Antibodies specific for nectin-4 and uses thereof

Abstract

The present invention relates to antibodies specific for nectin-4 and their use, inter alia, for the treatment of cancer.

Description

Technical Field

[0001] This invention relates to antibodies specific to nectin-4 and their uses. Background Technology

[0002] Nectin-4 is a surface molecule belonging to the nectin protein family, which contains four members. Nectins are cell adhesion molecules that play a crucial role in various biological processes (such as polarity, proliferation, differentiation, and migration) of epithelial cells, endothelial cells, immune cells, and nerve cells during development and adulthood. They are involved in several pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus, and measles virus.

[0003] PVRL4 / Nectin-4 collaborates with cadherins in the formation and maintenance of adhesion junctions. In fact, Nectin-4 is a type I transmembrane cell adhesion molecule composed of three Ig-like domains (VCC type) in its extracellular region. It is also a receptor for measles virus, mediating its endocytosis. Nectin-4 is expressed during fetal development, but its expression is decreased in adults compared to the widespread expression of other nectins in adult tissues.

[0004] Previous studies (see references 1, 2, 3, 4, 5, 6) have shown that nectin-4 is re-expressed as a tumor-associated antigen with carcinogenic properties in various cancers, including breast cancer. Specifically, Nectin-4 is a tumor-associated antigen primarily found in tumors with poor prognosis, accounting for 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively. In breast tumors, Nectin-4 is primarily expressed in triple-negative and ERBB2+ carcinomas. The detection of soluble Nectin-4 in the serum of patients with these cancers is associated with poor prognosis. Serum Nectin-4 levels increase during metastatic development and decrease after treatment.

[0005] These characteristics are associated with the limited expression of nectin-4 in their respective normal tissues, suggesting that nectin-4 may be a relevant therapeutic target for antibody-drug conjugates (ADCs) in these cancers, as recently reported in bladder cancer, breast cancer, and pancreatic cancer (References 5, 6).

[0006] Therefore, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME, such as Ha22-2mAb from Seattle Genetics) is an antibody-drug conjugate (ADC) targeting Nectin-4 and is currently undergoing clinical trials for the treatment of patients with solid tumors.

[0007] WO2017042210 and WO2018158398 also provide anti-Nectin-4 antibodies (i.e., N41 mAb and 14A5.2 mAb), which recognize epitopes with similar affinity to those recognized by Ha22-2 antibodies and can be used for cancer treatment.

[0008] However, these antibodies are for therapeutic applications only. In fact, none of them can be used for immunohistochemical (IHC) staining, and therefore none have diagnostic applications.

[0009] Monoclonal antibodies that can be used for IHC have high clinical value. In particular, in the field of oncology, IHC-validated mAbs are used to diagnose whether cancer is benign or malignant, determine tumor stage and grade, and identify cell type and metastatic origin to locate the primary tumor. IHC-validated mAbs are also widely used to predict treatment response, especially in breast cancer.

[0010] In this context, mAbs available for both therapeutic and diagnostic (i.e., targeted detection) purposes are clinically crucial because they allow for the determination of a patient's eligibility for treatment. Furthermore, the use of a unique and identical antibody in diagnosis, prognosis, and treatment offers significant value in terms of reliability and development costs.

[0011] Therefore, there is still a need to develop antinectin-4 that can be measured by immunohistochemistry (IHC) for diagnostic applications while also possessing excellent therapeutic properties. Invention Overview

[0013] This disclosure relates to antibodies specific to nectin-4 and their uses.

[0014] In particular, this article discloses an anti-Nectin-4 antibody that selectively binds to Nectin-4 in biological samples during IHC assays. More specifically, the antibody selectively binds to Nectin-4 in tissue sample sections, especially in fixed tissue samples and, more specifically, formaldehyde-fixed paraffin-embedded (FFPE) tissue samples. Therefore, this antibody can be used for diagnostic and / or prognostic purposes.

[0015] In some embodiments, the anti-Nectin-4 antibody of this disclosure binds to Nectin-4, particularly human Nectin-4 of SEQ ID NO:1, with a significant affinity binding constant (K). D The concentration of this antibody is less than 10 μg / mL, significantly less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL, or less than 5 ng / mL. Therefore, this antibody is typically used for therapeutic applications.

[0016] In a specific implementation, the anti-Nectin-4 mAb according to this disclosure competitively binds to Nectin-4 with the following reference mouse antibody mAb 5A12.2, which can be obtained from a hybridoma deposited in CNCM with accession number CNCM I-5407.

[0017] In a specific implementation, the anti-Nectin-4 antibody according to this disclosure comprises a heavy chain and a light chain containing six CDRs of antibody mAb 5A12.2, which can be obtained from a hybridoma deposited in CNCM with accession number CNCM I-5407.

[0018] Typically, the anti-Nectin-4 antibody disclosed herein comprises:

[0019] -HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:5, LCDR2 of SEQ ID NO:6 and LCDR3 of SEQ ID NO:7;

[0020] - A variable heavy chain (VH) domain and a variable light chain domain, which have at least 90% identity with the heavy chain and light chain of SEQ ID NO:8 and 9, respectively; and / or

[0021] - These are the variable heavy chain (VH) domain and the variable light chain domain, respectively, of SEQ ID NO:8 and 9.

[0022] In the specific implementation plan, the anti-Nectin-4 antibody is a human antibody, a chimeric antibody, or a humanized antibody.

[0023] In some embodiments of this disclosure, the anti-Nectin-4 antibody is conjugated to the cytotoxic portion.

[0024] Another aspect of this disclosure relates to a nucleic acid molecule encoding a heavy chain and / or light chain of any of the anti-Nectin-4 antibodies as described above.

[0025] This disclosure also relates to host cells containing such nucleic acids, particularly for the production of any of the anti-Nectin-4 antibodies as described above.

[0026] Another aspect of this disclosure relates to anti-Nectin-4 antibodies as defined above, used for treatment, particularly for cancer treatment, especially for breast cancer, ovarian cancer, or lung cancer. Typically, the cancer is metastatic cancer.

[0027] This disclosure also covers compositions comprising an anti-Nectin-4 antibody according to this disclosure and at least a pharmaceutically acceptable carrier.

[0028] Another aspect of this disclosure relates to an in vitro method for diagnosing cancer in a subject, comprising performing an IHC assay on a tissue sample from the subject using an anti-Nectin-44 antibody as disclosed herein, optionally wherein the tissue sample is a fixed tissue sample from the subject, particularly a fixed embedded tissue sample, and more particularly a formaldehyde-fixed paraffin-embedded (FFPE) tissue sample.

[0029] Another aspect of this disclosure relates to an in vitro method for determining the eligibility of a subject to treatment with an anti-Nectin-4 antibody as described herein, comprising performing an IHC assay on tissue sample sections from the subject using the anti-Nectin-4 antibody, optionally wherein the tissue sample sections are fixed tissues from the subject, particularly fixed embedded tissue sample sections, and more particularly formaldehyde-fixed paraffin-embedded (FFPE) tissue sections.

[0030] This disclosure also relates to a method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of an anti-Nectin-4 antibody as defined above. In one specific embodiment, the method of treating cancer includes a prior step of diagnosing cancer or determining a patient's eligibility for treatment with the anti-Nectin-4 antibody, including the use of the anti-Nectin-4 antibody in an IHC assay performed on tissue sample sections from the subject. In particular, this disclosure covers methods of treating patients who have or are suspected of having cancer, the methods comprising:

[0031] Step 1), which assesses Nectin-4 expression in tissue samples from subjects suspected of having cancer, or assesses the responsiveness of said cancer patients to anticancer treatments, includes:

[0032] (1a) Contact the tissue sample with the antibody or its antigen-binding fragment disclosed herein;

[0033] (1b) Detecting the binding of the antibody or its antigen-binding fragment to the tissue sample; and

[0034] (1c) Determining the expression of Nectin-4 in the tissue sample, wherein the expression level of Nectin-4 in the tissue sample is compared with a reference expression level of Nectin-4; and

[0035] Step 2), that is, when an increased Nectin-4 expression level is observed compared to the reference, the anticancer therapeutic agent is administered to the patient;

[0036] Optionally, the expression level of Nectin-4 was detected using immunohistochemistry (IHC), Western blotting, fluorescence-activated cell sorting (FACS), or enzyme-linked immunosorbent assay (ELISA).

[0037] Optionally, the anticancer therapeutic agent comprises an anti-Nectin-4 antibody or an antibody-drug conjugate of an anti-Nectin-4 antibody, typically an antibody or a variant thereof according to this disclosure. Invention Details

[0039] definition

[0040] As used herein, the term “Nectin-4” has its general meaning in the art and includes human Nectin-4, particularly the native sequence polypeptide, isotypes, chimeric polypeptides, all homologues, fragments, and precursors of human Nectin-4. The amino acid sequence of native Nectin-4 includes the NCBI reference sequence: NP_112178.2.

