drug units” as defined herein.

[0044] The term “antineoplastic agent,” as defined herein, refers to all agents that provide a therapeutic benefit and / or are therapeutically effective in treating neoplasms or cancers.

[0045] The term "chemotherapeutic agent" refers to all chemical compounds effective in inhibiting tumor growth. Non-exclusive examples of chemotherapeutic agents include alkylating agents; e.g., nitrogen mustard, ethyleneimine compounds, and alkyl sulfonates; antimetabolites, e.g., folic acid, purine, or pyrimidine antagonists; mitotic inhibitors, e.g., antitubulin agents, e.g., vinca alkaloids, auristatin, and podophyllotoxin derivatives; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, e.g., DNA sulcus binding agents; and growth factor receptor antagonists. Furthermore, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.

[0046] It is well known in the field that the terms "complementarity-determining region" and "CDR" refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region.

[0047] The precise amino acid sequence boundaries of a given CDR can be found in 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) JMB 273, 927-948 ("Chothia" numbering scheme), MACCALLUM et. al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," 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," Dev Comp Immunol. This can be easily determined using one of several well-known schemes, including those described in 2003 January; 27(1):55-77 ("IMGT" numbering scheme) and HONEGGER A. and PLICKTHUN A., "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70 (AHo numbering scheme).

[0048] The boundaries of a given CDR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based on the most common antibody region sequence length, with insertions adjusted by inserting letters, e.g., "30a," and deletions that appear in some antibodies. These two schemes result in differential numbering by placing certain insertions and deletions ("indels") at various positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0049] Therefore, unless otherwise specified, a given antibody or region, e.g., the terms “CDR” and “complementarity-determining region” of a variable region, as well as individual CDRs of the antibody or region (e.g., “CDR-H1, CDR-H2”), should be understood to encompass the complementarity-determining region as defined by any of the known schemes described herein. In some cases, schemes are specified for the identification of a particular CDR(s), such as CDRs defined by the Kabat, Chothia, or Contact methods.

[0050] As used herein, the term “conservative substitution” means a substitution of an amino acid and / or amino acid sequence known to those skilled in the art, which can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution 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 shown in Tables II and III. For example, such substitutions include the substitution of any other of these hydrophobic amino acids by any one of isoleucine (I), valine (V), and leucine (L); the substitution of aspartic acid (D) for glutamic acid (E) and vice versa; the substitution of glutamine (Q) for asparagine (N) and vice versa; and the substitution of serine (S) for threonine (T) and vice versa. Other substitutions may also be considered conserved, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A), as well as alanine (A) and valine (V), may be frequently interchangeable. Relatively hydrophobic methionine (M) may be frequently interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable where the distinct feature of the amino acid residue is its charge, and where the different pKs of these two amino acid residues are not important. Other changes may be considered "conservative" in certain environments (see, for example, Table III in this specification; pages 13-15 "Biochemistry" 2nd ED. Lubert Stryer ed (Stanford University); HENIKOFF et. al., PNAS 1992 Vol 89 10915-10919; LEI et. al., J Biol Chem 1995 May 19; 270(20):11882-6).Other substitutions are also acceptable and can be determined experimentally or according to known conservative substitutions.

[0051] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity, function, and / or causes cell destruction. This term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as fragments and / or variants thereof, including small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin. Examples of cytotoxic agents include auristatin, auristatin derivatives, auromycin, camptothecin (a topoisomerase 1 inhibitor), mytansinoids, lysine, lysine A-chain, combrestatin, duocalmycin, dorastatin, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxyanthracendione. Dione), actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, geronin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors, as well as glucocorticoids and other chemotherapeutic agents, and radioisotopes, for example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 or 213 , P 32 , and Lu 177 This includes, but is not limited to, radioactive isotopes of Lu.

[0052] Antibodies containing the antibody of the present invention can also be conjugated to any of the cytotoxic agents described above, and to anticancer prodrug activating enzymes capable of converting prodrugs into their active form.

[0053] As used herein, the term “diabody” refers to a small antibody fragment having two antigen-binding sites, which are the same polypeptide chain (V H -V L ) contains a light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) include. By using a linker that is too short to allow pairing between two domains on the same chain, those domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites. Diabodies are described more fully, for example, in EP404,097;WO93 / 11161; and HOLLINGER et. al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).

[0054] The term "homolog" refers to a molecule that exhibits homology to another molecule, for example, by having the same or similar sequence of chemical residues at corresponding positions.

[0055] The term “identical” or “sequence identity” refers to the degree of identity between two nucleic acids or two amino acid sequences when they are optimally aligned and compared with appropriate insertions or deletions.

[0056] The "percent identity" between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % identity = number of identical positions / total number of positions × 100). Sequence comparison and determination of percentage identity between two sequences can be achieved using the mathematical algorithms described below. Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, with the NWS gap dna CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Percentage identity between two nucleotide or amino acid sequences can also be determined using the algorithm of Meyers, et al., Comput. Appi. Biosci., 4:11-17 (1988), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties. Furthermore, percentage identity between two amino acid sequences can be determined using either the Blossum 62 matrix or the PAM250 matrix, as well as the algorithm of NEEDLEMAN, et al., J. Mol. Biol. 48:444-453 (1970), incorporated into the GAP program in the GCG software package, using either the Blossum 62 matrix or the PAM250 matrix, and 16, 14, 12, 10, 8, 6, or 4 gap weights and 1, 2, 3, 4, 5, or 6 length weights.

[0057] For example, a polynucleotide sequence may be identical to a reference polynucleotide sequence that is 100% identical to the reference sequence, or it may contain nucleotide changes up to a certain integer compared to the reference sequence, for example, it may be at least 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% identical. Such changes are selected from substitutions or insertions that include at least one nucleotide deletion, rearrangement, and transversion, and these changes may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence, or anywhere between those terminal positions, individually between nucleotides in the reference sequence or scattered as one or more consecutive bases within the reference sequence. The number of nucleotide changes can be calculated by multiplying the total number of nucleotides in the reference polynucleotide sequence described herein by the numerical percentage (divided by 100) of each percentage identity, and subtracting that product from the total number of nucleotides in the reference polynucleotide sequence, or n n ≤x n -(x ny Determined by ), in the formula, n n x is the number of nucleotide changes. n x is the total number of nucleotides in the reference polynucleotide sequence described herein (for example reference polynucleotide sequences, see the nucleic acid sequences in the "Sequence Listings"), 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%, and is a symbol in place of the multiplication operator. n The product of any non-integer with y is x nBefore subtraction, the fraction is rounded down to the nearest integer. Similarly, a polypeptide sequence may be identical to a polypeptide reference sequence described herein, i.e., 100% identical, or may include amino acid changes up to a certain integer compared to the reference sequence, resulting in a % identity of 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, substitutions including conserved and non-conserved substitutions, or insertions, and these changes may occur at the amino-terminal or carboxy-terminal position of the reference polypeptide sequence, or anywhere between those terminal positions, individually between amino acids in the reference sequence, or scattered as one or more consecutive groups within the reference sequence. The number of amino acid changes for a given % identity is obtained by multiplying the total number of amino acids in the polypeptide sequence encoded by the polypeptide reference sequence by the numerical percentage (divided by 100) of each % identity, and then subtracting that product from the total number of amino acids in the polypeptide reference sequence described herein, or n a ≤x a -(x ay Determined by ), in the formula, n a x is the number of amino acid changes. a y is the total number of amino acids in the reference polypeptide sequence, where 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%, and x is a symbol used in place of the multiplication operator. a The product of any non-integer with y is x a Before subtraction, fractions are rounded down to the nearest integer. Percent identity can be determined over the length of the array. As defined herein, the term “over 75% identical” includes identity over 75%, 80%, 85%, 95%, and 99%, as well as all distinct values ​​and distinct subranges within this range.

[0058] In one embodiment, the antibodies provided herein are “human antibodies.” As used herein, the term “human antibody” refers to an antibody whose entire sequence, including the light and heavy chain sequences containing complementarity-determining regions, is derived from human genes. In one embodiment, human monoclonal antibodies are prepared by trioma techniques, human B-cell techniques (see, e.g., KOZBOR, et. al., Immunol. Today 4: 72 (1983)), EBV transformation techniques (see, e.g., COLE et. al. MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)), or by yeast or phage display (see, e.g., MARKS et. al., J. Mol. Biol. 222:581 (1991)). In specific embodiments, human antibodies are produced in transgenic mice. Techniques for producing such partially to fully human antibodies are known in the art, and any such technique may be used. In one particularly preferred embodiment, a fully human antibody sequence is produced in a transgenic mouse engineered to express human heavy and light chain antibody genes. Exemplary descriptions of preparing transgenic mice and their offspring that produce human antibodies are found in patent application WO02 / 43478 and U.S. Patent No. 6,657,103 (Abgenix). B cells derived from the transgenic mouse producing the desired antibody can then be fused to create a hybridoma cell line for the continuous production of the antibody. See, for example, U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; as well as JAKOBOVITS, Adv. Drug Del. Rev. 31:33-42 (1998); GREEN, et. al., J. Exp. Med. 188:483-95 (1998).

[0059] As used herein, the term “humanized antibody” refers to a form of antibody containing sequences derived from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR region is a human immunoglobulin sequence. A humanized antibody also, where applicable, contains at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, CABILLY, U.S. Patent No. 4,816,567; QUEEN, et. al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

[0060] The terms “inhibit” or “inhibit of” mean, as used herein, to reduce by a measurable amount or to prevent completely.

[0061] 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 present invention, the mammal is a mouse. In another embodiment of the present invention, the mammal is a human.

[0062] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread to regional lymph nodes or distant sites, and include stage D disease under the AUA system and stage T×N×M+ under the TNM system.

[0063] The term "modified," as used herein, refers to the presence of a change in a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. Such a change or modification may be obtained by post-synthetic modification or co-translation of a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide, or by post-translational modification of a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide.

[0064] "Molecular recognition" refers to the chemical event that allows a host molecule to form a complex with a second molecule (i.e., a guest). This process occurs through non-covalent chemical bonding, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

[0065] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring variations that may be present in trace amounts. Monoclonal antibodies are highly specific to a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a number of antibodies against (or specific to) different epitopes. In one embodiment, a polyclonal antibody contains multiple monoclonal antibodies with different epitope specificity, affinity, or binding affinity within a single antigen containing multiple antigenic epitopes. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described by KOHLER et. al., Nature 256: 495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using techniques described, for example, CLACKSON et. al., Nature 352: 624-628 (1991) and MARKS et. al., J. Mol. Biol. 222: 581-597 (1991). These monoclonal antibodies typically conjugate at a Kd of at least about 1 μM, more commonly at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, and more preferably at least about 3 nM or better, as usually determined by ELISA.

[0066] "Non-natural amino acids," or otherwise written as "nnAA," refer to amino acids that are neither one of the 20 common amino acids nor pyrrolidine or selenocysteine. Other terms that may be used as synonyms for nnAA include "amino acids not naturally encoded," "unnatural amino acids," and "amino acids that do not exist in nature." Furthermore, the term nnAA includes, but is not limited to, amino acids that do not exist in nature but can be obtained synthetically or through the modification of non-natural amino acids.

[0067] "Pharmaceutical additives" include materials such as adjuvants, carriers, pH adjusters and buffering agents, tension adjusters, wetting agents, and preservatives.

[0068] "Pharmacologically acceptable" refers to a composition that is physiologically compatible with humans or other mammals, is non-toxic, inert, and / or in nature.

[0069] The term "polypeptide" refers to a polymer of at least approximately 4, 5, 6, 7, or 8 amino acids. Throughout this specification, standard three-letter (see Table II) or one-letter designations for amino acids are used. In this art, this term is often used interchangeably with "peptide" or "protein."

[0070] As used herein, the terms “single-chain Fv” or “scFv” or “single-chain” antibody refer to the V of the antibody. H Domain and V L This refers to antibody fragments containing domains, which are present within a single polypeptide chain. Generally, Fv polypeptides are V H Domain and V LThe sFv further includes a polypeptide linker between the domain and the sFv, which allows the sFv to form the desired structure for antigen binding. For an overview of sFv, see PLUCKTHUN, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0071] As used herein, the terms “specific,” “specifically binds,” and “binds specifically” refer to the selective binding of an antibody to a target antigen epitope. An antibody may be tested for binding specificity by comparing its binding to a suitable antigen with its binding to an unrelated antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds at least 2, 5, 7, and preferably 10 times more to a suitable antigen than to an unrelated antigen or antigen mixture. In one embodiment, a specific antibody is an antibody that binds only to the nectin-4 antigen but not to any other unrelated antigen. In another embodiment, the specific antibody is an antibody that binds to the human nectin-4 antigen but does not bind to non-human nectin-4 antigens 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 the human nectin-4 antigen but does not bind to non-human nectin-4 antigens having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percent identity to the amino acid sequence of the nectin-4 antigen. In another embodiment, the specific antibody is an antibody that binds to the human nectin-4 antigen and the mouse nectin-4 antigen, with a higher degree of binding to the human antigen. In another embodiment, the specific antibody is an antibody that binds to human nectin-4 antigens and primate nectin-4 antigens, with a higher degree of binding to human antigens. In yet another embodiment, the specific antibody binds to human nectin-4 antigens and any non-human nectin-4 antigens, with a higher degree of binding to human antigens or any combination thereof.

[0072] As used herein, “to treat” or “therapeutic” and grammatically related terms refer to any improvement of any outcome of a disease, e.g., extended survival, lower morbidity, and / or reduction of side effects which are byproducts of an alternative therapeutic modality; as readily understood in the art, complete eradication of the disease is preferred, though not a requirement of a treatment act.

[0073] The term "variant" refers to a molecule that exhibits a variation from a described type or standard, for example, a protein having one or more different amino acid residues at the corresponding position(s) of a specifically described protein (e.g., the Nectin-4 protein shown in Table IV). Analogs are an example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

[0074] The terms “isolated” or “biologically pure” refer to material that substantially or essentially does not contain the components that would normally accompany it when found in its native state. Therefore, isolated peptides according to the present invention preferably do not contain the materials normally associated with the peptide in their in-situ environment. For example, a polynucleotide is said to be “isolated” if it is substantially separated from contaminant polynucleotides that correspond to or are complementary to genes other than the nectin-4 gene, or that encode polypeptides other than the nectin-4 gene product or fragments thereof. Those skilled in the art can readily use nucleic acid isolation procedures to obtain isolated nectin-4 polynucleotides. A protein is said to be “isolated” if, for example, physical, mechanical, or chemical methods are used to isolate the nectin-4 protein from cellular components normally associated with that protein. Those skilled in the art can readily use standard purification methods to obtain isolated nectin-4 proteins. Alternatively, isolated proteins can be prepared by chemical means.

[0075] Appropriate “labels” include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, 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. Furthermore, the antibodies provided herein may be useful as antigen-binding components of fluorobodies (see, for example, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003)).

[0076] The “nectin-4 protein” and / or “nectin-4 related protein” of the present invention include those specifically identified herein (see Table IV), as well as allele variants, conserved substitution variants, analogs, and homologs that can be isolated / produced and characterized without excessive experimentation according to the methods outlined herein or readily available in the art. Fusion proteins combining parts of different nectin-4 proteins or fragments thereof, as well as fusion proteins of nectin-4 proteins with heterologous polypeptides, are also included. Such nectin-4 proteins are collectively referred to as nectin-4 related proteins, the proteins of the present invention, or nectin-4. The term "nectin-4 related protein" refers to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 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, 135, 140, 145, 150, 155, 160, 1 This refers to a polypeptide fragment or nectin-4 protein sequence containing 65, 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.