[0041] More specifically, the term “Nectin-4” includes human Nectin-4 of the following SEQ ID: 1.

[0042] MPLSLGAEMWGPEAWLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYEC RVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLED QNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQM TQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV

[0043] As used herein, the terms “antibody” or “immunoglobulin” have the same meaning and will be used equivalently in this disclosure.

[0044] As used herein, the term "antibody" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, that is, molecules containing antigen-binding sites that specifically bind to immune antigens. Therefore, the term antibody encompasses not only complete antibody molecules but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments.

[0045] In natural antibodies, two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda(1) and kappa(κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant regions of the light chain (CL) and heavy chain (CH) endow important biological properties such as antibody chain binding, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR).

[0046] The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of variable portions of a light chain and a heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed primarily of residues from the hypervariable region or complementarity-determining region (CDR). Occasionally, residues from the non-hypervariable region or frame region (FR) may participate in the antibody binding site or influence the structure of the entire domain and thus affect the binding site. The complementarity-determining region, or CDR, is the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, the antigen binding site typically comprises six CDRs, containing a set of CDRs from each of the V regions of the heavy and light chains. The frame region (FR) is the amino acid sequence inserted between the CDRs. The variable regions of the light and heavy chains typically contain four framework regions and three or fewer CDRs: FR1-FR1-FR2-FR2-FR3-FR3-FR4. As used herein, the term "antibody" collectively refers to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during immune responses in any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice) and non-mammalian species (e.g., shark immunoglobulins).

[0047] Residues in the variable domain of an antibody are typically numbered according to a systematic design devised by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used in this specification. Kabat residue naming does not always directly correspond to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat number, which corresponds to a shortening or insertion of structural components (whether framework or complementarity-determining region (CDR)) of the basic variable domain structure. For a given antibody, the correct Kabat number of the residues can be determined by comparing homologous residues in the antibody sequence with a "standard" Kabat number sequence. According to the Kabat numbering system, the CDRs of the variable regions in the heavy chain are located at residues 31-35 (H-CDR1), 50-65 (H-CDR2), and 95-102 (H-CDR3), while the CDRs of the variable regions in the light chain are located at residues 24-34 (L-CDR1), 50-56 (L-CDR2), and 89-97 (L-CDR3). Based on the V (variable) domains of the IMGT numbering, in the heavy chain, the CDR1-IMGT region contains positions 26-33; the CDR2-IMGT region contains positions 51-58; and the CDR3-IMGT region contains positions 97-109. In the light chain, the CDR1-IMGT region contains positions 27-32; the CDR2-IMGT region contains positions 50-52; and the CDR3-IMGT region contains positions 89-94.

[0048] In specific implementations, the antibodies provided herein are antibody fragments, and more particularly, any protein including the antigen-binding domain of the antibodies disclosed herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and biantibodies.

[0049] As used in this article, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to Nectin-4 are substantially free of antibodies that specifically bind to antigens other than Nectin-4). However, isolated antibodies that specifically bind to Nectin-4 may exhibit cross-reactivity with other antigens (such as related Nectin-4 molecules from other species). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0050] Antibody affinity refers to the strength with which an antibody binds to an epitope present on an antigen (such as Nectin-4 in this disclosure) through its antigen-binding site (complementary site). The apparent affinity binding constant of Nectin-4 (K...) D The binding can be assessed by flow cytometry by measuring the saturation curve of antibody binding and by determining the EC50 value. This is illustrated in the examples of cell lines expressing Nectin-4 (such as cancer cell lines) or cells expressing recombinant Nectin-4 (see specifically the Materials and Methods section).

[0051] As used in this article, the term "K" D "This is intended to represent the equilibrium dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). K" D The value relates to the antibody concentration (the amount of antibody required for a specific experiment), therefore K D The lower the K value (lower concentration), the higher the antibody affinity. D The value can also be determined using methods known in the art. For example, K used to determine mAb D The method for determining the value can be found in Harlow, et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc.; and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), all of which are incorporated herein by reference in their full text. This method is used to determine antibody K. D The method involves using surface plasmon resonance or biosensor systems such as or System. Typically, surface plasmon resonance analysis is used, employing... -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) Kd was measured at approximately 10 response units (RU) using a fixed antigen CM5 chip at 25°C. In short, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 pg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of conjugated protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two serial dilutions of Fab (0.78 nM–500 nM) were prepared in a solution containing 0.05% polysorbate 20 (TWEEN-20). TM The surfactant (PBST) was injected into PBS at a flow rate of approximately 25 μl / min at 25 °C. Binding rate (k... on ) and dissociation rate (k 0ff Using a simple one-to-one Langmuir combination model ( The evaluation software (version 3.2) calculates the equilibrium dissociation constant (Kd) by simultaneously fitting the bound and dissociation sensor maps. off / k on (See, for example, Chen et al. J. Mol. Biol. 293: 865-881, 1999). If the binding rate exceeds 10 by the above surface plasmon resonance analysis. 6 M -1 s -1 The binding rate can then be determined using fluorescence quenching (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) on antigen-antibody (Fab form) in 20 nM anti-pH 7.2 PBS at 25 °C, with increased antigen concentration, measured in a spectrometer, such as an Aviv Instruments spectrometer equipped with a stop-flow spectrometer or an 8000-series SLM-AMINCO spectrometer with a stirring cuvette. TM A stirred cuvette is used in a spectrophotometer (ThermoSpectronic).

[0052] The term “Kassoc” or “Ka” as used in this article refers to the binding rate of a specific antibody-antigen interaction, while the term “Kdis” or “Kd” as used in this article refers to the dissociation rate of a specific antibody-antigen interaction.

[0053] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to formulations of antibody molecules that are single-molecule compositions. Monoclonal antibody compositions exhibit single-molecule binding specificity and affinity for specific epitopes.

[0054] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope present on an antigen (such as Nectin-4). In this disclosure, although relatively low detectable reactivity with non-Nectin-4 proteins or structures (such as other proteins present on cancer cells or other cell types) is typically intended to represent antibodies or proteins that bind to human Nectin-4, as described in the examples, which is commonly expressed in cancer cell lines (such as the SUM190 cancer cell line), preferably having the specificity described in the examples and... Figure 2 The apparent affinity binding constant (K0) measured in the medium is less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL, or less than 5 ng / mL. D (or EC50). Typically, the epigenetic affinity binding constant (K) is... D The concentration should be not less than 0.05 ng / ml, especially 0.1 ng / ml. Typically, the epigenetic affinity binding constant (K0) is... D (It can be determined as shown in the examples) is 0.1 ng-10 μg / ml. In some embodiments, it is expressed as 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less K. D Recombinant peptides that bind to antigens. Typically, K D Not less than 0.05 pM, especially 0.1 pM. Typically, K... D The range is 0.1 pM to 10 nM.

[0055] Antibodies that “cross-competitively bind to Nectin-4” with reference antibody mAb5A12.2 are intended to represent antibodies that bind to Nectin-4, with an epigenetic affinity binding constant (K). D (or EC50) less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL or less than 5 ng / mL, as in the examples and Figure 2 The antibody, which is "not cross-competing with specific antigens," is defined as having a K+ level of 100 nM or greater. D or 1μM or larger K D or 10 μM or larger K D Antibodies that bind to the antigen. In some embodiments, these antibodies, which do not cross-react with the antigen, exhibit substantially undetectable binding to these proteins in standard binding assays.

[0056] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” in this document.

[0057] "Selective binding" typically refers to an antibody binding more strongly to a target (e.g., an epitope) than to another target, indicating that the antibody is specific to that target. If an antibody has a higher affinity for a first target than for a second target, it binds more strongly to the first target than to the second. Typically, if an antibody binds to the first target with a dissociation constant (Kd) or EC50 lower than that of the second target, as described above, the antibody binds more strongly to the first target than to the second target. Most specifically, the reagent does not bind to the second target to a relevant degree, or has relatively low detectable reactivity with non-Nectin-4 proteins or structures (such as other proteins present on cancer cells or other cell types) (typically, in IHC assays, it does not have detectable staining, particularly by qualitative visual examination via quantitative analysis, where staining levels are below reference values).

[0058] Selectivity can also be further demonstrated by, for example, a ratio of approximately 10:1, approximately 20:1, approximately 50:1, approximately 100:1, 10,000:1 or greater of the affinity of binding to a specific antigen versus the affinity of binding to other unrelated molecules (in this case, the specific antigen is the Nectin-4 peptide).

[0059] The selective binding of antibodies in the IHC assays disclosed herein can be further evaluated as described in the examples for tissue samples (particularly for cancer tissue samples expressing Nectin-4). Typically, the selective Nectin-4 binding of the antibodies disclosed herein in the tissue sample portion of an IHC assay can be qualitatively assessed by identifying the nature of the Nectin-4 staining. Intercellular junction staining is observed in cancer / tumor tissue samples having anti-Nectin-4 antibodies as disclosed herein when compared to a similar IHC assay performed on a control negative tissue sample (i.e., not expressing Nectin-4). Indeed, since proteins are transmembrane cell adhesion molecules, identifiable membrane staining is an indicator of selective binding. In some embodiments, the selective anti-Nectin-4 antibodies of this disclosure exhibit staining of non-Nectin-4 antigens that are essentially undetectable. Thus, in such embodiments, typically, the selective anti-Nectin-4 antibodies only exhibit intercellular junction staining and have essentially undetectable intracellular staining.