[0077] II.) Antibodies Another aspect of the present invention provides antibodies that bind to nectin-4 as disclosed herein. In one embodiment, antibodies that bind to nectin-4 (Table IV) and other nectin-4-related proteins.

[0078] As is well known in the art, the nectin-4 antibody of the present invention is particularly useful in cancer (see, for example, Table I) for prognostic assays, imaging, diagnostic and therapeutic methodologies. In one embodiment, a nectin-4 binding assay is disclosed herein for use in cancer detection, for example, in immunoassays. Similarly, such nectin-4 antibody is useful in the treatment and / or prognosis of cancer (e.g., cancers shown in Table I) insofar as nectin-4 is also expressed or overexpressed in these other cancers (e.g., in combination with therapeutic agents, for example, in ADCs). Furthermore, intracellularly expressed antibodies (e.g., single-chain antibodies) are therapeutically useful when treating cancers in which the expression of nectin-4 and other targets is involved.

[0079] Various methods for preparing antibodies, specifically monoclonal antibodies, are well known in the field. For example, antibodies can be prepared by immunizing a suitable mammalian host using nectin-4-related proteins, peptides, or fragments in isolated or immunoconjugated forms (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). Furthermore, nectin-4 fusion proteins, such as nectin-4 GST fusion proteins, can also be used. In certain embodiments, a GST fusion protein containing all or most of the amino acid sequence of nectin-4 is produced and then used as an immunogen to generate a suitable antibody. In other embodiments, nectin-4-related proteins are synthesized and used as an immunogen.

[0080] Furthermore, naked DNA immunization techniques known in the field are used (with or without purified nectin-4-related protein or nectin-4-expressing cells) to generate an immune response to the encoded immunogen (see DONNELLY et. al., 1997, Ann. Rev. Immunol. 15: 617-648 for an overview).

[0081] Preferred methods for generating Nectin-4 antibodies are further illustrated by the examples provided herein. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers, e.g., BSA, KLH, or other carrier proteins are also well known in the art. In some situations, direct conjugation using, for example, carbodiimide reagents is used; in other cases, conjugation reagents, e.g., supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of Nectin-4 immunogen is often carried out by injection over a suitable period of time and by the use of appropriate adjuvants, as understood in the art. During the immunization schedule, antibody titers may be obtained to determine the validity of antibody formation.

[0082] Nectin-4 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines secreting the desired monoclonal antibody can be prepared using the standard hybridoma technique of Kohler and Milstein or modifications to immortalize antibody-producing B cells, as is generally known. Immortalized cell lines secreting the desired antibody are screened by immunoassays in which the antigen is a nectin-4 related protein. Once a suitable immortalized cell culture is identified, the cells can be expanded, and the antibody can be produced from the in vitro culture or from ascites fluid.

[0083] The antibodies or fragments of the present invention may also be produced by recombinant means. Regions that specifically bind to a desired region of the nectin-4 protein may also be produced for chimeric antibodies or complementarity-determining region (CDR) graft antibodies of multiple species origins. Humanized or human nectin-4 antibodies may also be produced and are preferred for use in a therapeutic context. Methods for humanizing mouse and other non-human antibodies by substituting one or more non-human antibody CDRs for corresponding human antibody sequences 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, Proc. Natl. Acad. Sci. USA 89: 4285 and SIMS et al., 1993, J. Immunol. 151: 2296.

[0084] In one embodiment, the human monoclonal antibody of the present invention may be prepared using VelocImmune mice (Regeneron, Tarrytown, NY) in which the genomic sequence containing endogenous mouse variable segments at the immunoglobulin heavy chain (VH, DH, and JH segments) and / or kappa light chain (VK and JK) loci is entirely or partially replaced with a human genomic sequence containing unreorganized germline variable segments at the human immunoglobulin heavy chain (VH, DH, and JH) and / or kappa light chain (VK and JK) loci. 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.

[0085] Furthermore, the human antibodies of the present invention can be generated using HuMAb mice (Medarex, Inc.) that contain a human immunoglobulin gene minilocus encoding unreorganized human heavy chain (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous mu and kappa chain loci (see, for example, LONBERG, et. al. (1994) Nature 368(6474): 856-859).

[0086] In another embodiment, the fully human antibody of the present invention may be produced using a mouse possessing a human immunoglobulin sequence on a transgene and a transchromosome, for example, a mouse possessing a human heavy chain transgene and a human light chain transchromosome. Such a mouse is referred to herein as a "KM mouse," and such a mouse is described in TOMIZUKA et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO02 / 43478 to TOMIZUKA et al.

[0087] The human monoclonal antibodies of the present invention may also be prepared using phage or yeast display methods to screen a library of human immunoglobulin genes. Such phage display methods for isolating human antibodies are well-established in the art. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 for LADNER et al.; U.S. Patents Nos. 5,427,908 and 5,580,717 for DOWER et al.; U.S. Patents Nos. 5,969,108 and 6,172,197 for MCCAFFERTY et al.; and U.S. Patents Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 for GRIFFITHS et al.

[0088] The human monoclonal antibodies of the present invention may also be prepared using SCID mice in which human immune cells have been reconstituted so that a human antibody response can be generated during immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0089] Furthermore, the human antibodies of the present invention may be produced using a technique that utilizes transgenic mice called Xenomouse (Amgen Fremont, Inc., formerly Abgenix, Inc.), which are engineered at human heavy and light chain loci and inactivated for antibody production. Exemplary descriptions of preparing transgenic mice that produce human antibodies can be found in U.S. Patent No. 6,657,103; U.S. Patents No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,661,016; and No. 5,545,806; as well as MENDEZ, et. al. Nature Genetics, 15: 146-156 (1998); KELLERMAN, SA & GREEN, LL, Curr. Opin. Biotechnol 13, 593-597 (2002).

[0090] Any of the above production methods yields an antibody having a specific ability to bind to nectin-4, or a homolog, fragment, or polypeptide sequence having 85, 90, 91, 92, 93, 94, 95, 96, 9, 98, or 99% sequence identity to nectin-4.

[0091] The binding affinity (K) of an antibody against nectin-4, its binding fragment, and an antibody-drug conjugate containing the same. D ) may be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, K D It can be between 5nM and 10nM; or between 1nM and 2nM. K DThis concentration may be between 1 micromolar and 500 micromolar or between 500 micromolar and 1 nM.

[0092] The binding affinity of an antigen-binding protein is determined by its association constant (Ka) and dissociation constant (Kd) (KD = Kd / Ka). Binding affinity can be measured, for example, by capturing a test antibody on a protein-A coated sensor surface and flowing nectin-4 over this surface using BIACORE. Alternatively, binding affinity can be measured, for example, by capturing a test antibody receptor on a protein-A coated needle and flowing nectin-4 over this surface using FORTEBIO. Those skilled in the art can identify other suitable assays known in the art for measuring binding affinity.

[0093] The manipulated antibodies of the present invention include (for example, to improve the properties of the antibody) V H and / or V L The present invention includes antibodies in which framework residues have been modified. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to “reverse mutagenerate” one or more framework residues into the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues different from the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. To return the framework region sequence to its germline configuration, the somatic mutation can be “reverse mutagenerated” into the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis (e.g., “reverse mutagenerated” from leucine to methionine). Such “reverse mutagenerated” antibodies are also intended to be included in the present invention.

[0094] Manipulation of the VH and / or VL may also be performed to alter the binding affinity to the antigen. For example, altering residues within the framework and / or CDR region to increase or decrease affinity to nectin-4 is also intended to be included by the present invention.

[0095] 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 and thereby reduce the potential immunogenicity of the antibody. This approach, also known as “deimmunization,” is described in more detail in U.S. Patent Application Publication 2003 / 0153043 by CARR et al.

[0096] Furthermore, instead of modifications made within the framework or CDR region, the antibodies of the present invention may be manipulated to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cell-mediated cytotoxicity. In addition, the nectin-4 antibodies of the present invention may be chemically modified (e.g., to allow the antibody to bind to one or more chemical portions) or to alter their glycosylation, or further to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below.

[0097] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, by increasing or decreasing it. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the nectin-4 antibody.

[0098] In another embodiment, the Fc hinge region of an antibody is mutated to reduce 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 such that the antibody has impaired staphylococcyl protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by WARD et al.

[0099] In another embodiment, the nectin-4 antibody is modified to increase its biological half-life. Various approaches are possible. For example, mutations may be introduced as described in U.S. Patent No. 6,277,375 for Ward. Alternatively, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope obtained from two loops of the CH2 domain in the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and No. 6,121,022 by PRESTA et al.

[0100] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(A).

[0101] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(B).

[0102] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(C).

[0103] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(D).

[0104] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(E).

[0105] In another embodiment, the nectin-4 antibody comprises the antibody heavy chain sequence shown in Table VI(F).

[0106] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(G).

[0107] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(H).

[0108] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(I).

[0109] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(J).

[0110] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(K).

[0111] In another embodiment, the nectin-4 antibody comprises the antibody light chain sequence shown in Table VI(L).

[0112] In another embodiment, the nectin-4 antibody includes the antibody heavy chain variable region sequence shown in Table VIII.

[0113] In another embodiment, the nectin-4 antibody includes the antibody light chain variable region sequence shown in Table IX.

[0114] In another embodiment, the nectin-4 antibody comprises the antibody CDR sequence shown in Table X.

[0115] In another embodiment, the nectin-4 antibody is conjugated to the therapeutic agent.

[0116] The reactivity of a nectin-4 antibody can be established by several 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. The nectin-4 antibody or a fragment thereof may be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes.

[0117] III.) Antibody-drug conjugates In another embodiment, the present invention provides an antibody-drug conjugate (ADC) comprising an antibody (preferably a nectin-4 antibody as disclosed herein) conjugated to a therapeutic agent. The therapeutic agent may be a cytotoxic agent, a cell proliferation inhibitor, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin), or a radioisotope (i.e., a radioconjugate). In another embodiment, the present invention further provides a method of using an ADC. In one embodiment, the ADC comprises any of the nectin-4 antibodies conjugated to a cytotoxic agent or a detectable agent by covalent bond or via an oxime bond.

[0118] In further embodiments, the ADC comprises a nectin-4 antibody conjugated to the therapeutic agent using an autohydrolyzable maleimide for cysteine ​​modification (see WO2013 / 173337).

[0119] In a further embodiment, the ADC comprises a nectin-4 antibody conjugated to a therapeutic agent using a cysteine ​​modification, further comprising reducing a cysteine ​​residue to form a sulfhydryl moiety.

[0120] In further embodiments, the ADC comprises a nectin-4 antibody conjugated to a therapeutic agent using a polypeptide moiety and a self-immolative moiety.

[0121] In further embodiments, the ADC comprises a nectin-4 antibody conjugated to a therapeutic agent, and the ADC has a high drug-to-antibody ratio (DAR).

[0122] As background, the use of antibody-drug conjugates for local delivery of cytotoxic or cell proliferation inhibitors in cancer treatment (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; NICULESCU-DUVAZ and SPRINGER (1997) Adv. Drg. Del. Rev. 26:151-172; U.S. Patent No. 4,975,278) enables targeted delivery of the drug portion to tumors and intracellular accumulation therein. In this context, systemic administration of these unconjugated drugs can produce unacceptable levels of toxicity to normal cells and tumor cells that need to be eliminated (BALDWIN et. al., (1986) Lancet pp. (Mar. 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. (ed.), pp. 475-506). This requires maximum efficacy with minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (ROWLAND et. al., (1986) above).Toxins used in antibody-toxin conjugates include bacterial toxins, such as diphtheria toxin; plant toxins, such as lysine; small molecule toxins, such as geldanamycin (MANDLER et. al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; MANDLER et. al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; MANDLER et. al. (2002) Bioconjugate Chem. 13:786-791); mytansinoids (EP1391213; LIU et. al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); and calicheamicin (LODE et. al. (1998) Cancer Res. 58:2928; HINMAN et. al. (1993) Cancer Res. 53:3336-3342) is included. Toxins can influence their cytotoxic and cell proliferation inhibitory effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

[0123] To date, the FDA has approved 12 ADCs, including gemtuzumab ozogamicin (MYLOTARG, Wyeth Pharmaceuticals), which was the first ADC approved by the FDA in 2000 (see, for example, Drago et al. 2021 Nature Reviews 18, 327-344; Mckertish et al. 2021 Biomedicines 9, 872; Khongorzui et al. 2020 Molecular Cancer Res. 18:3-19; Bross et al. 2001 Clin. Cancer Res. 7, 1490-1496; Hamann et al. 2002 Bioconjug. Chem. 13, 47-58; Lamb, 2017 Drugs 77, 1603-1610).

[0124] Further examples of commercially available antibody-drug conjugates include ADCETRIS (brentuximab vedotin, Seattle Genetics), ZEVALIN (registered trademark) (ibritumomab tiuxetan, Biogen / Idec), KADCYLA (registered trademark) (ado-trastuzumab emtansine, Genentech), BESPONSA (registered trademark) (inotuzumab ozogamicin, Pfizer / Wyeth), POLIVY (polatuzumab vedotin, Genentech / Roche), cantuzumab meltansine (Immunogen, Inc.), MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), and PADCEV (enfortumumab vedotin-ejfv, Seattle Genetics / Astellas (Agensys, Inc., Santa Monica, California.

[0125] Furthermore, therapeutic agents useful in the production of ADCs, including but not limited to chemotherapeutic agents, are described herein. Enzymatically active toxins and their fragments that may be used include the diphtheria A chain, an unbound active fragment of diphtheria toxin; the exotoxin A chain (derived from Pseudomonas aeruginosa); the lysine A chain; the abrin A chain; the modesine A chain; alpha-sarcin; Aleurites fordii protein; dianthin protein; Phytolaca americana protein (PAPI, PAPII, and PAP-S); momordica charantia inhibitors; curcin; crotin; sapaonaria officinalis inhibitors; geronin; mitogenin; restrictocin; phenomycin; enomycin; and tricothecene. See, for example, WO93 / 21232, published October 28, 1993. Various radionuclides are available for the production of radioconjugated antibodies. Examples include, 177 Lu, 89 Zr, 212 Bi, 131 I, 131 In, 90 Y and 186Re is included. Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein-coupling agents, e.g., N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antibodies (WO94 / 11026). Other antitumor agents that can be conjugated to the antibodies of the present invention include BCNU, streptozocin, vincristine, and 5-fluorouracil, a family of agents collectively known as the LL-E33288 conjugate described in U.S. Patents No. 5,053,394 and No. 5,770,710, as well as esperamicin (U.S. Patent No. 5,877,296).

[0126] Enzymatically active toxins and their fragments that can be used include the diphtheria A chain, an unbound active fragment of diphtheria toxin; the exotoxin A chain (derived from Pseudomonas aeruginosa); the lysine A chain; the abrin A chain; the modesine A chain; alpha-sarcin; Aleurites fordii protein; dianthin protein; Phytolaca americana protein (PAPI, PAPII, and PAP-S); momordica charantia inhibitors; curcin; crotin; sapaonaria officinalis inhibitors; geronin; mitogenin; restrictosin; phenomycin; enomycin; and trichothecenes. For example, lysine immunotoxins can be prepared as described in Vitetta et al (1987) Science, 238:1098. See, for example, WO93 / 21232 (published October 28, 1993).

[0127] The present invention further envisions the formation of an ADC between an antibody and a compound having nucleic acid degradation activity (e.g., a ribonuclease or DNA endonuclease, e.g., deoxyribonuclease; DNase).

[0128] For the selective destruction of tumors, antibodies may contain highly radioactive atoms. Various radioisotopes are available for the production of radioconjugated antibodies. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 88 Sm 53 , Bi 212 , P 32 Pb 212 , and radioactive isotopes of Lu. When a conjugate is used for detection, this is a radioactive atom for scintigraphy studies, e.g., tc 99m Or I 123or spin labeling for nuclear magnetic resonance (NMR) imaging (also known as MRI), which may again include iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0129] Radioactive labels or other labels can be incorporated into the conjugate by known methods. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using, for example, suitable amino acid precursors containing fluorine-19 instead of hydrogen. Labels, e.g., tc 99m or I 123 Re 186 Re 188 and In 111 It can be bound via cysteine ​​residues in the peptide. Yttrium-90 can be bound via lysine residues. The IODOGEN method (FRAKER et. al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (CHATAL, CRC Press 1989) describes other methods in detail.

[0130] The present invention provides, in particular, antibody-drug conjugate compounds for targeted delivery of therapeutic agents. The inventors have found that antibody-drug conjugate compounds have potent cytotoxic activity and / or cell proliferation inhibitory activity against cells expressing nectin-4 and its variants.

[0131] An antibody-drug conjugate compound comprises an antibody unit covalently linked to at least one drug unit. The drug unit may be covalently linked to the antibody unit directly or via a linker unit (-LU-).

[0132] In some embodiments, the antibody-drug conjugate compound is given by the following formula: Ab-(LU-D) p ADC Schema (I) or having a pharmaceutically acceptable salt or solvate thereof; where: Ab is an antibody unit, for example, the nectin-4 antibody of the present invention; • (LU-D) is the linker unit-drug unit portion, where: LU- is a linker unit, and ·-D is a drug unit that has cell proliferation inhibitory activity or cytotoxic activity against target cells; and • p is in the range of 1 to 20 or 1 to 50.

[0133] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] ADC Schema (II) or having a pharmaceutically acceptable salt or solvate thereof, where: • Ab is an antibody unit, for example, the nectin-4 antibody of the present invention; and · [ka] This is a linker unit (LU), where: ·-A- is a stretcher unit, • a is 0, 1, 2, or 3. ·each [ka] It is an independent amino acid unit, • w is an integer in the range of 0 to 12. · [ka] It is a self-sacrificing spacer unit, • y is 0, 1, or 2; ·-D is a drug unit that has cell proliferation inhibitory activity or cytotoxic activity against target cells; and • p is an integer between 1 and 20 or between 1 and 50.

[0134] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] ADC Schema (III) or [ka] ADC Schema (IV) or having a pharmaceutically acceptable salt or solvate thereof, where: Ab is an antibody unit, for example, the nectin-4 antibody of the present invention; Each R is independently selected from N, CH, or C; ·R' is C or CH W is [ka] Selected from.

[0135] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] ADC schema (V) [ka] ADC schema (VI) It has, Ab is an antibody unit, for example, the anti-nectin-4 antibody of the present invention. Each R is selected independently from N, CH, or C. W is [ka] [ka] Selected from. ·X b This is a spacer portion selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptides. b is 0, 1, or 2. ·Y b It is a polypeptide moiety containing approximately 1 to 6 amino acids, which are natural and / or non-natural amino acids. ·Z b teeth, [ka] This self-sacrificing aspect includes, but is not limited to, these examples. D is a drug unit that has cell proliferation inhibitory activity or cytotoxic activity against target cells. • p is an integer between 1 and 20 or between 1 and 50.

[0136] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] ADC Schema (VII) or having a pharmaceutically acceptable salt or solvate thereof, where: Ab is an antibody unit, for example, the nectin-4 antibody of the present invention; ·R 1 teeth, [ka] And here, R 2 These are unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; ·R x and R y Each of them is R and LR z Selected independently from, however, R x and R y One of them is NRz When it is, the other is R; ·R 5 is H or CR’3, where each R’ is independently H or F; ·R 6 is H or CH2CN; ·LU is a linker unit; and ·R is H or C1-C3 alkyl; and ·i is an integer within the range of 1 to about 20.

[0137] In some embodiments, the antibody-drug conjugate compound has the following formula:

Chemical formula

Chemical formula

[0138] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] ADC schema (IX) or having a pharmaceutically acceptable salt or solvate thereof, where: Ab is an antibody unit, for example, the nectin-4 antibody of the present invention; R 1 teeth, [ka] And here, R 2 These are unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R x and R y Each of them is R and LR z Selected independently from, however, R x and R y One of them is NR z If so, the other is R; R 5 is H or CR'3, where each R' is independently H or F; R 6 is H or CH2CN; LU is a linker unit; and R is H or C1-C3 alkyl; and k is an integer in the range of 1 to approximately 20.

[0139] In some embodiments, the antibody-drug conjugate compound is given by the following formula: [ka] It has, ·R 1 teeth, [ka] And here, R 2 These are substituted or unsubstituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; ·R 3 is H or C1-C3 alkyl; ·R 5 is H or CR'3, where each R' is independently H or F; ·R 6 is H or CH2CN; Ab is an antibody unit, for example, the anti-nectin-4 antibody of the present invention. Each R is selected independently from N, CH, or C. J is the conjugation part. ·X b This is a spacer portion selected from the group consisting of alkyl, heteroalkyl, polyethylene glycol (PEG), and peptides. b is 0, 1, or 2. ·Y b It is a polypeptide moiety containing approximately 1 to 6 amino acids, which are natural and / or non-natural amino acids. ·Z b teeth, [ka] This self-sacrificing aspect includes, but is not limited to, these examples.

[0140] For a composition comprising a plurality of antibodies, the drug load is indicated by p, which is the average number of drug molecules per antibody. The drug load can range from 1 to 24 drugs (D) per antibody. The average number of drugs per antibody in a preparation of the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay and HPLC. The quantitative distribution of the antibody-drug conjugate with respect to p can also be determined. In some examples, the separation, purification and characterization of a homogeneous antibody-drug conjugate where p is a certain value from other antibody-drug conjugates having other drug loads can be achieved by means such as reverse phase HPLC or electrophoresis. In an exemplary embodiment, p is from 2 to 8. In some embodiments, p is from 2 to 24.

[0141] The generation of the antibody-drug conjugate compound can be achieved by any technique known to those skilled in the art. Briefly stated, the antibody-drug conjugate compound comprises a nectin-4 antibody.

[0142] In one embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(A).

[0143] In another embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(B).

[0144] In another embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(C).

[0145] In another embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(D).

[0146] In another embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(E).

[0147] In another embodiment, the nectin-4 ADC comprises the heavy chain antibody sequence shown in Table VI(F).

[0148] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(G).

[0149] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(H).

[0150] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(I).

[0151] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(J).

[0152] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(K).

[0153] In another embodiment, the nectin-4 ADC comprises the antibody light chain sequence shown in Table VI(L).

[0154] In another embodiment, the nectin-4 ADC includes the antibody heavy chain variable region sequence shown in Table VIII.

[0155] In another embodiment, the nectin-4 ADC includes the antibody light chain variable region sequence shown in Table IX.

[0156] In another embodiment, the nectin-4 ADC comprises the antibody CDR sequence shown in Table X.

[0157] In another embodiment, the Nectin-4 antibody described above is conjugated to a therapeutic agent.

[0158] In one embodiment, the therapeutic agent is a drug-linker (DL) payload shown in Figure 10.

[0159] In one embodiment, the DL payload is shown in Figure 10(A) and has the following chemical structure: [ka] or has a pharmaceutically acceptable salt or solvate form thereof.

[0160] In one embodiment, the DL payload is shown in FIG. 10(B) and has the following chemical structure: [Chemical Formula] or has a pharmaceutically acceptable salt or solvate form thereof.

[0161] In one embodiment, the DL payload is shown in FIG. 10(C) and has the following chemical structure: [Chemical Formula] or has a pharmaceutically acceptable salt or solvate form thereof.

[0162] In one embodiment, the DL payload is shown in FIG. 10(D) and has the following chemical structure: [Chemical Formula] or has a pharmaceutically acceptable salt or solvate form thereof.

[0163] In one embodiment, the DL payload is shown in FIG. 10(E) and has the following chemical structure: [Chemical Formula] or has a pharmaceutically acceptable salt or solvate form thereof.

[0164] In one embodiment, the DL payload is shown in FIG. 10(F) and has the following chemical structure: [Chemical Formula] or has a pharmaceutically acceptable salt or solvate form thereof.

[0165] In one embodiment, the DL payload is shown in FIG. 10(G) and has the following chemical structure: [Chemical Formula] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0166] In one embodiment, the DL payload is shown in Figure 10(H) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0167] In one embodiment, the DL payload is shown in Figure 10(I) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0168] In one embodiment, the DL payload is shown in Figure 10(J) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0169] In one embodiment, the DL payload is shown in Figure 10(K) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0170] In one embodiment, the DL payload is shown in Figure 10(L) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0171] In one embodiment, the DL payload is shown in Figure 10(M) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0172] In one embodiment, the DL payload is shown in Figure 10(N) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0173] In one embodiment, the DL payload is shown in Figure 10(O) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0174] In one embodiment, the DL payload is shown in Figure 10(P) and has the following chemical structure: [ka] Or it may have a pharmaceutically acceptable salt or solvate form thereof.

[0175] Several different reactions are available for the covalent bonding of drugs and / or linkers to binding agents. This is often achieved by the reaction of amino acid residues of binding agents, such as antibody molecules, which include the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various parts of aromatic amino acids. One of the most commonly used nonspecific methods for covalent bonding is the carbodiimide reaction, which links the carboxyl (or amino) group of a compound to the amino (or carboxyl) group of an antibody.

[0176] Furthermore, 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 are also available for drug binding to binding agents. This method involves periodic acid oxidation of a glycol or hydroxyl group-containing drug, which then reacts with the binding agent to form an aldehyde. Binding occurs via the formation of a Schiff base with the amino group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently bind a drug to a binding agent. Other techniques are known to those skilled in the art and are within the scope of this invention.

[0177] In certain embodiments, an intermediate that is a precursor of the linker is reacted with a drug under appropriate conditions. In certain embodiments, a reactive group on the drug and / or intermediate is used. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with a nectin-4 antibody under appropriate conditions.

[0178] IV.) Linker Unit Typically, antibody-drug conjugate compounds include a linker unit between the drug unit and the antibody unit. In some embodiments, the linker is cleavable under intracellular conditions, so that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is incleavable, and the drug is released, for example, by antibody degradation.

[0179] In a preferred embodiment, the linker is conjugated to a nectin-4 antibody as described herein.

[0180] In some embodiments, the linker can be cleaved by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). The linker may be a peptidyl linker cleaved by intracellular peptidase or protease enzymes, including but not limited to lysosome or endosomal proteases. The linker may also be cleaved by cleavage agents present in the extracellular environment (e.g., near the cell membrane or in tissue space). The linker may be a peptidyl linker cleaved by extracellular peptidase or protease enzymes, including but not limited to cathepsin family enzymes or matrix metalloproteinases.

[0181] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, this is a pH-sensitive linker that can be hydrolyzed under acidic conditions. For example, acid-unstable linkers that can be hydrolyzed in lysosomes (e.g., oximes, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) may be used (see, for example, U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; DUBOWCHIK AND WALKER, 1999, Pharm. Therapeutics 83:67-123; NEVILLE et. al., 1989, Biol. Chem. 264:14653-14661).

[0182] In yet another embodiment, the linker can be cleaved under reducing conditions known in the art (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; WAWRZYNCZAK et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW VOGEL ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935). The linker can also be cleaved under reducing conditions found intracellular (or extracellular). For example, in a preferred embodiment, the NO bond of a particular linker can be formally reduced and cleaved to result in linker cleavage.

[0183] In yet other specific embodiments, the linker may be a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0184] In yet another embodiment, the linker unit is incapable of cleavage, and the drug is released by antibody degradation (see PCT publication number WO2012 / 166560 (Ambrx, Inc.), which is incorporated herein by reference in its entirety for all purposes).

[0185] Typically, linkers are substantially insensitive to the extracellular environment. As used herein, “substantially insensitive to the extracellular environment” means that, with respect to the linker, about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5% or less, about 3% or less, or about 1% or less of the linker in a sample of the antibody-drug conjugate compound is cleaved when the antibody-drug conjugate compound is present in the extracellular environment (e.g., in plasma). Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0186] In other non-exclusive embodiments, the linker facilitates intracellular relocation, as is known in the art.

[0187] Various exemplary linkers that may be used in conjunction with the compositions and methods of the present invention are described in WO2004 / 010957, U.S. Patent Application Publication 2006 / 0074008, U.S. Patent Application Publication 20050238649 and U.S. Patent Application Publication 2006 / 0024317 (each of which is incorporated herein by reference in whole for all purposes).

[0188] For the purposes of this disclosure, a “linker 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 linker unit is of formula: [ka] It has, Here, -A- is a stretcher unit, 〇a is either 0 or 1, Each [ka] It is an independent amino acid unit, 〇w is an integer in the range of 0 to 12. 〇 [ka] It is a self-sacrificing spacer unit, and 〇y is 0, 1, or 2.

[0189] 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, if 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.

[0190] V.) Stretcher Unit The stretcher unit (A) is, if present, the amino acid unit ( [ka] ) If present, spacer unit ( [ka] The antibody unit can be linked to (-D) or to the drug unit (-D). Useful functional groups that may be present in the nectin-4 antibody, either naturally or through chemical manipulation, include, but are not limited to, keto, aldehyde, sulfhydryl, amino, hydroxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl. Suitable functional groups are keto, aldehyde, sulfhydryl, and amino. In one example, the keto group is on a non-natural amino acid (nnAA) incorporated into the antibody of the present invention. In a further example, the aldehyde group is on an nnAA incorporated into the antibody of the present invention. In another example, the sulfhydryl group can be produced by reduction of an intramolecular disulfide bond in the nectin-4 antibody. In another embodiment, the sulfhydryl group can be produced by reaction of the amino group of the lysine moiety of the nectin-4 antibody with 2-iminothiolane (Traut reagent) or other sulfhydryl-producing reagent. In a particular embodiment, the nectin-4 antibody is a recombinant antibody and is manipulated to possess one or more lysines. In certain other embodiments, the recombinant nectin-4 antibody is engineered to possess additional sulfhydryl groups, such as additional cysteine.

[0191] In one embodiment, the stretcher unit forms a bond with a sulfur atom of the antibody unit. The sulfur atom may originate from a sulfhydryl group of the antibody. In a particular embodiment, the stretcher unit is linked to the antibody unit via a disulfide bond between the sulfur atom of the antibody unit and the sulfur atom of the stretcher unit. In yet another embodiment, the stretcher contains a reactive site that can form a bond with a primary or secondary amino group of the antibody. Examples of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0192] In some embodiments, the stretcher contains a reactive site that is reactive to the (-CHO) group of a modified carbohydrate that may be present on the antibody. For example, the carbohydrate may be mildly oxidized using a reagent, such as sodium periodate, and the resulting (-CHO) unit of the oxidized carbohydrate may be condensed with a stretcher containing a functional group, such as a hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, carboxylic acid hydrazine, and aryl hydrazide, for example, as described by Kaneko et al., 1991, Bioconjugate Chem. 2:133-41.