[0060] Specificity can also be controlled by a competitive assay in which anti-Nectin-4 mAb is pre-incubated with the recombinant soluble Nectin-4 Igv domain prior to the IHC assay. Therefore, there is a lack of detectable staining indicators of the selectivity of the anti-Nectin-4 antibody disclosed herein.

[0061] Staining can also be quantitatively analyzed, thus by multiplying the percentage of positive cells (P) (maximum 100%) by the staining intensity (I) (from 0 (no expression) to 3 (maximum expression, as defined in (M-Rabet et al. “Nectin-4: a new prognostic biomarker for efficient therapeutic targeting of primary and metastatic triple-negative breast cancer”. Ann Oncol. 2017 Apr 1; ​​28(4):769-776)). Formula: QS = P x I. The maximum score is 300. 'Nectin-4-high' (QS>100) and 'Nectin-4-low' (QS<100) can be distinguished. 'Nectin-4-high' refers to the group potentially suitable for treatment with 5A12.2-derived ADCs. Therefore, in one embodiment, the anti-Nectin4 antibody as disclosed herein exhibits a QS score of at least 100, as described above (see also Rabet et al., 2017, implementation of the reference assay).

[0062] The term "identity" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. When positions in two compared sequences are occupied by the same bases or the same amino acid residues, the corresponding molecules are identical at that position. The percentage of identity between two sequences corresponds to the number of common matching positions in the two sequences divided by the number of positions compared and multiplied by 100. Typically, two sequences are compared when they are aligned to give the maximum identity. Identity can be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) stacking program or any sequence comparison algorithm (such as BLAST, FASTA, or CLUSTALW).

[0063] Functional variants of the reference molecule according to this disclosure exhibit functionality substantially equal to or superior to that of the corresponding reference molecule (e.g., 5A12.2 mAb). "Substantially equal to" herein means that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the corresponding functional property of the reference molecule.

[0064] In one aspect, this disclosure relates to anti-Nectin-4 antibodies, said antibodies having at least one and more specifically both of the following properties:

[0065] (i) with an apparent affinity binding constant (K0) less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL or less than 5 ng / mL. D (or EC50) combined with Nectin-4, especially Nectin-4 in people with SED ID NO: 1;

[0066] (ii) It binds to Nectin-4 in immunohistochemical (IHC) assays. More specifically, the antibody selectively binds to Nectin-4 in biological sample sections in IHC assays.

[0067] Anti-Nectin-4 antibodies of this disclosure that possess such advantageous properties can be screened among anti-Nectin-4 antibodies using IHC assays and / or affinity assays as described in the examples.

[0068] In some embodiments, cancer is a carcinoma, lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), germ cell tumor, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, or various types of head and neck cancer. Typically, cancers according to this disclosure include locally or metastatic breast cancer, ovarian cancer, bladder cancer, urothelial carcinoma, pancreatic cancer and lung cancer or carcinoma.

[0069] Immunohistochemistry (IHC) is a routine laboratory technique for visualizing proteins in tissues or tissue sections using antibodies conjugated to enzymes or fluorescent labels. Typically, the IHC assay is performed on fixed tissue sections. Fixation can be achieved using formaldehyde (typically 4%) or neutral buffered formalin (typically 10%). More specifically, the tissue sections are fixed and embedded. In one specific embodiment, the IHC assay is a formaldehyde-fixed paraffin-embedded (FFPE) IHC assay. IHC techniques are further described in the section on immunohistochemical staining methods. Thomas Boenisch, ed. (3rd ed. 2001).

[0070] "Biosample" refers to a collection of similar cells obtained from a subject or patient. A biosample can be a tissue or cell sample. The source of a tissue or cell sample can be solid tissue from fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Biosamples can also be obtained from in vitro tissue or cell cultures. Tissue samples may contain compounds that are not inherently mixed with the tissue itself, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of biosamples described herein include, but are not limited to, tumor biopsies, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples. In a specific embodiment, a biosample is a tissue sample, particularly a fixed tissue sample, and more particularly a paraffin-embedded tissue sample.

[0071] In some implementations, the biological sample comes from a subject who has cancer, is susceptible to cancer, or is being tested for cancer. More specifically, the tissue is also a cancer tissue sample. "Cancer tissue" herein refers to tissue obtained from a subject with cancer and taken from an organ or tissue affected by cancer. In other implementations, the biological sample comes from a subject with the aforementioned cancers, particularly those with localized or metastatic breast cancer, ovarian cancer, bladder cancer, urothelial carcinoma, pancreatic cancer, and lung cancer or tumors.

[0072] As used herein, “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue” refers to a sample, cell, tissue, standard, or level used for comparative purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or disease-free portion (e.g., tissue or cells) of the same subject or individual. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be healthy and / or disease-free cells or tissue adjacent to diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or disease-free portion (e.g., tissue or cells) of an individual who is not a subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and / or cells of an individual who is not a subject or individual.

[0073] The term “detection” includes any means of detection, including direct and indirect detection. The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease, or condition (e.g., cancer). For example, “diagnosis” can refer to the identification of a specific type of cancer. “Diagnosis” can also refer to the classification of a specific subtype of cancer, for example, by histopathological criteria, or by molecular characteristics (e.g., a subtype characterized by the expression of one or a combination of biomarkers (e.g., a specific gene or a protein encoded by said gene)).

[0074] The terms “level of expression” or “expression level” are generally used interchangeably and typically refer to the amount of polynucleotides, mRNA, or amino acid products or proteins in a biological sample. “Expression” generally refers to the process of converting information encoded by a gene into a structure present and manipulated in the cell. Therefore, according to this disclosure, “expression” of a gene (e.g., the Nectin-4 gene) can refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translational modified proteins should also be considered expressed, whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of proteins, such as by proteolysis. In some embodiments, “level of expression” refers to the amount of protein (e.g., Nectin-4) in a biological sample as determined using methods known in the art or described herein, including but not limited to immunohistochemistry (IHC), Western blotting (e.g., protein blotting), immunofluorescence (IF), and flow cytometry (e.g., FACS). TM) or enzyme-linked immunosorbent assay (ELISA).

[0075] "Increased expression", "increased expression level", "increased level", "enhanced expression", "enhanced expression level" or "enhanced level" refers to an increased expression or increased level of a biomarker in an individual relative to a control, such as an individual without a disease or condition (e.g., cancer) or an internal control (e.g., a housekeeper biomarker).

[0076] "Reduced expression," "reduced expression level," "reduced level," "decreased expression," "reduced expression level," or "decreased level" refers to a reduced expression or reduced level of a biomarker in an individual relative to a control, such as an individual without a disease or symptom (e.g., cancer) or an internal control (e.g., a housekeeping biomarker). In some implementations, reduced expression is little or no expression.

[0077] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. A primary antibody is typically the first antibody used in an immunohistochemical assay. A primary antibody may be the only antibody used in an immunohistochemical assay. As used herein, the term "secondary antibody" refers to an antibody that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and subsequent reagents (if any). A secondary antibody is typically the second antibody used in an immunohistochemical assay.

[0078] Reference antibody mAb 5A12.2

[0079] The antibodies disclosed in this article include the reference mouse monoclonal antibody 5A12.2, which was produced by hybridoma and deposited on March 20, 2019, with the corresponding accession number CNCM I-5407 at the Microbial Culture Collection Center (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) in accordance with the provisions of the Budapest Treaty.

[0080] In a specific embodiment, the anti-Nectin-4 antibody of the present invention, preferably a humanized anti-Nectin-4 antibody, comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise six CDRs of antibody mAb 5A12.2, and the mAb 5A12.2 can be obtained from a hybridoma deposited in CNCM with accession number CNCM I-5407.

[0081] This disclosure also relates to any antibody that includes the corresponding VH and VL regions of the aforementioned reference antibody, particularly humanized antibodies.

[0082] This disclosure also relates to the hybridoma that can be obtained in CNCM with accession number CNCM I-5407.

[0083] mAb 5A12.2 includes:

[0084] -HCDR1 of SEQ ID NO:2, HCDR2 of SEQ ID NO:3, HCDR3 of SEQ ID NO:4, LCDR1 of SEQ ID NO:5, LCDR2 of SEQ ID NO:6, and LCDR3 of SEQ ID NO:7; and

[0085] - Variable heavy chain (VH) domain and variable light chain domain, which have at least 90% identity with the heavy chain and light chain of SEQ ID NO:8 and 9, respectively.

[0086] The reference antibody mAb 5A12.2 binds to the IgV-like distal domain (the domain used for immunizing mice) in the extracellular region of Nectin-4. Typically, this reference antibody does not cross-competitively bind to Nectin-4 with Ha22-2 mAb (Seattle Genetics) or any antibodies disclosed in WO 02017042210 and WO 2018158398 (particularly N41 mAb and 14A5.2 mAb), indicating that the antibodies of the present invention bind to different epitopes. Cross-competition assays can be performed as shown in the examples (see the Materials and Methods section).

[0087] Other antibodies disclosed herein include those having amino acids mutated through deletion, insertion, or substitution, but possessing at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% identity in the CDR region with the reference mAb 5A12.2. Typically, according to this disclosure, antibodies may have 1, 2, 3, or 4 amino acid changes (including deletions, insertions, or substitutions) in one or more CDRs compared to the CDR sequence of the reference antibody mAb 5A12.2.

[0088] In some embodiments, the antibody of this disclosure is a mutant variant of reference mAb 5A12.2 having 100% identical 6 CDR regions to the corresponding 6 CDR regions of said reference mAb 5A12.2, and said mutant variant antibody comprises a mutant amino acid sequence wherein no more than 1, 2, 3, 4 or 5 amino acids in the FR1, FR2, FR3 and FR4 regions have been mutated by amino acid deletion, insertion or substitution when compared with the corresponding framework regions of the reference antibody.

[0089] Functional variant antibodies

[0090] In yet another embodiment, the functional variant antibody of this disclosure has a full-length heavy chain and light chain amino acid sequence; or a variable region heavy chain and light chain amino acid sequence; or all six CDR region amino acid sequences that are homologous to or more specifically identical to the corresponding amino acid sequence of the antibody mAb 5A12.2 described above (as shown in SEQ ID NO: 2-7), and wherein said functional variant antibody retains the desired functional properties of the original mAb 5A12.2 antibody.

[0091] Referring to mAb 5A12.2, functional variants of the antibody, particularly VL, VH, or CDR used in the context of monoclonal antibodies of this disclosure, still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of its affinity (typically via K). D Or in conjunction with EC50 assessment, such as by flow cytometry, e.g. on cell lines expressing Nectin-4 such as cancer cell lines) and / or the selectivity of the parent antibody (e.g., mAb5A12.2), and in some cases, such monoclonal antibodies of this disclosure can bind with greater affinity, selectivity and / or specificity than the parent Ab (e.g., mAb 5A12.2).

[0092] In some embodiments, the anti-Nectin4 antibody, a functional variant of the reference mAb 5A12.2 antibody disclosed herein, has a variable heavy chain (VH) domain and a variable light chain domain having at least 90% identity with the amino acid sequences SEQ ID NO: 8 and 9, respectively.

[0093] The desired functional properties of the original mAb 5A12.2 antibody can be selected from the following group:

[0094] i. Its apparent affinity binding constant (Ki) is less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL, or less than 5 ng / mL (typically 0.05 ng-10 μg / mL, especially 0.1 ng-1 μg / mL). D (or EC50) binds to Nectin-4, especially human Nectin-4 bound to SEQ ID NO: 1;

[0095] ii. It binds to Nectin-4 in immunohistochemical (IHC) assays. More specifically, the antibody selectively binds to Nectin-4 in biological sample sections during IHC assays.

[0096] For example, this disclosure relates to a functional variant antibody of reference mAb 5A12.2, which comprises a variable heavy chain (V H ) and variable light chains (V LThe sequence, wherein the CDR sequence, i.e., the 6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 and the corresponding CDR sequences of reference mAb5A12.2 (as defined in SEQ ID NO: 2-7 respectively) share at least 60%, 70%, 90%, 95% or 100% sequence identity with the corresponding CDR sequences of reference mAb5A12.2, wherein the functional variant antibody specifically binds to Nectin-4, and the antibody exhibits at least one of the following functional properties:

[0097] (i) with an apparent affinity binding constant (Ki) of less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL, or less than 5 ng / mL (typically 0.05 ng-10 μg / mL, especially 0.1 ng-1 μg / mL). D (or EC50) combined with Nectin-4;

[0098] (ii) It binds to Nectin-4 in immunohistochemical (IHC) assays. More specifically, the antibody selectively binds to Nectin-4 in biological sample sections in IHC assays.

[0099] It further relates to a functional variant antibody of the mAb 5A12.2 reference antibody, comprising at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding heavy and light chain variable regions of the mAb 5A12.2 reference antibody (as shown in SEQ ID NO 8 and 9, respectively); the functional variant antibody specifically binds to Nectin-4 and exhibits at least one of the following functional properties:

[0100] (i) with an apparent affinity binding constant (Ki) of less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL, or less than 5 ng / mL (typically 0.05 ng-10 μg / mL, especially 0.1 ng-1 μg / mL). D (or EC50) combined with Nectin-4;

[0101] (ii) It binds to Nectin-4 in immunohistochemical (IHC) assays. More specifically, the antibody selectively binds to Nectin-4 in biological sample sections in IHC assays.

[0102] In various embodiments, the antibody may exhibit one or both of the desired functional properties described above. The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody. Preferably, the antibody or protein is a humanized human antibody, more preferably a humanized silent antibody.

[0103] As used herein, the term "silent" antibody refers to an antibody that shows no or low ADCC activity when measured in an in vitro ADCC activity assay that measures cell lysis of target cells.

[0104] In one embodiment, the term "no or low ADCC activity" means that the ADCC activity exhibited by the silent antibody is less than 50% of that observed by the corresponding wild-type (non-silent) antibody, such as wild-type human IgG1 antibody, for example, less than 10%. Preferably, no detectable ADCC activity is observed in the in vitro ADCC activity assay using the silent antibody compared to the control Fab antibody.

[0105] Silent effector functions can be acquired through mutations in the constant Fc region of an antibody and have been described in the art: Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 181(2008):6664-69, Strohl, CO Biotechnology 20(2009):685-91). Examples of silencing IgG1 antibodies include mutations that reduce ADCC at positions 234, 235, and / or 331 of the IgG1 Fc amino acid sequence (EU number). Another silencing IgG1 antibody contains the N297A mutation, which produces aglycone-free or non-glycosylated antibodies.

[0106] The sequence of a CDR variant may differ from the CDR sequence of the parent antibody sequence by most conserved substitutions, such as all or 10 or more substitutions in the variant, for example, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or more substitutions being conserved amino acid residue substitutions. In the context of this disclosure, a conserved substitution can be defined as a substitution within the amino acid class reflected as follows:

[0107] Aliphatic residues I, L, V, and M.

[0108] Cycloalkenyl-related residues F, H, W, and Y.

[0109] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0110] Negatively charged residues D and E.

[0111] Polar residues C, D, E, H, K, N, Q, R, S, and T.

[0112] Positively charged residues H, K, and R.

[0113] Small residues A, C, D, G, N, P, S, T, and V.

[0114] Very small residues A, G, and S.

[0115] The residues involved in the turn are A, C, D, E, G, H, K, N, Q, R, S, P, and the residue T involved in the formation.

[0116] Flexible residues Q, T, K, S, G, P, D, E, and R.

[0117] Further conserved substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Compared to the CDR of this reference antibody, the variant CDRs largely retain conservation in hydropathic / hydrophilic properties and residue weight / size. The importance of the hydropathic amino acid index in conferring biological functions of protein interactions is generally understood in the art. It is accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Based on their hydrophobic and charge characteristics, each amino acid was assigned a hydrophilic index, which are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also be measured, or alternatively, by a similarity score, such as using a BLAST procedure (e.g., BLAST 2.2.8, available from NCBI, using standard settings BLOSUM62, Open Gap = 11, and Extended Gap = 1). Suitable variants typically exhibit at least about 70% identity with the parent peptide. According to this disclosure, at least 70% identity between the first and second amino acid sequences means that the first and second sequences have 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity. According to this disclosure, at least 50% identity between the first amino acid sequence and the second amino acid sequence means that the first sequence and the second amino acid sequence have 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity.

[0118] In some embodiments, the antibodies disclosed herein are chimeric antibodies, typically chimeric mouse / human antibodies. The term "chimeric antibody" refers to a monoclonal antibody comprising the VH and VL domains of an antibody derived from a non-human animal, and the CH and CL domains of a human antibody. Any animal (e.g., mouse, rat, hamster, rabbit, etc.) can be used as the non-human animal. Specifically, the mouse / human chimeric antibody may comprise the VH and VL domains of this reference antibody.

[0119] In some embodiments, the antibody of this disclosure is a humanized antibody. In a specific embodiment, the antibody of this disclosure is a humanized antibody comprising the six CDRs of this reference antibody. As used herein, the term "humanized antibody" refers to an antibody in which the framework region (FR) has been modified to comprise the FR of a donor immunoglobulin from a different species (e.g., the human species) compared to the parental immunoglobulin (e.g., mouse CDR).