[0193] VI.) Amino Acid Units Amino acid unit ( [ka] If present, the stretcher unit is connected to the spacer unit if one exists; if the spacer unit does not exist, the stretcher unit is connected to the drug portion; and if neither the stretcher unit nor the spacer unit exists, the antibody unit is connected to the drug unit.

[0194] In certain embodiments, the amino acid unit may include natural amino acids. In other embodiments, the amino acid unit may include non-natural amino acids.

[0195] In some embodiments, the amino acid unit may be enzymatically cleaved by one or more enzymes, including cancer or tumor-associated proteases, to release a drug unit (-D) that is protonated in vivo upon release to provide a drug (D) in one embodiment.

[0196] In one embodiment of the amino acid unit, the amino acid unit is valine-citrulline (vc or Val-Cit). In another embodiment, the amino acid unit is phenylalanine-lysine. In yet another embodiment of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another embodiment, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinoline carboxylate-lysine, cyclohexylalanine-lysine, isonepecotic acid-lysine, beta-alanine-lysine, glycineserine-valinglutamine, and isonepecotic acid.

[0197] VII.) Spacer Unit Spacer unit ( [ka] The spacer unit, if present, connects to the amino acid unit, and if the amino acid unit is present, it connects to the drug unit. Alternatively, the spacer unit connects to the stretcher unit, and if the amino acid unit is absent, it connects to the drug unit. The spacer unit also connects to the drug unit, and if neither the amino acid unit nor the stretcher unit is present, it connects to the antibody unit. The spacer unit is of two common types: non-self-sacrificing or self-sacrificing. Examples of possible spacers 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.

[0198] Other examples of self-sacrificing spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (HAY et. al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzyl acetals.

[0199] Spacers that undergo cyclization during amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (RODRIGUES et. al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (STORM et. al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (AMSBERRY et. al., 1990, J. Org. Chem. 55:5867), may be used. The 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-sacrificing spacer.

[0200] VIII.) Drug Unit The drug portion (D) may be any cytotoxic agent, cell proliferation inhibitor, or immunomodulatory (e.g., immunosuppressive) agent. D is a drug unit (portion) having atoms that can form bonds with a spacer unit, with an amino acid unit, with a stretcher unit, or with an antibody unit. In some embodiments, drug unit D has a nitrogen atom that can form a bond with a spacer unit. As used herein, the terms “drug unit” and “drug portion” are synonymous and are interchangeable.

[0201] Useful classes of cytotoxic agents, cell proliferation inhibitors, or immunomodulators include, for example, antitubulins, DNA sulcus binding agents, DNA replication inhibitors, and alkylating agents.

[0202] In some embodiments, the drug is auristatin, for example, auristatin E (also known in the art as a derivative of dorastatin-10) or a derivative thereof. The auristatin may be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E may be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.

[0203] In some embodiments, the drug unit is calicheamycin, camptothecin, mytansinoid, or anthracycline. In some embodiments, the drug is a taxane, a topoisomerase inhibitor, or a vinca alkaloid.

[0204] In some typical embodiments, suitable cytotoxic agents include, for example, DNA ligulators (e.g., enediyne and lexitropsin, CBI compounds; see also U.S. Patent No. 6,130,237), duocalmycin, taxanes (e.g., paclitaxel and docetaxel), puromycin, and vinca alkaloids. Other cytotoxic agents include, for example, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epotilon A and B, estramustine, cryptophysin, semadotin, mytansinoids, discodermorid, eryuterobin, and mitoxantrone.

[0205] In some embodiments, the drug is an antitubulin. Examples of antitubulins include auristatin, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine). Other antitubulins include, for example, baccatin derivatives, taxane analogs (e.g., epotilon A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycin, semadotine, mytansinoids, combretastatin, discodermorid, and eryuterobin.

[0206] In certain embodiments, the cytotoxic agent is a mytansinoid, which is an antitubulin agent from another group. For example, in specific embodiments, the mytansinoid is mytansin or DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131).

[0207] In certain embodiments, the cytotoxic agent or cell growth inhibitor is drastatin. In certain embodiments, the cytotoxic agent or cell growth inhibitor is of the auristatin class, for example, drastatin-10, auristatin e, or auristatin PHE.

[0208] In a particular embodiment, the drug unit (D) has the following structural formula: [ka] an auristatin analog or a pharmaceutically acceptable salt thereof having the following properties: Here, R 1 teeth, [ka] And here, R 2These are unsubstituted or substituted C1-C6 alkyl, heteroalkyl, cycloalkyl, or cycloheteroalkyl; R a , R b and R c Each of them is H and NR x R y Selected from, however, 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 R, R r and LR z Selected independently from, however, R x and R y One of them is LR z or R r If so, the other is R; R 5 is H or CR'3, where each R' is independently H or F; R 6 is H or CH2CN; L is the linker; R r (C=O)-O-(CH2) p -R v Or (C=O)-(CH2) q -R v and; R v This is R, OR, NHR, NR2, an aryl group, or an amino acid; p is 0, 1, 2, 3, 4, 5, or 6; q is 0, 1, 2, 3, 4, 5, or 6; R z is a functional group or a reactive group; and R is either H or a C1-C3 alkyl group.

[0209] IX.) Drug Loading The drug load is denoted by p and is the average number of drug moieties per antibody in the molecule. The drug load can range from 1 to 24 drug moieties (D) per antibody. The ADC of the present invention involves collecting antibodies conjugated with drug moieties ranging from 1 to 24. The average number of drug moieties per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means, e.g., mass spectrometry and ELISA assays. The quantitative distribution of ADCs with respect to p can also be determined. In some examples, the separation, purification, and characterization of homogeneous ADCs, where p is a specific value from ADCs with other drug loads, can be achieved by means such as electrophoresis.

[0210] For some antibody-drug conjugates, p may be limited by the number of binding sites on the antibody. For example, if the binding is cysteinethiol, as in the exemplary embodiments above, the antibody may have only one or more cysteinethiol groups, or only one or more sufficiently reactive thiol groups through which the linker can bind. In certain embodiments, a higher drug load, e.g., p > 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the drug load for the ADCs of the present invention ranges from 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, for a given ADC, the optimal ratio of drug portion per antibody may be less than 8 and may range from approximately 2 to approximately 5. See U.S. Patent No. 7,498,298 (which is thus incorporated herein by reference in its entirety).

[0211] In certain embodiments, a drug moiety smaller than the theoretical maximum value of the drug moiety is conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with either the drug-linker intermediate or the linker reagent, as discussed below. Generally, antibodies do not contain a large number of free reactive cysteinethiol groups that can be linked to the drug moiety; in fact, most cysteinethiol residues in antibodies exist as disulfide crosslinks. In certain embodiments, the antibody may be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or complete reduction conditions to generate reactive cysteinethiol groups. In certain embodiments, the antibody is subjected to denaturation conditions to reveal reactive nucleophiles, such as lysine or cysteine.

[0212] The ADC load (drug-to-antibody ratio) can be controlled in different ways, for example, by (i) limiting a molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) applying partial or limited reduction conditions for cysteine ​​thiol modification, or (iv) manipulating the amino acid sequence of the antibody by recombinant techniques so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug bindings (e.g., thioMab or thioFab prepared as disclosed herein and in WO2006 / 034488 (whole of which is thus incorporated herein by reference)).

[0213] When more than one nucleophile reacts with the drug-linker intermediate or linker reagent, and then with the drug moiety reagent, the resulting product is understood to be a mixture of ADC compounds having a distribution of one or more drug moieties bound to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double 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, for example, hydrophobic interaction chromatography (see, e.g., 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, Mar. 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, Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, a homogeneous ADC having a single load value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0214] X.) Method for determining the cytotoxic effect of ADCs Methods for determining whether a drug or antibody-drug conjugate exerts cell proliferation inhibitory and / or cytotoxic effects on cells are known. Generally, the cytotoxic or cell proliferation inhibitory activity of an ADC can be measured by exposing mammalian cells expressing the target protein of the antibody-drug conjugate in 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 assays can be used to measure the viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of antibody-drug conjugates.

[0215] A thymidine uptake assay can be used to determine whether ADCs exert a cell proliferation inhibitory effect. For example, cancer cells expressing a target antigen at a density of 5,000 cells / well in a 96-well plate may be cultured for a period of 72 hours, with 0.5 μCi during the last 8 hours of the 72-hour period. 3 Can be exposed to H-thymidine. 3 H-thymidine uptake is measured in the presence and absence of ADC.

[0216] To determine cell damage, necrosis or apoptosis (programmed cell death) can be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane; cell swelling; and plasma membrane rupture. Apoptosis is typically characterized by membrane blebbing, cytoplasmic condensation, and activation of endogenous endonucleases. Determining either of these effects on cancer cells indicates that ADCs are useful in treating cancer.

[0217] Cell viability can be measured by determining the uptake of a dye in cells, such as neutral red, trypan blue, or ALAMAR® blue (see, e.g., PAGE et al., 1993, Intl. J. Oncology 3:473-476). In such assays, cells are incubated in a culture medium containing the dye, washed, and residual dye, reflecting the cell uptake of the dye, is measured spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (SKEHAN et al., 1990, J. Natl. Cancer Inst. 82:1107-12).

[0218] Alternatively, tetrazolium salts, such as MTT or CellTiter-Glo®, are used in quantitative assays for mammalian cell survival and proliferation by detecting living cells but not dead cells (see, for example, MOSMANN, 1983, J. Immunol. Methods 65:55-63).

[0219] Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods are available for the quantitative in vitro determination of DNA fragmentation. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0220] Apoptosis can also be determined by measuring morphological changes in cells. For example, loss of plasma membrane integrity, as with necrosis, 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 are described by Duke and Cohen, Current Protocols in Immunology (COLIGAN et al. eds., 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 cells can be observed for chromatin condensation and margination along the inner nuclear membrane. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane bleving, and cell shrinkage.

[0221] The presence of apoptotic cells can be measured in both the adherent and "suspension" compartments of the culture. For example, both compartments can be collected by removing the supernatant, trypsinizing the adherent cells, combining the preparations after a centrifugation wash step (e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., by measuring DNA fragmentation) (see, e.g., PIAZZA et. al., 1995, Cancer Research 55:3110-16).

[0222] In vivo, the efficacy of nectin-4 antibody therapeutic compositions can be evaluated in appropriate animal models. For example, xenograft models can be used in which cancer explants or passaged xenograft tissues are introduced into immunodeficient 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 capable of repeating the development of primary tumors, micrometastases, and the formation of osteoblastic metastases characteristic of late-stage disease. Efficacy can be predicted using assays that measure inhibition of tumorigenesis, tumor regression, or metastasis.

[0223] In vivo assays that evaluate the promotion of apoptosis are useful when evaluating therapeutic compositions. In one embodiment, xenografts from tumor-bearing mice treated with a therapeutic composition may be tested for the presence of apoptotic focus and compared to untreated control xenograft-bearing mice. The extent to which apoptotic focus is found in the tumors of the treated mice provides an indicator of the therapeutic efficacy of the composition.

[0224] The therapeutic composition used in carrying out the above-described method may be formulated into a pharmaceutical composition containing a carrier suitable for the desired delivery method. A suitable carrier may include any material that, when combined with the therapeutic composition, retains the antitumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of several standard pharmaceutical carriers, such as sterile phosphate-buffered saline or bacteriostatic water (see, in general, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

[0225] Therapeutic formulations may be solubilized 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, intravisceral, and orthotopic administration. Preferred formulations for intravenous injection comprise the therapeutic composition diluted in a solution of preservative-treated bacteriostatic water, sterile unpreservative-treated water, and / or in a polyvinyl chloride or polyethylene bag containing 0.9% sterile sodium chloride for injection, USP. Therapeutic protein preparations may be lyophilized, preferably under vacuum, and stored as sterile powder, which may then be reconstituted in bacteriostatic water (e.g., containing benzyl alcohol preservative) or sterile water before injection.

[0226] The dosage and administration protocols for cancer treatment using the methods described above vary depending on the method and target cancer, and generally depend on several other factors recognized in the field.

[0227] In one embodiment, the pharmaceutical composition of the present invention may contain more than one of the ADCs of the present invention due to modifications of the nectin-4 antibody. For example, the present invention includes a pharmaceutical composition comprising the ADC of the present invention, wherein the nectin-4 antibody is an antibody in which the C-terminal lysine is partially or completely removed, an antibody having an N-terminal post-translational modification, an antibody lacking heavy chain C-terminal lysine and having an N-terminal post-translational modification, and / or an antibody having heavy chain C-terminal lysine and lacking an N-terminal post-translational modification.

[0228] In preferred embodiments, the nectin-4 antibodies are shown in Tables VI and VII.

[0229] XI.) Treatment of cancer(s) expressing nectin-4 The identification of nectin-4 as a protein that is normally expressed in a limited set of tissues or cells, but is also expressed in cancers, such as those listed in Table I, opens up several therapeutic approaches to the treatment of such cancers.

[0230] Notably, targeted antitumor therapies have been effective even when the targeted protein is expressed on normal tissue or cells, and even on vital normal organ tissue. 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 still being able to survive. Examples of non-vital organs include the ovaries, breasts, and prostate.

[0231] The expression of a target protein in normal tissue, and even in important normal tissue, does not negate the usefulness of a targeting agent for that protein as a treatment for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not harmful in itself. Furthermore, organs considered unnecessary, such as the prostate and ovaries, can be removed without affecting mortality. Finally, some vital organs are not affected by normal organ expression due to immunoprivilege. Immunoprivilege organs are those protected from the blood by the blood-organ barrier and therefore inaccessible to immunotherapy. Examples of immunoprivilege organs are the brain and testes.

[0232] Therefore, therapeutic approaches that inhibit the activity of the nectin-4 protein are useful for patients with cancers that express nectin-4 (e.g., cancers shown in Table I). These therapeutic approaches generally fall into three classes. The first class modulates nectin-4 function, resulting in inhibition or delay of tumor cell growth or induction of tumor cell death, as it is associated with tumor cell growth. The second class includes various methods for inhibiting the binding or association of the nectin-4 protein with its binding partners or other proteins. The third class includes various methods for inhibiting the transcription of the nectin-4 gene or the translation of nectin-4 mRNA.

[0233] Therefore, cancer patients may be assessed for the presence and level of nectin-4 expression, 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. Immunohistochemical analysis of tumor biopsy or surgical specimens is preferred for this purpose, where applicable. Methods for immunohistochemical analysis of tumor tissue are well known in the art.

[0234] XII.) Nectin-4 ADC Cocktail The therapeutic methods of the present invention involve the administration of a single nectin-4 ADC, as well as combinations or cocktails of different antibodies (i.e., nectin-4 antibodies or antibodies that bind to another protein). Since these contain antibodies that target different epitopes, utilize different effector mechanisms, or directly combine cytotoxic antibodies with antibodies that depend on immunoeffector functionality, such antibody cocktails may have certain advantages. Such antibodies in combination may exhibit synergistic therapeutic effects. Furthermore, nectin-4 antibodies may be administered in conjunction with various chemotherapeutic and biologic agents, androgen blockers, immunomodulators (e.g., IL-2, GM-CSF, PD1, PD-L1), surgery, or radiation therapy, among other therapeutic modalities.

[0235] In a preferred embodiment, the nectin-4 antibody is administered in a conjugated form.

[0236] In a more preferred embodiment, the nectin-4 antibodies are shown in Tables VI and VII.