[0120] In some embodiments, the antibodies of this disclosure are selected from the group consisting of Fab, F(ab')2, Fab', and scFv. As used herein, the term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, wherein in a fragment obtained by treating IgG with the protease papain, about half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, wherein in a fragment obtained by treating IgG with the protease pepsin, the antigen-binding activity is slightly greater than that of Fab linked by disulfide bonds in the hinge region. The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of F(ab'). Single-chain Fv ("scFv") polypeptides are covalently linked VH:VL heterodimers, typically expressed by a gene fusion comprising VH and VL encoding genes linked by a linker encoding the peptide. The human scFv fragments disclosed herein include, preferably, CDRs that maintain an appropriate conformation through the use of gene recombination technology.

[0121] Functional variant antibodies with mutated amino acid sequences can be obtained by mutagenesis of the encoded nucleic acid molecule (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), followed by testing for the retained function of the encoded altered antibody using the functional assays described herein (i.e., the function described above).

[0122] Antibodies that cross-compete with reference mAb 5A12.2

[0123] Other antibodies possessing similar advantageous properties to the reference antibody mAb 5A12.2 disclosed herein can be identified based on their ability to cross-compete (e.g., competitively inhibit its binding) with the reference antibody mAb 5A12.2 in a statistically significant manner in a standard Nectin-4 competitive binding assay (see Reference 7).

[0124] In some implementations, the binding affinity of antibodies to Nectin-4 can be screened first, for example using phage display technology from a human recombinant antibody library or from transgenic mice expressing human variable region antibodies immunized with Nectin-4 antigen.

[0125] The ability of the test antibody to competitively bind to or inhibit the binding of the disclosed antibody to human Nectin-4 indicates that the test antibody can competitively bind to human Nectin-4; according to the non-limiting theory, such an antibody can bind to the same or related (e.g., structurally similar or spatially close) epitopes on human Nectin-4 with the competing antibody. As used herein, when a competitive antibody inhibits the binding of the disclosed antibody or antigen-binding fragment to Nectin-4 by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% in the presence of an equimolar concentration of competitive antibody, the antibody is considered to "competitively" bind.

[0126] In some embodiments, the antibody or antigen-binding fragment of this disclosure binds to one or more epitopes of Nectin-4. In some embodiments, the epitopes bound by the antibody or antigen-binding fragment of this invention are linear epitopes. However, the epitopes bound by the antibody or antigen-binding fragment of this invention are typically non-linear conformational epitopes.

[0127] In some embodiments, the antibodies according to this disclosure bind to epitopes different from those of Ha22-2mAb (Seattle Genetics) or any antibodies disclosed in WO2017042210 and WO2018158398 (especially N41 mAb and 14A5.2 mAb). Typically, the antibodies disclosed herein do not cross-competitively bind to Nectin-4 with Ha22-2 mAb (Seattle Genetics) or any antibodies disclosed in WO2017042210 and WO2018158398 (especially N41 mAb and 14A5.2 mAb). Cross-competition assays can be performed as shown in the examples (see Materials and Methods section).

[0128] In one embodiment, this disclosure provides an antibody that binds to the same epitope as the reference antibody of this disclosure (i.e., mAb 5A12.2).

[0129] The specific binding of the disclosed antibodies can be determined by any method known in the art. Many different competitive binding assays can be used for epitope binding. Immunoassays that can be used include, but are not limited to, competitive assay systems using techniques such as Western blotting, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, precipitin assay, gel diffusion precipitin assay, immunoradioassay, fluorescence immunoassay, protein A immunoassay, and complement fixation assay. Such assays are routine and well known in the art (see, for example, Ausubel et al., eds., 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

[0130] For example, to screen the ability of anti-Nectin-4 antibodies to bind to the same epitope or to compete with reference antibody mAb 5A12.2 for binding to cells (such as cancer cell lines expressing Nectin-4), a saturated concentration of the reference antibody can be used for staining. After washing, different doses of test anti-Nectin-4 mAbs are scanned to test their competitive potential against the reference antibody. mAbs competing with the reference antibody for the same binding site will not recognize Nectin-4 in the presence of this reference antibody. Data can be expressed as mean fluorescence intensity.

[0131] The antibodies disclosed herein can be produced using any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Typically, when the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the antibodies using standard techniques for producing peptides. For example, they can be synthesized using known solid-phase methods, preferably using commercially available peptide synthesis equipment (e.g., those manufactured by Applied Biosystems, Foster City, California) and according to the manufacturer's instructions. Alternatively, the antibodies disclosed herein can be synthesized using recombinant DNA techniques well known in the art. For example, antibodies as DNA expression products can be obtained after integrating the DNA sequence encoding the antibody into expression vectors and introducing these vectors into suitable eukaryotic or prokaryotic hosts to which the desired antibody will be expressed, and then isolating them using known techniques.

[0132] In one embodiment, this disclosure provides an isolated antibody that cross-blocks or is cross-blocked by reference mAb 5A12.2 to the binding of Nectin-4, wherein said antibody:

[0133] (i) with an apparent affinity binding constant (K0) less than 10 μg / mL, especially less than 1 μg / mL, less than 100 ng / mL, less than 10 ng / mL or less than 5 ng / mL. D (or EC50) combined with Nectin-4;

[0134] (ii) Binding of Nectin-4 in an immunohistochemical (IHC) assay. More specifically, the antibody selectively binds to Nectin-4 in biological sample sections during an IHC assay.

[0135] In a specific implementation, this disclosure provides an antibody that binds to the same epitope as the reference mAb 5A12.2 described herein.

[0136] In specific implementations, this disclosure provides antibodies that do not compete with Ha22-2mAb (Seattle Genetics) or any antibodies disclosed in WO2017042210 and WO2018158398 (especially N41 mAb and 14A5.2 mAb) for binding to Nectin-4.

[0137] Typically, antibodies that compete with reference mAb 5A12.2 according to this disclosure for binding to Nectin-4 retain at least a considerable proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of the affinity and / or selectivity of the reference antibody (e.g., mAb 5A12.2), and in some cases may be associated with greater affinity, selectivity, and / or specificity than the reference antibody (e.g., mAb 5A12.2).

[0138] In some implementations, the cross-blocking antibody that competes with reference mAb 5A12.2 for binding to Nectin-4 is a chimeric antibody, a humanized antibody, or a recombinant human antibody.

[0139] Production of monoclonal antibody-producing transfected tumors

[0140] The antibodies disclosed herein can be produced using any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Typically, when the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the antibodies using standard techniques for producing peptides. For example, they can be synthesized using known solid-phase methods, preferably using commercially available peptide synthesis equipment (e.g., those manufactured by Applied Biosystems, Foster City, California) and according to the manufacturer's instructions. Alternatively, the antibodies disclosed herein can be synthesized using recombinant DNA techniques well known in the art. For example, antibodies as DNA expression products can be obtained after integrating the DNA sequence encoding the antibody into expression vectors and introducing these vectors into suitable eukaryotic or prokaryotic hosts to which the desired antibody will be expressed, and then isolating them using known techniques.

[0141] Therefore, a further object of this disclosure relates to nucleic acid molecules encoding antibodies according to this disclosure as shown in Table 1. More specifically, the nucleic acid molecules encode the heavy or light chain of the antibody of this disclosure. More specifically, the nucleic acid molecules comprise a VH or VL coding region having at least 70%, 80%, 90%, 95%, or 100% identity with the corresponding nucleic acid encoding the heavy chain variable region (VH region) or light chain variable region (VL) of reference antibody 5A12.2.

[0142] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, granule, episome, artificial chromosome, bacteriophage, or viral vector. As used herein, the terms “vector,” “cloning vector,” and “expression vector” refer to a medium that can introduce a DNA or RNA sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Therefore, another object of the present invention relates to vectors containing the nucleic acids of this disclosure. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to induce or direct the expression of the antibody when administered to a subject. Examples of promoters and enhancers for expression vectors used in animal cells include the early promoter and enhancer of SV40, the LTR promoter and enhancer of Moloney mouse leukemia virus, the promoter and enhancer of immunoglobulin H chain, etc. Any expression vector for animal cells can be used, as long as it allows insertion and expression of a gene encoding the C region of a human antibody. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, etc. Other examples of plasmids include replication plasmids containing the origin of replication, or integration plasmids such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be produced using techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056, and WO 94 / 19478.

[0143] A further aspect of the invention relates to host cells transfected, infected, or transformed by nucleic acids and / or vectors as described above. As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. Host cells that accept and express the introduced DNA or RNA are "transformed."

[0144] The nucleic acids disclosed herein can be used to produce the antibodies disclosed herein in suitable expression systems. The term "expression system" refers to a host cell and compatible vector under suitable conditions, for example, used to express proteins encoded by exogenous DNA carried by the vector and introduced into the host cell. Common expression systems include *E. coli* host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include *E. coli*, *Kluyveromyces* or yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells with a defective dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al.; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells").