[0237] Nectin-4 ADC preparations are administered via any route capable of delivering the antibody to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, and intradermal. Treatment generally involves repeated administration of Nectin-4 ADC preparations via acceptable routes of administration, such as intravenous injection (IV), in doses typically within a range 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 per kg of body weight. Generally, doses of MAb in the range of 10–1000 mg per week are effective and well-tolerated.

[0238] Based on clinical experience with Herceptin® (trastuzumab) in the treatment of metastatic breast cancer, an acceptable dosing regimen is an initial loading dose of approximately 4 mg / kg patient body weight IV followed by weekly doses of approximately 2 mg / kg IV of the MAb preparation. Preferably, the initial loading dose is administered as an infusion over 90 minutes or longer. Provided that the initial dose has been well tolerated, a regular maintenance dose is administered as an infusion over 30 minutes or longer. As will be understood by those skilled in the art, numerous factors may influence the ideal dose regimen in particular. Such factors include, for example, the binding affinity and half-life of the antibody used, the degree of nectin-4 expression in the patient, the degree of circulating shed nectin-4 antigen, the desired steady-state antibody concentration level, the frequency of treatment, and the influence of chemotherapeutic agents or other drugs used in combination with the treatment method of the present invention, as well as the health status of the particular patient.

[0239] If necessary, patients should be assessed for levels of nectin-4 in a given sample (e.g., levels of circulating nectin-4 antigen and / or nectin-4 expressing cells) to aid in determining the most effective drug regimen. Such assessments are also used for monitoring purposes throughout treatment and, in combination with assessments of other parameters (e.g., urine cytology and / or immunocytology levels in bladder cancer treatment, or by analogy, serum PSA levels in prostate cancer treatment) are useful in determining therapeutic success.

[0240] An object of the present invention is to provide a nectin-4 ADC that inhibits or delays the growth of tumor cells expressing nectin-4. A further object of the present invention is to provide a method of using such nectin-4 ADC, in particular in combination with other drugs or immunologically active treatments, to inhibit angiogenesis and other biological functions and thereby reduce tumor growth in mammals, preferably humans.

[0241] XIII.) Combination Therapy In one embodiment, a synergistic effect exists when tumors, including human tumors, are treated with nectin-4 ADC in combination with chemotherapeutic agents, radiation, or a combination thereof. In other words, the inhibition of tumor growth by nectin-4 ADC is enhanced beyond what would be expected when combined with chemotherapeutic agents, radiation, or a combination thereof. The synergistic effect may be demonstrated, for example, by higher inhibition of tumor growth with the combination treatment than what would be expected from treatment with nectin-4 ADC alone, or from the additive effect of treatment with nectin-4 ADC and chemotherapeutic agents or radiation. Preferably, the synergistic effect is demonstrated by cancer remission, which would not be expected from treatment with nectin-4 ADC alone, or from treatment using an additive combination of nectin-4 ADC and chemotherapeutic agents or radiation or immunotherapy, such as CAR-T or NK cell therapy.

[0242] A method for inhibiting tumor cell growth using Nectin-4 ADC and a combination of chemotherapy or radiation, or both, includes the step of administering Nectin-4 ADC before, during, or after initiating chemotherapy or radiation therapy, and any combination thereof (i.e., before and during chemotherapy and / or radiation therapy, before and after initiation, during and after initiation, or before, during and after initiation). For example, Nectin-4 ADC is typically administered between 1 day and 60 days, preferably between 3 days and 40 days, and more preferably between 5 days and 12 days, before initiating radiation therapy and / or chemotherapy. However, depending on the treatment protocol and the specific patient's needs, this method is carried out in a manner that provides the most effective treatment and ultimately extends the patient's lifespan.

[0243] The administration of chemotherapeutic agents can be achieved in various ways, including systemic administration via parenteral and enteral routes. In one embodiment, nectin-4 ADC and the chemotherapeutic agent are administered as separate molecules. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechloretamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (Taxol), docetaxel (Taxotere), aldezleukin, and aspergillol. This includes laginase, busulfan, carboplatin, cladribine, dacarbazine, phloxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobromane, plicamycin, streptozocin, tamoxifen, teniposide, testactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

[0244] The radiation source used in combination with Nectin-4 ADC can be either external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT). In one embodiment, the radiation therapy is boron neutron capture therapy. In one embodiment, the radiation is proton-boron fusion therapy.

[0245] The above treatment regimens may be further combined with additional cancer treatment agents and / or regimens, such as additional chemotherapy, cancer vaccines, signaling inhibitors, agents useful in treating abnormal cell growth or cancer, antibodies (e.g., anti-CTLA-4 antibodies described in WO / 2005 / 092380 (Pfizer)) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

[0246] The above-mentioned chemotherapeutic agents may be used when mammals are subjected to further chemotherapy. In addition, growth factor inhibitors, biological response modifiers, anti-hormone therapies, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and anti-androgens may be used. For example, anti-hormones, e.g., anti-estrogens, e.g., Nolvadex (tamoxifen) or anti-androgens, e.g., Casodex (4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-'-(trifluoromethyl)propionanilide) may be used.

[0247] The above treatment approach can be combined with any one of a wide range of surgical, chemotherapy, or radiotherapy regimens. The treatment approach of the present invention may allow for the use of reduced doses and / or less frequent administration of chemotherapy (or other therapies), which is advantageous for all patients, especially those who do not have sufficient tolerance to the toxicity of chemotherapy agents.

[0248] XIV.) Kits / Manufactured Products For use in the laboratory, prognostic, prophylactic, diagnostic, and therapeutic applications described herein, the kits are within the scope of the invention. Such a kit may comprise a carrier, package, or container compartmentated to receive one or more containers, e.g., vials, tubes, etc., each container(s) may comprise one of the separate elements used in the method, together with a label or accompanying document containing instructions for use, e.g., instructions for use described herein. For example, a container(s) may comprise one or more nectin-4 antibodies of the Disclosure (see Tables VI and VII). The kit may comprise a container comprising a drug unit. The kit may comprise all or part of a diagnostic assay for detecting nectin-4 ADCs and / or cancer and / or other immunological disorders.

[0249] The kit of the present invention typically includes the above-mentioned container, as well as one or more other containers associated therewith containing commercially and user-desirable materials, such as buffers, diluents, filters, needles, and syringes; a carrier, package, container, vial, and / or tube label listing the contents and / or instructions for use, and a document containing instructions for use.

[0250] Labels may be present on or with the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, e.g., prognostic, prophylactic, diagnostic or laboratory application, and may also indicate instructions for in vivo or in vitro use, such as those described herein. Instructions and other information may also be present on the accompanying documentation(s) or label(s) included with or on the kit. Labels may be on or associated with the container. Labels may be on the container if the letters, numbers or other characters constituting the label are molded into or etched onto the container itself; labels may be associated with the container if they are present, for example, as accompanying documentation within a receptacle or carrier that also holds the container. Labels may indicate that the composition is used to diagnose, treat, prevent or prognose a condition, e.g., cancer or other immunological disorder.

[0251] The terms "kit" and "manufactured product" can be used as synonyms.

[0252] In another embodiment of the present invention, a product(s) containing a composition, for example, the Nectin-4 ADC of the present disclosure. The product typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers may be formed from a variety of materials, for example, glass, metal or plastic. Containers may hold one or more Nectin-4 ADCs and / or one or more therapeutic doses of Nectin-4 ADCs.

[0253] Alternatively, the container may hold a composition effective for treating, diagnosing, prognosing, or preventing a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). The active agent in the composition may be the nectin-4 antibody or ADC of this disclosure.

[0254] The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. This may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and / or accompanying documentation with indications and / or instructions for use.

[0255] Exemplary Embodiments 1) An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. 2) The antibody or antigen-binding fragment according to claim 1, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72. 3) The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 28. 4) The antibody or antigen-binding fragment thereof according to claim 2, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 34. 5) The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 5. 6) The antibody or antigen-binding fragment thereof according to claim 2, wherein the light chain comprises the sequence shown in SEQ ID NO: 17. 7) The antibody or antigen-binding fragment according to claim 1, comprising a heavy chain variable region comprising an amino acid sequence homologous to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the heavy chain variable region amino acid sequence shown in SEQ ID NO: 28, and a light chain variable region comprising an amino acid sequence homologous to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the light chain variable region amino acid sequence shown in SEQ ID NO: 34. 8) The antibody or antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 9) The antibody or its antigen-binding fragment according to claim 1, wherein the antibody is a fully human antibody. 10) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 1. 11) The antibody or antigen-binding fragment thereof according to claim 1, which is conjugated to a drug via a linker. 12) The antibody or antigen-binding fragment thereof according to claim 11, wherein the drug is an auristatin analog. 13) The antibody or antigen-binding fragment thereof according to claim 11, further comprising a stretcher unit. 14) The antibody or antigen-binding fragment according to claim 11, further comprising a spacer unit. 15) The antibody or antigen-binding fragment thereof according to claim 11, further comprising an amino acid unit. 16) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable additive. 17) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16. 18) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 16. 19) A method for treating cancer in a subject, comprising the step of administering the kit described in claim 17 to the subject in a therapeutically effective dose. 20) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment according to any one of claims 1 to 15. 21) The method according to any one of claims 18 to 20, wherein the subject is a human subject. 22) The method according to claim 21, wherein cancer is as shown in Table I. 23) The method according to claim 22, further comprising the step of administering a radioactive or chemotherapeutic agent. 24) An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57. 25) The antibody or antigen-binding fragment according to claim 24, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75. 26) The antibody or antigen-binding fragment thereof according to claim 24, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 29. 27) The antibody or antigen-binding fragment thereof according to claim 24, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 35. 28) The antibody or antigen-binding fragment thereof according to claim 24, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 6. 29) The antibody or antigen-binding fragment thereof according to claim 24, wherein the light chain comprises the sequence shown in SEQ ID NO: 18. 30) The antibody or antigen-binding fragment according to claim 24, comprising a heavy chain variable region having 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 heavy chain variable region amino acid sequence shown in SEQ ID NO: 29, and a light chain variable region having 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 light chain variable region amino acid sequence shown in SEQ ID NO: 35. 31) The antibody or antigen-binding fragment according to claim 24, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 32) The antibody or its antigen-binding fragment according to claim 24, wherein the antibody is a fully human antibody. 33) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 24. 34) The antibody or antigen-binding fragment thereof according to claim 24, which is conjugated to a drug via a linker. 35) The antibody or antigen-binding fragment thereof according to claim 34, wherein the drug is an auristatin analog. 36) The antibody or antigen-binding fragment thereof according to claim 34, further comprising a stretcher unit. 37) The antibody or antigen-binding fragment according to claim 34, further comprising a spacer unit. 38) The antibody or antigen-binding fragment according to claim 34, further comprising an amino acid unit. 39) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 24 to 38 and a pharmaceutically acceptable additive. 40) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 24 to 39. 41) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 39. 42) A method for treating cancer in a subject, comprising the step of administering the kit described in claim 40 to the subject in a therapeutically effective dose. 43) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment according to any one of claims 24 to 39. 44) The method according to any one of claims 41 to 43, wherein the subject is a human subject. 45) The method according to claim 43, wherein cancer is as shown in Table I. 46) The method according to claim 43, further comprising the step of administering a radioactive or chemotherapeutic agent. 47) An antibody or its antigen-binding fragment containing a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60. 48) The antibody or antigen-binding fragment according to claim 47, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequence shown in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78. 49) The antibody or antigen-binding fragment thereof according to claim 47, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 30. 50) The antibody or antigen-binding fragment thereof according to claim 47, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 36. 51) The antibody or antigen-binding fragment thereof according to claim 47, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 8. 52) The antibody or antigen-binding fragment thereof according to claim 47, wherein the light chain comprises the sequence shown in SEQ ID NO: 20. 53) The antibody or antigen-binding fragment according to claim 47, comprising a heavy chain variable region having 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 heavy chain variable region amino acid sequence shown in SEQ ID NO: 30, and a light chain variable region having 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 light chain variable region amino acid sequence shown in SEQ ID NO: 36. 54) The antibody or antigen-binding fragment according to claim 47, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 55) The antibody or its antigen-binding fragment according to claim 47, wherein the antibody is a fully human antibody. 56) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 47. 57) The antibody or antigen-binding fragment thereof according to claim 47, which is conjugated to a drug via a linker. 58) The antibody or antigen-binding fragment thereof according to claim 47, wherein the drug is an auristatin analog. 59) The antibody or antigen-binding fragment thereof according to claim 47, further comprising a stretcher unit. 60) The antibody or antigen-binding fragment according to claim 47, further comprising a spacer unit. 61) The antibody or antigen-binding fragment thereof according to claim 47, further comprising an amino acid unit. 62) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 47 to 61 and a pharmaceutically acceptable additive. 63) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 47 to 62. 64) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 62. 65) A method for treating cancer in a subject, comprising the step of administering the kit according to claim 63 to the subject in a therapeutically effective dose. 66) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 47 to 65. 67) The method according to any one of claims 64 to 66, wherein the subject is a human subject. 68) The method according to claim 66, wherein cancer is as shown in Table I. 69) The method according to claim 66, further comprising the step of administering a radioactive or chemotherapeutic agent. 70) An antibody or an antigen-binding fragment thereof comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63. 71) The antibody or antigen-binding fragment according to claim 70, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequence shown in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81. 72) The antibody or antigen-binding fragment thereof according to claim 70, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 31. 73) The antibody or antigen-binding fragment thereof according to claim 70, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 37. 74) The antibody or antigen-binding fragment thereof according to claim 70, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 10. 75) The antibody or antigen-binding fragment thereof according to claim 70, wherein the light chain comprises the sequence shown in SEQ ID NO: 22. 76) The antibody or antigen-binding fragment according to claim 70, comprising a heavy chain variable region having an amino acid sequence homologous to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the heavy chain variable region amino acid sequence shown in SEQ ID NO: 31, and a light chain variable region having an amino acid sequence homologous to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the light chain variable region amino acid sequence shown in SEQ ID NO: 37. 77) The antibody or antigen-binding fragment according to claim 70, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 78) The antibody or its antigen-binding fragment according to claim 70, wherein the antibody is a fully human antibody. 79) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 70. 80) The antibody or antigen-binding fragment thereof according to claim 70, which is conjugated to a drug via a linker. 81) The antibody or antigen-binding fragment thereof according to claim 70, wherein the drug is an auristatin analog. 82) The antibody or antigen-binding fragment thereof according to claim 70, further comprising a stretcher unit. 83) The antibody or antigen-binding fragment according to claim 70, further comprising a spacer unit. 84) The antibody or antigen-binding fragment thereof according to claim 70, further comprising an amino acid unit. 85) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 70 to 84 and a pharmaceutically acceptable additive. 86) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 70 to 84. 87) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 85. 88) A method for treating cancer in a subject, comprising the step of administering the kit of claim 86 to the subject in a therapeutically effective dose. 89) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 70 to 88. 90) The method according to any one of claims 87 to 89, wherein the subject is a human subject. 91) The method according to claim 89, wherein cancer is as shown in Table I. 92) The method according to claim 89, further comprising the step of administering a radioactive or chemotherapeutic agent. 93) An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. 94) The antibody or antigen-binding fragment according to claim 93, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequence shown in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84. 95) The antibody or antigen-binding fragment thereof according to claim 93, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 32. 96) The antibody or antigen-binding fragment thereof according to claim 93, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 38. 97) The antibody or antigen-binding fragment thereof according to claim 93, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 12. 98) The antibody or antigen-binding fragment thereof according to claim 93, wherein the light chain comprises the sequence shown in SEQ ID NO: 24. 99) The antibody or antigen-binding fragment according to claim 93, comprising a heavy chain variable region having 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 heavy chain variable region amino acid sequence shown in SEQ ID NO: 32, and a light chain variable region having 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 light chain variable region amino acid sequence shown in SEQ ID NO: 38. 100) The antibody or antigen-binding fragment according to claim 93, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 101) The antibody or its antigen-binding fragment according to claim 93, wherein the antibody is a fully human antibody. 102) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 93. 103) The antibody or antigen-binding fragment thereof according to claim 93, which is conjugated to a drug via a linker. 104) The antibody or antigen-binding fragment thereof according to claim 93, wherein the drug is an auristatin analog. 105) The antibody or antigen-binding fragment thereof according to claim 93, further comprising a stretcher unit. 106) The antibody or antigen-binding fragment according to claim 93, further comprising a spacer unit. 107) The antibody or antigen-binding fragment according to claim 93, further comprising an amino acid unit. 108) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 93 to 107 and a pharmaceutically acceptable additive. 109) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 93 to 108. 110) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 108. 111) A method for treating cancer in a subject, comprising the step of administering the kit according to claim 109 to the subject in a therapeutically effective dose. 112) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment according to any one of claims 93 to 107. 113) The method according to any one of claims 110 to 112, wherein the subject is a human subject. 114) The method according to claim 112, wherein cancer is as shown in Table I. 115) The method according to claim 112, further comprising the step of administering a radioactive or chemotherapeutic agent. 116) An antibody or its antigen-binding fragment containing a heavy chain variable region including a complementarity-determining region (CDR) having the sequence shown in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69. 117) The antibody or antigen-binding fragment according to claim 116, further comprising a light chain variable region including a complementarity-determining region (CDR) having the sequence shown in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87. 118) The antibody or antigen-binding fragment thereof according to claim 116, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 33. 119) The antibody or antigen-binding fragment thereof according to claim 116, wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 39. 120) The antibody or antigen-binding fragment thereof according to claim 116, wherein the heavy chain comprises the sequence shown in SEQ ID NO: 14. 121) The antibody or antigen-binding fragment thereof according to claim 116, wherein the light chain comprises the sequence shown in SEQ ID NO: 26. 122) The antibody or antigen-binding fragment according to claim 116, comprising a heavy chain variable region having 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 heavy chain variable region amino acid sequence shown in SEQ ID NO: 33, and a light chain variable region having 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 light chain variable region amino acid sequence shown in SEQ ID NO: 39. 123) The antibody or antigen-binding fragment according to claim 116, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv. 124) The antibody or its antigen-binding fragment according to claim 116, wherein the antibody is a fully human antibody. 125) Recombinantly produced antibody or antigen-binding fragment thereof according to claim 116. 126) The antibody or antigen-binding fragment thereof according to claim 116, conjugated to a drug via a linker. 127) The antibody or antigen-binding fragment thereof according to claim 116, wherein the drug is an auristatin analog. 128) The antibody or antigen-binding fragment according to claim 116, further comprising a stretcher unit. 129) The antibody or antigen-binding fragment according to claim 116, further comprising a spacer unit. 130) The antibody or antigen-binding fragment according to claim 116, further comprising an amino acid unit. 131) A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 116 to 130 and a pharmaceutically acceptable additive. 132) A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 116 to 131. 133) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 131. 134) A method for treating cancer in a subject, comprising the step of administering the kit described in claim 132 to the subject in a therapeutically effective dose. 135) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment according to any one of claims 116 to 130. 136) The method according to any one of claims 133 to 135, wherein the subject is a human subject. 137) The method according to claim 135, wherein cancer is as shown in Table I. 138) The method according to claim 135, further comprising the step of administering a radioactive or chemotherapeutic agent or CAR-T therapy or NK cell therapy. 139) An antibody-drug conjugate (ADC) comprising a nectin-4 antibody or an antigen-binding fragment conjugated to a drug-linker (DL) payload, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR region containing the amino acid sequence shown in any of SEQ ID NOs. 52 to 69. 140) The ADC according to claim 139, further comprising a nectin-4 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a light chain CDR region having an amino acid sequence shown in any of SEQ ID NOs. 70 to 87. 141) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 142) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 143) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 144) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 145) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 146) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 147) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 148) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 149) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 150) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 151) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 152) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 153) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 154) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 155) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 156) The DL payload has the following chemical structure: [ka] The ADC according to claim 139 or 140, including the ADC described in claim 139 or 140. 157) A pharmaceutical composition comprising the ADC according to any one of claims 139 to 156 and a pharmaceutically acceptable additive. 158) A kit comprising the ADC according to any one of claims 139 to 156. 159) A kit comprising the pharmaceutical composition according to claim 157. 160) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective dose of an ADC according to any one of claims 139 to 156. 161) A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 157. 162) The method according to claim 160, wherein the subject is a human. 163) The method according to claim 161, wherein the subject is a human. 164) The method according to claim 160, wherein cancer is as shown in Table I. 165) The method according to claim 161, wherein cancer is as shown in Table I. 166) The method according to claim 160, further comprising the step of administering a radioactive or chemotherapeutic agent or CAR-T therapy or NK cell therapy. 167) The method according to claim 161, further comprising the step of administering a radioactive or chemotherapeutic agent or CAR-T therapy or NK cell therapy. [Examples]

[0256] Various aspects of the present invention are further described and illustrated by several embodiments that follow below, none of which are intended to limit the scope of the invention.

[0257] (Example 1) Methods for generating antibodies Nectin-4 antibodies were generated by a de novo discovery campaign using a yeast-display human Fab antibody library. After several rounds of enrichment and subsequent clonal screening, clones that specifically recognized human nectin-4 expressed on cancer cells were identified. Subsequently, the variable heavy and light chains of the antibody were sequenced from DNA isolated from the yeast clones. To recombinantly express the nectin-4 antibody, the antibody variable heavy and light chain sequences were cloned upstream of the human heavy chain IgG1 and human light chain Igκ constant regions, respectively. Signal peptides were inserted upstream of the heavy and light chains to enable antibody secretion. The complete anti-nectin-4 antibody human heavy and light chain cassettes were cloned downstream of the CMV promoter / enhancer in the cloning vector. Polyadenylation sites were included downstream of the MAb coding sequence. Recombinant anti-nectin-4 antibody heavy and light chain expression constructs were transfected into CHO cells.

[0258] Stable transfected Chinese hamster ovary (CHO) cells underwent a selection and harvesting process to generate a stable pool expressing recombinant antibodies and Fc variants. For antibody production, a fed-batch production process with a typical culture duration of 8–15 days was used for the stable transfected pool before harvesting the culture medium. Alternatively, transiently transfected cells were cultured for a typical duration of 3–15 days before harvesting the culture medium. Subsequently, protein-A affinity purification was performed on the harvested cell culture medium, and the purified material was buffer-exchanged to phosphate-buffered saline (PBS) or other preferred antibody formulation buffer. The quality of the recombinant antibodies was evaluated by size-exclusion chromatography, SDS-PAGE, and other methods known in the art.

[0259] The obtained nectin-4 antibodies are shown in Tables VI and VII, and include (SEQ ID NO: 4) to (SEQ ID NO: 27).

[0260] (Example 2) Nectin-4 antibody binding assay The binding affinity of the Nectin-4 antibody of the present invention was evaluated using the following protocol. Briefly, tumor cell lines were harvested and the cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). The cells were plated in a 96-well round-bottom plate and incubated with antibody (10 μg / mL) on ice for 1 hour. For cell binding specificity determination, antibody was added at 10 μg / mL. After incubation, the cells were pelleted by centrifugation at 200 × g for 5 minutes, washed twice with FACS buffer, protected from light, and counterstained with R-PE-labeled goat anti-human Fc gamma fragment-specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice for a further 1 hour. The labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

[0261] The results indicate that the nectin-4 antibody specifically binds 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 Figure 1).

[0262] (Example 3) Nectin-4 antibody binding assay In another example, the binding affinity of the Nectin-4 antibody of the present invention was evaluated using the following protocol. Briefly, tumor cell lines were harvested and the cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). The cells were plated in a 96-well round-bottom plate and incubated with the antibody (10 μg / mL) on ice for 1 hour. For cell binding specificity determination, the antibody was added at a 3-fold dilution of 10 μg / mL. After incubation, the cells were pelleted by centrifugation at 200 × g for 5 minutes, washed twice with FACS buffer, protected from light, and counterstained with R-PE-labeled goat anti-human Fc gamma fragment-specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice for a further 1 hour. The labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

[0263] The results indicate that the nectin-4 antibody specifically binds to nectin-4 on the T-47D breast cancer cell line (see Figure 2 and Table XII).

[0264] (Example 4) Nectin-4 antibody binding assay In another example, the binding affinity of the Nectin-4 antibody of the present invention was evaluated using the following protocol. Briefly, tumor cell lines were harvested and the cells were resuspended in FACS buffer (2% FBS + 5 mM EDTA in PBS). The cells were plated in a 96-well round-bottom plate and incubated with the antibody (10 μg / mL) on ice for 1 hour. For cell binding affinity determination, the antibody was added at a 3-fold dilution of 10 μg / mL. After incubation, the cells were pelleted by centrifugation at 200 × g for 5 minutes, washed twice with FACS buffer, protected from light, and counterstained with R-PE-labeled goat anti-human Fc gamma fragment-specific secondary antibody (Jackson Immuno Research; West Grove, PA) on ice for a further 1 hour. The labeled cells were then washed twice with FACS buffer and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific; Carlsbad, CA).

[0265] The results show that the nectin-4 antibodies, in their respective ADC forms, specifically bind to nectin-4 on the NCI-H292 lung cancer cell line and also bind relatively well to NCI-H292 (see Figure 3 and Table XIII).

[0266] (Example 5) In vitro cytotoxicity of nectin-4 ADC In vitro cytotoxicity of nectin-4 ADC was determined using the following protocol. Briefly, tumor cell lines were harvested, plated in 384-well white flat-bottom plates, and reattached for 2–4 hours while incubating at 37°C. Cells were then treated with ADC or free payload test material over dose titration (up to 500 nM at 5-fold dilutions). After 5 days of treatment, residual cell viability was determined by CellTiter Glo assay based on manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells, and dose-response curves were fitted using a 4-parameter logistic equation with GraphPad Prism software (version 9; La Jolla, CA).

[0267] The results indicate that nectin-4 ADC exhibits in vitro cytotoxic effects against multiple cancer cell lines, including nectin-4-positive lung adenocarcinoma NCI-H322 cells (see Figure 4(A)) and nectin-4-positive PC3-nectin-4 recombinant prostate cancer cells (see Figure 4(B)), but not against nectin-4-negative cells (see Figure 4(C)). See also Table XIV.

[0268] In another set of experiments using the protocol shown above, the in vitro cytotoxic efficacy (IC) was observed. 50 This is demonstrated using the same Nectin-4 Ab conjugated to nine different linkers / payloads. 50 (nM) values ​​are shown in Table XV. These results further confirm that nectin-4 ADCs utilizing multiple linkers / payloads have an in vitro cytotoxic effect against the nectin-4-positive cell line PC3-nectin-4.

[0269] (Example 6) In vitro cytotoxicity of Nectin-4 ADC payload In vitro cytotoxicity of the Nectin-4 ADC payload was determined using the following protocol. Briefly, tumor cell lines were harvested, plated in 384-well white flat-bottom plates, and reattached for 2–4 hours while incubated at 37°C. Cells were then treated with ADC or free payload test material over dose titration (up to 500 nM at 5-fold dilutions). After 5 days of treatment, residual cell viability was determined by the CellTiter Glo assay based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells, and dose-response curves were fitted using a 4-parameter logistic equation with GraphPad Prism software (version 9; La Jolla, CA).

[0270] The results show in vitro cytotoxicity of nectin-4 ADCs (black symbols) presenting three different payload entities compared to their respective isotype control ADCs (white symbols) and their respective free payloads (dotted lines) in the nectin-4 positive breast cancer cell line Sum190PT (see Figure 5).

[0271] (Example 7) Bystander activity of nectin-4 ADC compared to enfortumab vedotin The bystander activity of nectin-4 ADC compared to commercially available enfortumab vedotin (PADCEV) was determined using the following protocol. Briefly, a co-culture model of nectin-4-expressing PC3-nectin-4 recombinant cells labeled with Cell Trace Violet (Invitrogen; Waltham, MA) and nectin-4-nonexpressing SU-DHL-1 cells was performed in 24-well plates. The ratio of target-positive cells to target-negative cells was 1:2. After 96 hours of treatment with the ADC test material, SU-DHL-1 cells were harvested, collected in 96-well round-bottom plates, subsequently stained with Fixable Viability Dye (FVD) eFluor780 (Invitrogen) and APC-Annexin V (BioLegend; San Diego, CA), and then analyzed by flow cytometry. ADC-treated SU-DHL-1 cell monocultures served as a control to demonstrate non-target-specific cell death or its absence. Early apoptotic (Annexin V mono-positive), late apoptotic (FVD eFluor780 mono-positive), and necrotic (Annexin V and FVD eFluor780 double-positive) cells were considered non-viable.

[0272] The results show the bystander activity of representative ADCs (black squares) compared to enfortumab vedotin (black circles) and to no treatment (white triangles). Bystander activity is determined by graphing cell death in nectin 4-negative cell lines when co-cultured with nectin 4-expressing cell lines (Figure 6(A)). Nonspecific activity of representative ADCs is absent in target-negative cell single cultures compared to enfortumab vedotin (Figure 6(B)).

[0273] (Example 8) In vivo efficacy of nectin-4 ADCs using multiple payloads in an HT1-376 xenograft model. The in vivo efficacy of Nectin-4 ADC was investigated using the following protocol. Briefly, an HT-1376 cell suspension was mixed with Matrigel in a 1:1 ratio. 5,000,000 viable cells were subcutaneously injected into the posterior flank of female BALB / c nude mice. The average tumor size was approximately 100 mm. 3 Once the mice reached a certain stage, they were randomized into five groups. Each group received a single dose of the test substance at 5 mg / kg. The study was concluded on day 28.

[0274] The results show that all nectin-4 ADCs inhibited tumor growth compared to the control group (see Figure 7).

[0275] (Example 9) In vivo efficacy of nectin-4 ADC in the Sum190PT breast cancer xenograft model Further in vivo efficacy of Nectin-4 ADC (Ab5-ADC2) was investigated using the following protocol. Briefly, Sum190PT cell suspension was mixed with Cultrex ECM in a 1:1 ratio. 3,000,000 viable cells were subcutaneously injected into the posterior flank of female NSG mice. The average tumor size was approximately 130 mm. 3 Once the mice reached a certain stage, they were randomized into groups of 5 mice each. The test substance was administered twice, 1 week apart, at a dose of 10 mg / kg. The study was concluded on day 28.

[0276] The results show the in vivo efficacy of nectin-4 ADCs (black circles) compared to each isotype control ADC (white squares) and PBS group (white circles) in a nectin-4 positive Sum190PT breast cancer xenograft model (see Figure 8).

[0277] (Example 10) In vivo efficacy of nectin-4 ADC compared to PADCEV in patient-derived head and neck cancer models. Further in vivo efficacy of nectin-4 ADC (Ab5-ADC2) compared to commercially available enfortumab vedotin (PADCEV) was investigated using the following protocol. Briefly, tumors derived from patients with head and neck squamous cell carcinoma were grown in vivo as patient-derived xenografts (PDX) in immunodeficient mice. Tumor fragments (2-3 mm in diameter) were collected from stock mice and used for subcutaneous inoculation into female NOD / SCID mice. The average tumor size was approximately 150 mm. 3 Once the mice reached a certain stage, they were randomized into groups of 5 mice each. The test substance was administered as a single dose or in two doses of 5 mg / kg or 10 mg / kg, separated by 2 weeks.