[0145] The present invention also relates to a method for producing recombinant host cells expressing antibodies according to the present disclosure, the method comprising the steps of: (i) introducing a recombinant nucleic acid or vector as described above into competent host cells in vitro or in vitro; (ii) culturing the obtained recombinant host cells in vitro or in vitro; and (iii) optionally, selecting cells that express and / or secrete the antibodies. These recombinant host cells can be used to produce the antibodies of the present invention.

[0146] The antibodies disclosed herein may be appropriately isolated from the culture medium using conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0147] In some embodiments, the human chimeric antibodies of this disclosure can be generated by: obtaining nucleic acid sequences encoding the VL and VH domains as described above; constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cells having genes encoding human antibody CH and human antibody CL; and expressing the encoding sequences by introducing the expression vector into animal cells. The CH domain, as a human chimeric antibody, can be any region belonging to human immunoglobulins, but those of the IgG class are suitable, and any subclass of IgG, such as IgG1, IgG2, IgG3, and IgG4, can also be used. Additionally, the CL domain, as a human chimeric antibody, can be any region belonging to Ig, and those of the κ or λ class can also be used. Methods for generating chimeric antibodies involve conventional recombinant DNA, and gene transfection techniques are well known in the art (see Morrison SL et al. (1984) and patent documents US5,202,238; and US5,204,244).

[0148] The humanized antibody of the present invention can be prepared by: obtaining nucleic acid sequences encoding the CDR domain as described above; constructing a humanized antibody expression vector by inserting them into an expression vector having genes encoding (i) the same heavy chain constant region and heavy chain variable frame region as human antibodies and (ii) the same light chain constant region and light chain variable frame region as human antibodies; and expressing the genes by introducing the expression vector into a suitable cell line. The humanized antibody expression vector can be of any type where the gene encoding the antibody heavy chain and the gene encoding the antibody light chain reside in different vectors, or where both genes reside in the same vector (tandem type). Tandem-type humanized antibody expression vectors are preferred in terms of ease of construction, ease of introduction into cell lines, and balance between the expression levels of the antibody H chain and L chain in the cell line. Examples of tandem-type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0149] Methods for generating humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-transplantation (EP 239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA et al. (1994)) and strand substitution (U.S. Patent No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0150] The Fab disclosed herein can be obtained by treating an antibody that specifically reacts with an AMH with a protease (papain). Alternatively, Fab can be produced by inserting DNA encoding the antibody Fab into a vector for a prokaryotic expression system or a eukaryotic expression system, and introducing the vector into a prokaryote or eukaryote (as appropriate) to express the Fab.

[0151] The F(ab')2 disclosed herein can be obtained by treating an antibody that specifically reacts with AMH with a protease (pepsin). Furthermore, F(ab')2 can be prepared by binding the following Fab' with a thioether bond or a disulfide bond.

[0152] The Fab' disclosed herein can be obtained by treating F(ab')2, which reacts specifically with AMH, with the reducing agent dithiothreitol. Alternatively, Fab' can be produced by inserting DNA encoding an antibody-encoding Fab' fragment into a prokaryotic or eukaryotic expression vector, and then introducing the vector into a prokaryote or eukaryote (as appropriate) for expression.

[0153] The scFv of this disclosure can be produced by: obtaining cDNA encoding the VH and VL domains as described above, constructing DNA encoding scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote (as the case may be) to express scFv.

[0154] To generate humanized scFv fragments, a well-known technique called CDR transplantation can be used, which involves selecting complementarity-determining regions (CDRs) from donor scFv fragments and transplanting them onto a human scFv fragment framework with a known three-dimensional structure (see, for example, WO98 / 45322, WO87 / 02671, US5,859,205, US5,585,089, US4,816,567, EP0173494).

[0155] The engineered antibodies disclosed herein include antibodies in which framework residues within the VH and / or VL are modified to, for example, improve antibody properties. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to “reverse mutate” one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence of the derived antibody. These residues can be identified by comparing the antibody framework sequence with the germline sequence of the derived antibody. To restore the germline conformation of the framework region sequence, the somatic mutation can be “reverse mutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such “reverse mutated” antibodies are also covered in this disclosure. Another type of framework modification involves mutating one or more residues within, or even one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0156] Fc Engineering

[0157] The antibodies disclosed herein may be characterized by one or more of the functional or structural features described above, or any combination of selected functional and structural features.

[0158] The antibodies of this invention can be any isotype. The choice of isotype is typically guided by the desired effector function (e.g., ADCC silencing). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Any of the human light chain constant regions, κ or λ, can be used. If desired, the class of the disclosed antibodies can be converted by known methods. Typically, class-switching techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Thus, for various therapeutic uses, the effector function of the disclosed antibodies can be altered by isotype conversion to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In some embodiments, the antibodies disclosed in this invention are full-length antibodies. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG4 antibody. In some embodiments, the Nectin-4 specific IgG4 antibody is a stable IgG4 antibody. Examples of suitable stabilized IgG4 antibodies are those in which the arginine at position 409 of the constant region of the human IgG4 heavy chain (as indicated in the EU index of Kabat et al. above) is replaced by lysine, threonine, methionine, or leucine, preferably lysine (described in WO 2006033386) and / or antibodies in which the hinge region contains the Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142.

[0159] In some embodiments, the antibodies of this disclosure do not contain an Fc portion that induces antibody-dependent cytotoxicity (ADCC). The terms “Fc domain,” “Fc portion,” and “Fc region” refer to the C-terminal segment of an antibody heavy chain, such as about amino acids (aa)230 to about aa450 from the human γ heavy chain or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε, and μ of human antibodies), or its naturally occurring allotypes. Unless otherwise stated, this disclosure uses the generally accepted Kabat amino acid numbering for immunoglobulins throughout (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD). In some embodiments, the antibodies of this disclosure do not contain an Fc domain capable of substantially binding to the FcgRIIIA (CD16) polypeptide. In some embodiments, the antibodies of this disclosure lack an Fc domain (e.g., lack CH2 and / or CH3 domains) or contain an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the antibodies of this disclosure comprise or are composed of Fab, Fab', Fab'-SH, F(ab')2, Fv, biantibodies, single-chain antibody fragments, or multispecific antibodies comprising or composed of multiple different antibody fragments. In some embodiments, the antibodies of this disclosure are not linked to a toxic moiety. In some embodiments, one or more amino acids selected from amino acid residues may be substituted with different amino acid residues, such that the antibody exhibits altered C2q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551.

[0160] Another modification of the antibodies contemplated in this disclosure is polyethylene glycol (PEG) oxidation. PEGylation of antibodies can, for example, increase their biological (e.g., serum) half-life. To PEGylate an antibody, typically, the antibody or a fragment thereof is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are linked to the antibody or antibody fragment. PEGylation can be carried out by acylation or alkylation with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG used to derive other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this disclosure. See, for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0161] Another modification of the antibody anticipated herein is to couple or fuse at least the antigen-binding region of the disclosed antibody with a serum protein (e.g., human serum albumin) or a fragment thereof to increase the half-life of the resulting molecule.

[0162] In some embodiments, this disclosure also provides multispecific antibodies. Exemplary forms of multispecific antibody molecules of this disclosure include, but are not limited to, (i) two antibodies cross-linked by chemical heterocoupling, one having specificity for Nectin-4 and the other for a second antigen; (ii) a single antibody comprising two distinct antigen-binding regions; (iii) a single-chain antibody comprising two distinct antigen-binding regions, such as two scFvs tandemly linked by an additional peptide linker; and (iv) a dual variable domain antibody (DVD-Ig), wherein each light chain and heavy chain contains two variable domains tandemly linked by short peptide bonds (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM(v) Chemically linked bispecific (Fab')2 fragments; (vi) Tandab, a fusion of two single-chain biantibodies, producing a tetravalent bispecific antibody with two binding sites against each target antigen; (vii) flexibody, a combination of scFv and biantibody, producing a multivalent molecule; (viii) a so-called "docking and locking" molecule, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, produces a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (ix) a so-called Scorpion molecule, comprising, for example, two scFvs fused to the two ends of a human Fab-arm; and (x) biantibody. Another exemplary form of bispecific antibody is an IgG-like molecule with complementary CH3 domains to force heterodimerization. This molecule can be prepared using known techniques, such as those known as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A / S) technologies. In some embodiments, the DuoBody technology is typically used, and bispecific antibodies are obtained or can be obtained via controlled Fab-arm exchange. In vitro methods for producing bispecific antibodies via controlled Fab-arm exchange are described in WO2008119353 and WO2011131746 (both Genmab A / S). In an exemplary method described in WO2008119353, a bispecific antibody is formed by an exchange of “Fab-arms” or “half-molecules” (exchange of the heavy chain and the linked light chain) between two monospecific antibodies, both containing an IgG4-like CH3 region, during incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms, which may contain different sequences.In another exemplary method described in WO2011131746, the bispecific antibody of this disclosure is prepared by a method comprising the steps of: a) providing a first antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a second CH3 region; wherein the sequences of the first and second CH3 regions are different, and such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between each of the first and second CH3 regions; c) incubating the first antibody and the second antibody together under reducing conditions; and d) obtaining the bispecific antibody, wherein the first antibody is an antibody of this disclosure and the second antibody has a different binding specificity, or vice versa. The reducing conditions may be provided, for example, by adding a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) may further include, for example, restoring the conditions to a non-reducing or less reducing state by removing the reducing agent (e.g., by desalting). Preferably, the sequences of the first and second CH3 regions are distinct, containing only a few fairly conserved asymmetric mutations, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction between each of the first and second CH3 regions. Further details regarding these interactions and how they are implemented are provided in WO2011131746, which is incorporated herein by reference in its entirety. The following are exemplary embodiments of combinations of these asymmetric mutations, optionally with one or both Fc-regions having the IgG1 type.