[0278] The results show that nectin-4 ADC (circle) demonstrates better efficacy and complete tumor eradication compared to enfortumab vedotin (square) in a head and neck cancer model derived from nectin-4-positive patients (see Figure 9).

[0279] (Example 11) Use of chimeric antigen receptor (CAR) T-cell therapy in cancers expressing nectin-4 Generally speaking, T cells help detect and fight infections and diseases in the body, such as cancer. Many cancers can hide from T cells, and therefore, if T cells cannot "see" the cancer, it can grow throughout the body. One promising form of immunotherapy in cancer is known as CAR-T therapy.

[0280] In CAR-T therapy, chimeric antigen receptors (CARs) are designed to recognize specific markers expressed in cancer (e.g., nectin-4). Studies have shown that when CARs bind to specific antigens, an immune response is induced, allowing T cells to recognize cancer and inhibit its growth.

[0281] As a non-limiting example, blood is collected from patients with cancer expressing nectin-4. T cells are isolated from the patient's blood and genetically engineered to generate CAR-T cells. The CAR-T cells are cultured and expanded using techniques known in the field. Finally, the CAR-T cells are injected into the patient's bloodstream. See JIN, et. al., Cancer Cell Int., 21:83 (2021).

[0282] The trial will first demonstrate safety and then confirm efficacy at repeated doses. The trial is open-label, comparing standard chemotherapy with standard treatment plus Nectin-4 CAR-T cells. As understood, one non-limiting criterion that may be used in connection with patient enrollment is the concentration of Nectin-4 in the tumor, as determined by standard detection methods known in the art.

[0283] (Example 12) Use of natural killer (NK) cell therapy in cancers expressing nectin-4 Similar to CAR-T therapy, natural killer (NK) cell therapy is a form of immunotherapy that has shown promise in treating cancer (e.g., cancers expressing nectin-4). Unlike T cells, NK cells are not adapted to specific antigens. However, NK cells can recognize and attack cancer cells, although they do not live long enough or multiply rapidly enough to completely fight cancer cells. However, research has 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 enables a more robust immune response. One advantage of NK cell therapy compared to CAR-T therapy is the lack of side effects. In some cases, NK cells are also enhanced with CARs to better adapt them to fight cancer. See LU, et. al., Frontiers in Oncology, vol. 11, Art. 720501 (Aug. 2021).

[0284] As a non-limiting example, several strategies may be used 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 NK92 cell lines. After isolation from the aforementioned sources, NK cells are stimulated with cytokines, e.g., IL-2, IL-15, and / or IL-18. Furthermore, NK cells may be modified ex vivo to express CARs, enabling them to recognize specific tumor-associated antigens, e.g., nectin-4. Finally, NK cells are injected into the patient's bloodstream. See MEHTA, et. al., Int. J. of Hematology, 107:262-270 (2018).

[0285] The trial will first demonstrate safety and then confirm efficacy at repeated doses. The trial is open-label, comparing standard chemotherapy with standard treatment plus nectin-4 NK cells. As understood, one non-limiting criterion that may be used in connection with patient enrollment is the concentration of nectin-4 in the tumor, as determined by standard detection methods known in the art.

[0286] (Example 13) Characterization assays for nectin-4 antibodies and nectin-4 ADCs The nectin-4 antibody and nectin-4 ADC composition of the present invention were further characterized using assays known in the art.

[0287] The results shown in Figures 11 and 12 confirm a better safety profile and stronger efficacy, resulting in an improved therapeutic range compared to other nectin-4 antibodies and nectin-4 ADCs known in the field.

[0288] (Example 14) In vitro safety evaluation of nectin-4 ADC across multiple primary cultures of normal human cells. In vitro cytotoxicity of nectin-4 ADC was determined using the following protocol. Briefly, primary cells derived from normal humans, including corneal epithelial cells, adult dermal fibroblasts, and adult epidermal keratinocytes, were harvested, plated in 384-well white flat-bottom plates, and reattached for 2–4 hours while incubated at 37°C. The cells were then treated with ADC or free payload test material over dose titration (up to 1000 nM at 5-fold dilutions). After 5 or 6 days of treatment, cell viability was determined by the CellTiter Glo assay based on the manufacturer's instructions (Promega; Madison, WI). Data were normalized to untreated control cells, and dose-response curves were fitted using a 4-parameter logistic equation with GraphPad Prism software (version 9; La Jolla, CA).

[0289] The results show that nectin-4 ADC Ab5-ADC2 has a significantly weaker cytotoxic effect than enfortumab vedotin in vitro against multiple normal primary cells, e.g., human corneal epithelial cells (see Figure 11(A)), adult dermal fibroblasts (see Figure 11(B)), and adult epidermal keratinocytes (see Figure 11(C)). See also Table XVI, which shows the toxicological parameters of ADC and total IgG at repeated doses up to 18 mg / kg and therefore provides evidence of increased HNSTD in NHP as an outcome of the improved in vitro safety profile described.

[0290] Another set of experiments using the protocol shown above will show flow cytometry histograms of the cells described above: human corneal epithelial cells (see Figure 12(A)), adult dermal fibroblasts that do not express nectin-4 and exhibit toxicity liability due to untargeted ADC uptake (see Figure 12(B)), and adult epidermal keratinocytes that express nectin-4 and exhibit on-target toxicity tendency to nectin-4 ADCs (see Figure 12(C)).

[0291] (Example 15) Cell cycle analysis of Ab5-ADC2 In this experiment, the cell cycle analysis of Ab5-ADC2 was determined using the following protocol. Briefly, HT-1376 tumor cells were harvested, plated in 6-well plates, and incubated overnight at 37°C. The following day, the cells were treated with 5 nM Ab5-ADC2 for 72 hours and then treated for propidium iodide staining with FxCycle PI / RNase Staining Solution (Invitrogen, Waltham, MA, USA). DNA content was measured by flow cytometry, and cell cycle data modeling was analyzed using the Watson Pragmatic algorithm along with FlowJo software (version 10; BD Biosciences, Franklin Lakes, NJ, USA).

[0292] The results show a combined percentage (%) cell cycle analysis of HT-1376 cells in the G2 and sub-G1 phases after 72 hours of treatment with Ab5-ADC2 (see Figure 13(A)). Figure 13(B) shows a representative flow cytometry histogram of Ab5-ADC2-treated cells, showing an increase in the cell population in both the sub-G1 and G2 phases of the cell cycle compared to untreated control cells, as expected for this payload class.

[0293] (Example 16) Immunogenic cell death (ICD) analysis of free payloads compared to MMAE In this experiment, ICD analysis of the free payload compared to MMAE was determined using the following protocol. Briefly, NCI-H292 tumor cells were harvested, plated in 100 mm culture dishes, and incubated overnight at 37°C. The following day, the cells were treated with a 10 nM free payload for 48 hours and then treated for live cell staining with immunogenic cell death (ICD) markers: anti-calreticulin, anti-HSP70, and anti-HMGB1. All marker antibodies were labeled with AlexaFluor 488 (Novus Biologicals, Centennial, CO, USA). Cells were analyzed by flow cytometry using an Attune NxT flow cytometer to determine %ICD marker positivity compared to isotype control antibody staining.

[0294] The results show, as expected for this payload class, an increase in immunogenic cell death markers to a similar degree with both payloads (see Figure 14).

[0295] (Example 17) Complement-dependent cell-mediated cytotoxicity (CDC) analysis of Ab5-ADC2 and Ab5 In this experiment, the CDC analysis of Ab5-ADC2 was determined using the following protocol. Briefly, HT-1376 tumor cells were harvested, plated in a 96-well white flat-bottom plate, and incubated with the indicated serially diluted test material on ice for 1 hour to allow binding of the antibody or ADC to the cells. The cells were then opsonized with baby rabbit complement at 37°C for 1 hour, and residual cell viability after cell lysis due to CDC activity was determined by CellTiter-Glo 2.0 Assay (Promega).

[0296] The results show that both Ab5-ADC2 and enfortumab lack significant CDC activity (see Figure 15).

[0297] (Example 18) Antibody-dependent cell-mediated cytotoxicity (ADCC) analysis of Ab5-ADC2 and Ab5 In this experiment, ADCC analysis of Ab5-ADC2 was determined using the following protocol. Briefly, NCI-H292 tumor cells were harvested, plated in a 96-well white flat-bottom plate, and incubated overnight at 37°C. The following day, the tumor cells were co-incubated for 6 hours with engineered effector cells (6:1 ratio of effector cells to target cells) in the presence of the indicated antibody or ADC test material, and ADCC activity was measured using the commercially available ADCC Reporter Bioassay, V Variant kit (Promega, Madison, WI, USA).

[0298] The results indicate that the positive control (Her2 Ab) showed strong ADCC activity, but Ab5-ADC2 did not show significant ADCC activity (see Figure 16).

[0299] (Example 19) Antibody-dependent cell-mediated phagocytosis (ADCP) analysis of Ab5-ADC2 and Ab5 In this experiment, the ADCP analysis of Ab5-ADC2 was determined using the following protocol. Briefly, tumor cells were harvested, plated in a 384-well white flat-bottom plate, and incubated overnight at 37°C. The following day, the tumor cells were co-incubated for 6 hours with engineered effector cells (in a 6:1–7.5:1 ratio of effector cells to target cells) in the presence of the indicated antibody or ADC test material, and ADCP activity was measured using a commercially available FcγRI ADCP bioassay kit (Promega).

[0300] The results show that weaker ADCP activity was observed for Ab5-ADC2 in nectin-4 expressing SUM190PT and HT-1376 cancer cell lines compared to the assay control reagent (isotype control Ab in RAMOS cells expressing the target of isotype control Ab) (see Figure 17).

[0301] Overall, the lack of knowledge regarding CDC, ADCC, and ADCP ruled out any significant contribution of FcR-mediated effector functional activity to the mechanism of action of Ab5-ADC2.

[0302] (Example 20) Pharmacokinetic profiles of ADCs and Ab5-ADC2 free payloads in cynomolgus monkeys In this experiment, the pharmacokinetic profiles of Ab5-ADC2 and its corresponding free payload were determined using the following protocol. Briefly, cynomolgus monkeys were administered Ab5-ADC2, an ADC test item, at different dose levels for two separate doses. Blood was collected at different time points and processed into serum for total antibody and ADC quantification, and into plasma for free payload analysis. Total antibody and ADC concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA) by capturing with soluble recombinant nectin-4 and detecting either biotin-labeled anti-human IgG or biotin-labeled anti-payload followed by the binding of streptavidin-HRP, respectively. TMB was used as a colorimetric substrate, and the color reaction was stopped with H2SO4 solution. Absorbance was measured on a plate reader at 450 nm to 630 nm. Calibration curves were generated by plotting the response of serially diluted test samples against the calibration sample concentration (average absorbance of each calibration curve sample minus the average absorbance of the blank) and fitting them using a four-parameter logistic model. All data were analyzed using SoftMax Pro 7.0 software.

[0303] For free payload quantification, the payload was extracted from 20 μL of cynomolgus monkey K2EDTA plasma by protein precipitation and then detected and quantified using an AB SCIEX TRIPLE QUAD® 6500+ mass spectrometer with an internal standard. TK parameters are summarized in Table XVI.

[0304] The results show linear PK profiles for total IgG, ADC, and free payload without significant accumulation. Notably, the total IgG and ADC curves overlap at each dose level throughout the 21-day dosing cycle with a half-life of approximately 5 days (Table XVI), which is achieved via the highly stable conjugation technique used to generate Ab5-ADC2. Importantly, repeated dosing of 18 mg / kg Ab5-ADC2 was well tolerated in non-human primates, achieved through increased conjugation stability and better tumor-targeted payload accumulation while preserving normal tissue (see Figure 18).

[0305] (Example 21) In vivo efficacy of nectin-4 ADC compared to enfortumab vedotin in patient-derived cervical cancer model PDX36. Further in vivo efficacy of nectin-4 ADC (Ab5-ADC2) compared to commercially available enfortumab vedotin was investigated using the following protocol. Briefly, PDX (in this case, PDX36) derived from patients with cervical cancer was grown in vivo in immunodeficient mice. Tumor fragments (2-3 mm in diameter) were collected from stock mice and used for subcutaneous inoculation into female NOD / SCID mice. The average tumor size was approximately 150 mm. 3 Once the mice reached a certain level, they were randomized into groups of 5 mice each. The test substance was administered at doses of 2.5, 5, or 10 mg / kg.

[0306] The results show that Ab5-ADC2 (dark circle) exhibits better efficacy and tumor inhibition compared to enfortumab vedotin (dark triangle) in patient-derived cervical cancer models, which leads to longer study-to-study survival in tumor-carrying mice treated with Ab5-ADC2 (see Figures 19(A) and 19(B)).

[0307] (Example 22) In vivo efficacy of nectin-4 ADC in multiple patient-derived cervical cancer models (PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) Further in vivo efficacy of nectin-4 ADC (Ab5-ADC2) was investigated in multiple cervical cancer models using the following protocol. Briefly, PDX (in this case, PDX10, PDX12, PDX13, PDX16, PDX34, and PDX36) derived from patients with cervical cancer were grown in vivo in immunodeficient mice. Tumor fragments (2-3 mm in diameter) were collected from stock mice and used for subcutaneous inoculation into female NOD / SCID mice. The average tumor size was approximately 150 mm. 3 Once the mice reached a certain level, they were randomized into groups of 5 mice each. The test substance was administered at doses of 2.5, 5, or 10 mg / kg.

[0308] The results show that Ab5-ADC2 (dark circle) exhibits significant efficacy and tumor inhibition compared to vehicle controls in multiple patient-derived cervical cancer models (see Figures 20(A)–20(F)).

[0309] (Example 23) Nectin-4 expression in patient-derived cervical cancer models via immunohistochemistry The expression characterization of Nectin-4 in patient-derived cervical cancer xenograft models was evaluated using the following protocol. Briefly, tissue samples were fixed in 10% buffered neutral formalin, processed, embedded in paraffin wax, and then prepared as 4 μm tissue sections. After deparaffinization and re-wetting, the sections were processed for antigen retrieval. The antigen-retrieved tissue sections were then incubated with mouse anti-Nectin-4 primary antibody or IgG (antibody control). The tissue sections conjugated with the primary antibody were washed and detected using an enzyme-labeled secondary antibody. The conjugated secondary antibody was stained using a Leica Bond Refine Polymer Detection system. The stained tissue sections were then scanned, imaged, and evaluated using an Aperio ScanScope CS (Aperio;Vista, CA) optical microscope. All stained sections were scanned using a NanoZoomer-HT 2.0 Image system at 40× magnification to generate images.

[0310] The results indicate 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 their respective figures, with images shown in high resolution (21(A), (C), (E), (G), (I), and (K)) and low resolution (21(B), (D), (F), (H), (J), and (L)) (see 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 inhibition of tumor growth in all models.

[0311] (Example 24) Analysis of free payload concentration of Ab5-ADC2 or enfortumab vedotin via LC-MS / MS The free payload concentrations of Ab5-ADC2 or enfortumab vedotin in multiple tissues of mice carrying nectin-4-positive breast cancer tumors were evaluated using the following protocol. Briefly, either Ab5-ADC2 or enfortumab vedotin was administered to a Sum190PT inflammatory breast cancer xenograft mouse model. Tumor, normal tissue, and plasma were collected and analyzed for payload concentrations by LC-MS / MS.

[0312] The results in Figure 22(A) show the free payload concentration compared to MMAE. The results in Figure 22(B) show the payload concentration of Ab5-ADC2 compared to enfortumab vedotin. Notably, Ab5-ADC2 was able to deliver more payload to nectin-4 expressing tumors while reducing payload exposure in normal tissue, which underlies a better safety profile compared to enfortumab vedotin.

[0313] (Example 25) Stability and DAR retention analysis Ab5-ADC2 Stability assessment and DAR retention analysis were performed using the following protocol. Briefly, Sprague Dawley rats, 10-12 weeks old upon arrival, were acclimatized for 1 week, and then administered a single intravenous dose of Ab5-ADC2 or enfortumab vedotin. Blood samples were collected at various time points after administration: 1 hour, 4 hours, 3, 7, 14 days, and 21 days. The collected blood in EDTA-coated tubes was processed into plasma by centrifugation at 10,000 g at 4°C for 10 minutes. The plasma was divided into aliquots and stored frozen at -80°C until analysis. Plasma samples were diluted in TBS and then combined with biotin-labeled capture anti-human antibody-coated beads, followed by mixing with streptavidin-coated DynaBeads (Invitrogen) and incubated for approximately 2 hours. Affinity-captured ADCs on the beads were subjected to a magnet to collect the complexes, then washed, and the ADCs were separated. Affinity-purified ADCs were tested on an Agilent QTOF 6550 B or equivalent under MassHunter B.07.00. Peaks were integrated, extracted, and spectra were deconvolved using the Maximum Entropy algorithm. The deconvolved spectra were exported as CSV files and imported into a DAR Calculator. DAR was determined for each sample and plotted over time.

[0314] The results in Figure 23(A) show the conjugation stability and drug-to-antibody ratio (DAR) of Ab5-ADC2 compared to enfortumab vedotin over time after IV injection. The results in Figure 23(B) show the deconvoluted MS profiles of the heavy and light chains of affinity-purified Ab5-ADC2 from plasma 1 hour and 21 days after injection in Sprague Dawley rats. Together, these results demonstrate that Ab5-ADC2 is highly stable in circulation, which allows for increased payload delivery to tumors beyond normal tissue, as demonstrated in Figure 22. Importantly, this allows for an HNSTD (highest non-severely toxic dose) of 18 mg / kg in non-human primates, as shown in Figure 18.

[0315] (Example 26) Human clinical trials on the treatment of human cancers via the use of nectin-4 antibodies and nectin-4 ADCs. Nectin-4 antibodies and nectin-4 ADCs, which specifically accumulate in tumor cells and are used in the treatment of certain tumors as well as other immunological disorders and / or other diseases (see Table I), are synthesized according to the present invention. In relation to each of these indications, two clinical approaches are successfully explored.

[0316] I.) Adjuvant therapy: In adjuvant therapy, patients are treated with nectin-4 antibody and nectin-4 ADC in combination with chemotherapeutic agents, pharmaceutical agents, or biopharmaceutical agents, or combinations thereof. Primary cancer targets are treated under standard protocols by adding nectin-4 antibody and nectin-4 ADC. The protocol design addresses efficacy, which includes, but is not limited to, reduction in tumor burden of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce the usual doses of standard chemotherapeutic agents and other biopharmaceuticals. These dose reductions enable further and / or extended treatment by reducing dose-related toxicity of chemotherapeutic agents or biopharmaceuticals.

[0317] II.) Monotherapy: In relation to the use of nectin-4 antibodies and nectin-4 ADCs in monotherapy for tumors, nectin-4 antibodies and nectin-4 ADCs are administered to the patient without chemotherapeutic agents, pharmaceutical agents, or biological agents. In one embodiment, monotherapy is clinically implemented in terminally ill cancer patients with extensive metastatic disease. The protocol design addresses efficacy, which is evaluated by the following examples, including but not limited to reduction in tumor burden of primary or metastatic lesions, increased progression-free survival, overall survival, improved patient health, disease stabilization, and the ability to reduce the usual doses of standard chemotherapeutic agents and other biological agents.

[0318] Dosage The dosage regimen may be adjusted to provide the optimal desired response. For example, a single nectin-4 antibody(s) and nectin-4 ADC injection may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent need of the treatment situation. “Dosage unit form” as used herein refers to physically separate units suitable as a single dose for the mammalian subject being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are governed by and directly depend on (a) the unique characteristics of the nectin-4 antibody(s) and nectin-4 ADC, the individual mechanical structures of the irradiation mechanism (reactor) and the specific therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations in the field of formulating such compounds for the treatment of susceptibility in an individual.

[0319] Clinical Development Program (CDP) CDP will track and develop the treatment of cancer(s) and / or immunological disorders (see Table I) using the nectin-4 antibodies and nectin-4 ADCs of this disclosure. The trials will first demonstrate safety and then confirm efficacy at repeated doses. The trials will be open-label and compare standard chemotherapy with standard treatment plus nectin-4 antibodies(s) and nectin-4 ADCs. As understood, one non-limiting criterion that may be used in connection with patient enrollment is the concentration of nectin-4 antibodies(s) and nectin-4 ADCs in the tumor, as determined by standard detection methods known in the art.

[0320] The present invention is not limited in scope by the embodiments disclosed herein, which are intended as sole examples of individual aspects of the invention, and any functionally equivalent invention is within its scope. Various modifications to the models, methods, and lifecycle methodologies of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and teachings and are likewise intended to fall within the scope of the invention. Such modifications or other embodiments may be implemented without departing from the true scope and spirit of the invention. [Table 1] [Table 2] [Table 3] Table IV. Nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of human nectin cell adhesion molecule 4 (nectin-4). [ka] Table V. Amino acid sequence of human nectin cell adhesion molecule 4 (nectin-4) (SEQ ID NO: 3). Signal peptides are underlined. [ka] Tables VI(A) to VI(F). Sequences of antibody heavy chains. Table VI(A).CL.CDNA sequence (SEQ ID NO: 4) and amino acid sequence (SEQ ID NO: 5) of the Z heavy chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(B).CL.X heavy chain cDNA sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7). Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(C).CL.cDNA sequence (SEQ ID NO: 8) and amino acid sequence (SEQ ID NO: 9) of the N heavy chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(D).CL.I shows the cDNA sequence (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 11) of the heavy chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(E).CL. cDNA sequence (SEQ ID NO: 12) and amino acid sequence (SEQ ID NO: 13) of the B heavy chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(F).CL.D shows the cDNA (SEQ ID NO: 14) and amino acid sequence (SEQ ID NO: 15) of the heavy chain. Nucleotide sequences encoding the variable region are underlined. [ka] Tables VI(G) to VI(L). Sequence of antibody light chains. Table VI(G).CL.Z light chain cDNA sequence (SEQ ID NO: 16) and amino acid sequence (SEQ ID NO: 17). Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(H).CL.X shows the cDNA sequence (SEQ ID NO: 18) and amino acid sequence (SEQ ID NO: 19) of the light chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(I).CL.cDNA sequence (SEQ ID NO: 20) and amino acid sequence (SEQ ID NO: 21) of the N light chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(J).CL.I shows the cDNA sequence (SEQ ID NO: 22) and amino acid sequence (SEQ ID NO: 23) of the light chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(K).CL.B light chain cDNA sequence (SEQ ID NO: 24) and amino acid sequence (SEQ ID NO: 25). Nucleotide sequences encoding the variable region are underlined. [ka] Table VI(L).CL.D shows the cDNA sequence (SEQ ID NO: 26) and amino acid sequence (SEQ ID NO: 27) of the light chain. Nucleotide sequences encoding the variable region are underlined. [ka] Table VII(A)-VII(F). Amino acid sequences of antibody heavy chains. Table VII(A).CL. Amino acid sequence of the Z heavy chain (SEQ ID NO: 28). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(B).CL.X heavy chain amino acid sequence (SEQ ID NO: 29). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(C).CL.N heavy chain amino acid sequence (SEQ ID NO: 30). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(D).CL.I heavy chain amino acid sequence (SEQ ID NO: 31). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(E).CL.B heavy chain amino acid sequence (SEQ ID NO: 32). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(F).CL.D heavy chain amino acid sequence (SEQ ID NO: 33). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(G)-VII(L). Amino acid sequences of antibody light chains. Table VII(G).CL.Z light chain amino acid sequence (SEQ ID NO: 34). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(H).CL.X shows the amino acid sequence of the light chain (SEQ ID NO: 35). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(I).CL.N light chain amino acid sequence (SEQ ID NO: 36). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(J).CL.I shows the amino acid sequence of the light chain (SEQ ID NO: 37). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(K).CL.B light chain amino acid sequence (SEQ ID NO: 38). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VII(L).CL.D light chain amino acid sequence (SEQ ID NO: 39). Variable regions are underlined, and Kabat CDR regions are enclosed in boxes. [ka] Table VIII. Amino acid sequence of the antibody heavy chain variable region. [ka] [ka] Table IX. Amino acid sequence of the antibody light chain variable region. [ka] [Table 10-1] [Table 10-2] Tables XI(A) to XI(F). Alignment of amino acid variable heavy chain regions with corresponding germline sequences. Table XI(A).CL.Alignment of the amino acid sequence of the heavy chain variable region (SEQ ID NO: 88) with the corresponding upper V (SEQ ID NO: 89), D (SEQ ID NO: 90), and J (SEQ ID NO: 91) germline sequences. The Kabat CDR region is enclosed. [ka] Table XI(B).CL.X shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 92) and its corresponding upper V (SEQ ID NO: 93), D (SEQ ID NO: 94), and J (SEQ ID NO: 95) germline sequences, with the Kabat CDR region enclosed. [ka] Table XI(C).CL. Alignment of the amino acid sequence of the N heavy chain variable region (SEQ ID NO: 96) with the corresponding upper V (SEQ ID NO: 97), D (SEQ ID NO: 98), and J (SEQ ID NO: 99) germline sequences. The Kabat CDR region is enclosed. [ka] Table XI(D).CL.I shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 100) and its corresponding upper V (SEQ ID NO: 101), D (SEQ ID NO: 102), and J (SEQ ID NO: 103) germline sequences, with the Kabat CDR region enclosed. [ka] Table XI(E).CL. Alignment of the amino acid sequence of the B heavy chain variable region (SEQ ID NO: 104) with the corresponding upper V (SEQ ID NO: 105), D (SEQ ID NO: 106), and J (SEQ ID NO: 107) germline sequences. The Kabat CDR region is enclosed. [ka] Table XI(F).CL.D shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 108) and its corresponding upper V (SEQ ID NO: 109), D (SEQ ID NO: 110), and J (SEQ ID NO: 111) germline sequences, with the alignment. The Kabat CDR region is enclosed. [ka] Tables XI(G) to XI(L). Alignment of amino acid variable light chain regions with corresponding germline sequences. Table XI(G).CL.Z light chain variable region amino acid sequence (SEQ ID NO: 112) and corresponding upper V (SEQ ID NO: 113) and J (SEQ ID NO: 114) germline sequences are aligned. The Kabat CDR region is enclosed. [ka] Table XI(H).CL.X shows the amino acid sequence of the light chain variable region (SEQ ID NO: 115) and its corresponding upper V (SEQ ID NO: 116) and J (SEQ ID NO: 117) germline sequences, with alignment. The Kabat CDR region is enclosed. [ka] Table XI(I).CL. Alignment of the amino acid sequence of the N light chain variable region (SEQ ID NO: 118) with the corresponding upper V (SEQ ID NO: 119) and J (SEQ ID NO: 120) germline sequences. The Kabat CDR region is enclosed. [ka] Table XI(J).CL.I shows the amino acid sequence of the light chain variable region (SEQ ID NO: 121) and its corresponding upper V (SEQ ID NO: 122) and J (SEQ ID NO: 123) germline sequences, with alignment. The Kabat CDR region is enclosed. [ka] Table XI(K).CL.B light chain variable region amino acid sequence (SEQ ID NO: 124) and corresponding upper V (SEQ ID NO: 125) and J (SEQ ID NO: 126) germline sequences are aligned. The Kabat CDR region is enclosed. [ka] Table XI(L).CL.D shows the amino acid sequence of the light chain variable region (SEQ ID NO: 127) and its corresponding upper V (SEQ ID NO: 128) and J (SEQ ID NO: 129) germline sequences, with the alignment. The Kabat CDR region is enclosed. [ka] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

Claims

1. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NOs. 52, 53, and 54.

2. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO:

57.

3. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO:

60.

4. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO:

63.

5. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO:

66.

6. An antibody or its antigen-binding fragment comprising a heavy chain variable region including a complementarity-determining region (CDR) having the sequences shown in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO:

69.

7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, conjugated to a drug via a linker.

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

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

10. The DL payload has the following chemical structure: 【Chemical 104】 The ADC according to claim 9, including the following:

11. The DL payload has the following chemical structure: 【Chemistry 105】 The ADC according to claim 9, including the following:

12. The DL payload has the following chemical structure: 【Chemistry 106】 The ADC according to claim 9, including the following:

13. The DL payload has the following chemical structure: 【Chemistry 107】 The ADC according to claim 9, including the following:

14. The DL payload has the following chemical structure: 【Chemistry 108】 The ADC according to claim 9, including the following:

15. The DL payload has the following chemical structure: 【Chemistry 109】 The ADC according to claim 9, including the following:

16. The DL payload has the following chemical structure: 【Chemical 110】 The ADC according to claim 9, including the following:

17. The DL payload has the following chemical structure: 【Chemistry 111】 The ADC according to claim 9, including the following:

18. The DL payload has the following chemical structure: 【Chemistry 112】 The ADC according to claim 9, including the following:

19. The DL payload has the following chemical structure: 【Chemistry 113】 The ADC according to claim 9, including the following:

20. The DL payload has the following chemical structure: 【Chemistry 114】 The ADC according to claim 9, including the following:

21. The DL payload has the following chemical structure: 【Chemical 115】 The ADC according to claim 9, including the following:

22. The DL payload has the following chemical structure: 【Chemistry 116】 The ADC according to claim 9, including the following:

23. The DL payload has the following chemical structure: 【Chemistry 117】 The ADC according to claim 9, including the following:

24. The DL payload has the following chemical structure: 【Chemistry 118】 The ADC according to claim 9, including the following:

25. The DL payload has the following chemical structure: 【Chemical 119】 The ADC according to claim 9, including the following:

26. A pharmaceutical composition comprising the ADC according to any one of claims 9 to 25 and a pharmaceutically acceptable additive.

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

28. A kit comprising the pharmaceutical composition described in claim 26.

29. A method for treating cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of an ADC according to any one of claims 9 to 25.

30. A method for treating cancer in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to claim 26 to the subject.

31. The method according to claim 29, wherein the subject is a human being.

32. The method according to claim 30, wherein the subject is a human being.

33. The method according to claim 29, wherein the cancer is as shown in Table I.

34. The method according to claim 30, wherein the cancer is as shown in Table I.

35. The method according to claim 29, further comprising the step of administering a radioactive or chemotherapeutic agent or CAR-T therapy or NK cell therapy.

36. The method according to claim 30, further comprising the step of administering a radioactive or chemotherapeutic agent or CAR-T therapy or NK cell therapy.

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