[0163] Antibody-drug conjugates

[0164] In some embodiments, the antibody of the present invention is conjugated to a therapeutic component, i.e., a drug. The therapeutic component may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleaved peptide, or a radioisotope. Such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”.

[0165] In some implementations, the antibody is conjugated to the cytotoxic moiety. The cytotoxic moiety may be selected, for example, from the group consisting of: paclitaxel; cytochalasin B; bacitracin D; ethidium bromide; emetine; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthradinone; microtubule inhibitors (such as maytansine) or their analogues or derivatives; antimitotic agents (such as monomethylolpropionate E or F) or their analogues or derivatives; salitonin 10 or 15. Or analogues thereof; irinotecan or analogues thereof; mitoxantrone; sclerosomycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; chachomycin or analogues or derivatives thereof; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, aminopyrimidine, hydroxyurea, asparaginase, gemcitabine or cladribine; alkyl Chemicals such as dichloromethyldiethylamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; docalamycin A, docalamycin SA, receramide (CC-1065) or their analogues or derivatives; antibiotics such as actinomycetes. Bleomycin, daunorubicin, doxorubicin, idarubicin, glabricin, mitomycin, mitoxantrone, proprammycin, benzomycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepine (PDB); diphtheria toxin and related molecules such as diphtheria A chain and its active fragments and hybrid molecules, ricin such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, shiga-like toxins such as SLTI, SLT II, SLT, IV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, aloin, saponins, sucralose root toxin, gellingin, absinthecin A chain, sucralose root toxin A chain, alpha-sarcin, Aleurites fordii protein, caryophyllin protein, American pokeweed protein (such as PAPI, PAPII and PAP-S), momordica charantia inhibitor, lacrimal, croton toxin, sapaonaria officinalis inhibitor, white tree toxin, mitogellin, localized aspergillin, phenolmycin and enoxacin toxin; ribonuclease (RNase); DNase I, staphylococcal endotoxin A; pokeweed antiviral protein; diphtheria toxin and Pseudomonas endotoxin.

[0166] In some embodiments, the antibody is conjugated to olipattin or its peptide analogs, derivatives, or prodrugs. Olipattin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and has anticancer (US 5663149) and antifungal activities (Pettit et al. (1998) Antimicrob. Agents and Chemother. 42:2961-2965). For example, olipattin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical olipattin derivatives include AFP, MMAF (monomethyl olipattin F), and MMAE (monomethyl olipattin E). Suitable olistatin and olistatin analogues, derivatives and prodrugs, as well as suitable adapters for conjugating olistatin to Abs, are described, for example, in U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0167] In some implementations, the antibody is conjugated to Mertansine (also known as emtansine or DM1) or its peptide analogs, derivatives, or prodrugs. Mertansine is a tubulin inhibitor, meaning it inhibits microtubule assembly by binding to tubulin.

[0168] In some implementations, the antibody is conjugated with pyrrolo[2,lc][l,4]-benzodiazepine (PDB) or its analogues, derivatives, or prodrugs. Suitable PDBs and PDB derivatives, as well as related techniques, are described, for example, in Hartley JA et al, Cancer Res 2010; 70(17):6849-6858; Antonow D. et al, Cancer J 2008; 14(3):154-169; Howard PWet et al, Bioorg Med ChemLett 2009; 19:6463-6466 and Sagnou et al, Bioorg Med ChemLett 2000; 10(18):2083-2086.

[0169] In some embodiments, the antibody is conjugated to a cytotoxic moiety selected from the group consisting of: anthracycline, maytansine, chachomycosis, docamycosis, resveratrol (CC-1065), salipodoxomil 10, salipodoxomil 15, irinotecan, monomethylolpropionate E, monomethylolpropionate F, PDB, or any analogue, derivative, or prodrug thereof.

[0170] In some embodiments, the antibody is conjugated with anthracycline or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with maytansine or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with chachiomycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with docalamycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with resveratrol (CC-1065) or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salivarius toxin 10 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salivarius toxin 15 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate E or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate F or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with pyrrolo[2,lc][1,4]-benzodiazepine (PDB) or its analogues, derivatives, or prodrugs. In some implementations, the antibody is conjugated with irinotecan or its analogues, derivatives, or prodrugs.

[0171] In some embodiments, the antibody is conjugated to a nucleic acid or a nucleic acid-related molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., siRNA molecule), or an immunostimulatory nucleic acid (e.g., a DNA molecule containing an immunostimulatory CpG motif). In some embodiments, the antibody is conjugated to an aptamer or a ribozyme.

[0172] In some implementations, antibodies are conjugated, for example, as fusion proteins to cleavage peptides (e.g., CLIP, magganin 2, melittin, cephalosporin, and P18).

[0173] In some implementations, the antibody is conjugated to cytokines such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansistatin, and TNFα.

[0174] In some embodiments, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody may be conjugated to a chelating agent linker (e.g., DOTA, DTPA, or thiacetam) that allows the antibody to complex with the radioisotope. The antibody may also, or optionally, contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include... 3 H, 14 C 15 N、 35 S, 90 Y、"Tc、 125 I, 131 I, 186 Re、 213 Bi、 225 Ac and 227 For therapeutic purposes, radioactive isotopes that emit beta or alpha particle radiation, such as 131I, 90Y, 211At, 212Bi, 67Cu, 186Re, 188Re, and 212Pb, can be used.

[0175] The techniques used to conjugate molecules with antibodies are well known in the art (see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy” in Monoclonal Antibodies And Cancer Therapy (edited by Reisfeld et al., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery” in Controlled Drug Delivery (edited by Robinson et al., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies '84: Biological And Clinical Applications (edited by Pinchera et al., 1985); and “Analysis, Results, and Future Prospective of the Therapeutic Use” in Monoclonal Antibodies For Cancer Detection And Therapy (edited by Baldwin et al., Academic Press, 1985). "of Radiolabeled Antibody In Cancer Therapy"; and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, for example, PCT Publication WO 89 / 12624). Typically, nucleic acid molecules are covalently linked to lysine or cysteine ​​residues on the antibody via N-hydroxysuccinimide or maleimide functional groups, respectively.It has been reported that using engineered cysteine ​​or incorporating non-natural amino acids can improve the homogeneity of conjugates (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K. et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101–16106.; Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G. et al. (2010). Engineered thio-trastuzumab-DM1 conjugate with An improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769–4778.). Junutula et al. (2008) developed a cysteine-based site-specific conjugate called “THIOMAB” (TDC), which reportedly showed an improved therapeutic index compared to conventional conjugation methods. The conjugation of non-natural amino acids incorporated into antibodies with ADCs has been explored; however, the generality of this approach has not been determined (Axup et al., 2012). In particular, those skilled in the art can also envision Fc-containing peptides engineered with tags containing acyl donor glutamine (e.g., peptide tags or Q-tags containing Gin) or with endogenous glutamine that makes the peptide reactive through peptide engineering (e.g., through amino acid deletion, insertion, substitution, or mutation on the peptide). Then, transglutaminase can be covalently crosslinked with an amine donor (e.g., a small molecule containing or linked to a reactive amine) to form a stable and homogeneous group of engineered Fc-containing peptide conjugates, wherein the amine donor is specifically conjugated to the Fc-containing peptide via a tag containing an acyl donor glutamine or an accessible / exposed / reactive endogenous glutamine site (WO 2012059882).

[0176] Pharmaceutical Composition

[0177] In another aspect, this disclosure provides compositions, such as pharmaceutical compositions, containing at least one antibody disclosed herein formulated with a pharmaceutically acceptable carrier. Such compositions may comprise one antibody or (e.g., two or more different) combinations of antibodies as described above. The pharmaceutical compositions disclosed herein may also be administered in combination therapy, i.e., in combination with other agents.

[0178] For example, the antibodies of this disclosure are typically combined with at least one antiviral agent, anti-inflammatory agent, or another antiproliferative agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section concerning the use of the antibodies of this disclosure.

[0179] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous administration.

[0180] Depending on the route of administration, active compounds (i.e., antibodies) can be coated with materials to protect them from acids and other natural conditions that can inactivate them. The form, route of administration, dosage, and regimen of a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, among other factors.

[0181] The pharmaceutical compositions disclosed herein can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular application.

[0182] Chimeric antigen receptor (CAR)

[0183] The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of the antibody disclosed herein. Typically, the chimeric antigen receptor comprises at least one VH and / or VL sequence of the antibody disclosed herein. The chimeric antigen receptor disclosed herein further comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0184] As used herein, the term "chimeric antigen receptor" or "CAR" has its general meaning in the art as referring to an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. CARs are characterized by the ability to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner by utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition provides CAR-expressing T cells with the ability to recognize antigens independently of antigen processing, thereby bypassing major mechanisms of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR).

[0185] In some embodiments, this disclosure provides a CAR comprising an antigen-binding domain, said antigen-binding domain comprising, or consisting of, or substantially comprising a single-chain variable fragment (scFv) of an N41 mab antibody. In some embodiments, the antigen-binding domain comprises a adaptor peptide. The adaptor peptide may be located between a light chain variable region and a heavy chain variable region.

[0186] In some implementations, the CAR comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from CD28, 4-1BB, and CD3ζ intracellular domains. CD28 is an important T cell marker in T cell co-stimulation. 4-1BB delivers effective co-stimulatory signals to T cells, promoting T lymphocyte differentiation and enhancing their long-term survival. CD3ζ binds to the TCR to generate signals and contains an activation motif (ITAM) based on the tyrosine of the immune receptor.

[0187] In some embodiments, the chimeric antigen receptor of this disclosure may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized by, for example, disulfide bridging, or converted into an acid addition salt and / or optionally dimerized or polymerized.

[0188] This disclosure also provides nucleic acids encoding the chimeric antigen receptors of this disclosure. In some embodiments, the nucleic acid is integrated into a vector as described above.

[0189] This disclosure also provides a host cell containing nucleic acid encoding the chimeric antigen receptor of this disclosure. Although the host cell can be any cell type, can be derived from any type of tissue, and can be at any developmental stage, the host cell is, for example, a T cell isolated from peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). In some embodiments, the T cell can be any T cell, such as cultured T cells, like primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. If obtained from mammals, the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cell can be any type of T cell and can be at any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells such as Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, etc. The T cell can be CD8+ T cells or CD4+ T cells.

[0190] The T cell populations prepared as described above can be used in methods and compositions of adoptive immunotherapy according to known techniques, or in variations thereof that are obvious to those skilled in the art based on this disclosure. See, for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; also see U.S. Patent No. 4,690,915 by Rosenberg. Adoptive immunotherapy for cancer refers to a treatment method in which immune cells with anti-tumor responsiveness are administered to a host carrying a tumor, with the aim of directly or indirectly mediating the regression of the established tumor. Infusion of lymphocytes, particularly T lymphocytes, falls into this category. Currently, most adoptive immunotherapies are autologous lymphocyte therapy (ALT), which uses the patient's own immune cells for treatment. These therapies involve treating the patient's own lymphocytes to enhance immune cell-mediated responses or to recognize specific antigens or foreign substances in the body, including cancer cells. Treatment is accomplished by removing the patient's lymphocytes and exposing these cells in vitro to biologics and drugs to activate the cells' immune function. Once the autologous cells are activated, these ex vivo activated cells are re-infused into the patient to enhance the immune system, thereby treating the cancer. In some implementations, cells are formulated by first harvesting them from their culture medium, then washing and concentrating them in a therapeutically effective amount in a suitable medium and container system (“pharmaceutically acceptable” carrier). Suitable infusion media can be any isotonic formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% glucose solution or Ringer's lactate may also be used. The infusion media may be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition depends on the relative representativeness of T cells with the desired specificity, the recipient's age and weight, the severity of the targeted disease, and the immunogenicity of the targeted Ag. These cell amounts can be as low as approximately 10-1. 3 / kg, preferably 5×10 3 / kg; up to 10 7 / kg, preferably 10 8 / kg. The cell count will depend on the end use of the composition, as well as the cell types contained therein. For example, if cells specific to a particular Ag are required, the population will contain more than 70%, typically more than 80%, 85%, and 90-95% of such cells. For the uses described herein, the cell volume is typically 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. A clinically relevant number of immune cells can be spread across multiple infusions that accumulate to equal or exceed the total number of cells required.

[0191] In particular, the cells of this disclosure are especially suitable for treating cancer. Therefore, another object of this disclosure relates to a method of treating cancer in a subject in need, comprising administering a therapeutically effective amount of the cell population of the present invention to the subject.

[0192] Uses and methods of the present invention

[0193] The antibodies or proteins disclosed herein have diagnostic and therapeutic uses both in vitro and in vivo. For example, these molecules can be administered to cells in a culture, either in vitro or in vivo, or to a subject, either in vivo, to treat, prevent, or diagnose a disease.

[0194] The method is particularly suitable for the treatment, prevention, or diagnosis of Nectin-4 related diseases, and is especially suitable for the treatment, prevention, or diagnosis of cancer.

[0195] This disclosure also relates to methods for preparing medicaments for the prevention or treatment of cancer, said medicaments comprising the anti-Nectin-4 antibody or CAR described in the preceding sections.

[0196] As used herein, “Nectin-4 related disorders” includes disorders and / or diseases associated with or characterized by abnormal Nectin-4 expression levels. In some embodiments, “Nectin-4 related disorders” includes disorders involving cells expressing Nectin-4.

[0197] In some embodiments, this disclosure provides a method for killing cells expressing Nectin-4 by contacting cells with the antibody of this disclosure. In some embodiments, this disclosure provides a method for killing cells expressing Nectin-4 by contacting the cells with the antibody of this disclosure in the presence of effector cells capable of inducing an Fc-mediated effector cell response (such as a CDC, ADCC, or ADCP response). In this embodiment, the antibody is typically full-length and is an isotype that causes a CDC or ADCC response, such as an IgG1 isotype. In some embodiments, this disclosure provides a method for killing cells expressing Nectin-4 by contacting cells with the ADC of this disclosure.

[0198] In some embodiments, the antibodies of this disclosure are particularly suitable for treating cancer. Cancer cells overexpressing Nectin-4 are actually good targets for the antibodies of this disclosure because each cell can bind more antibodies. Therefore, in one aspect, a condition involving cells expressing Nectin-4 is cancer, i.e., a neoplastic condition, such as a condition characterized by the presence of tumor cells expressing Nectin-4, including, for example, conditions in which the cells originate from solid tumors or hematologic malignancies. In particular, the antibodies of this disclosure can be used to treat hyperproliferative diseases associated with Nectin-4 expression, overexpression, or activation. In particular, the antibodies of this disclosure are particularly suitable for treating breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, and lung cancer or cervical cancer. The term “breast cancer” as used herein includes, but is not limited to, all types of breast cancer at all stages of development, such as metastatic breast cancer or cervical cancer. In particular, breast cancer is selected from triple-negative breast cancer (TNBC), which is distinguished by negative immunohistochemical staining for estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2), and accounts for 15% of all breast cancers. The term “ovarian cancer” as used herein includes, but is not limited to, all types of ovarian cancer at all stages of development, such as metastatic ovarian cancer or cervical cancer. The term “lung cancer” as used in this article includes, but is not limited to, all types of lung cancer at all stages of development, such as metastatic lung cancer, non-small cell lung cancer, or small cell lung cancer.

[0199] In some implementations, the antibodies disclosed herein are particularly suitable for treating metastatic cancers.

[0200] As used herein, the term “treatment” refers to preventive or prophylactic treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having the disease, and subjects who are ill or diagnosed with the disease or medical condition, including suppression of clinical relapse. Treatment may be administered to subjects with a medical condition or who may eventually have the condition to prevent, cure, delay the onset of one or more symptoms of the condition or relapse, reduce the severity of one or more symptoms of the condition or relapse, or alleviate one or more symptoms of the condition or relapse, or to prolong the subject’s survival beyond what would be expected without such treatment. “Treatment regimen” refers to a mode of treatment for the disease, such as the dosage pattern used during treatment. Treatment regimens may include induction regimens and maintenance regimens. The phrase “induction regimen” or “induction period” refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of the disease. The general goal of an induction regimen is to provide the subject with a high level of the drug at the initial stage of the treatment regimen. An induction regimen may take the form of a (partial or complete) “loading regimen,” which may include administering a larger dose of the drug than the physician would use during a maintenance regimen, administering the drug more frequently than the physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a subject's remission during disease treatment, such as maintaining the subject's remission for a long period (months or years). Maintenance regimens can be continuous treatment (e.g., administration of medication at regular intervals (e.g., weekly, monthly, yearly, etc.)) or intermittent treatment (e.g., treatment interruption, intermittent treatment, relapse treatment, or treatment to achieve specific predetermined criteria (e.g., disease manifestations, etc.)).

[0201] As used herein, the term "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dose and time period. Therapeutic effective amounts of the disclosed antibodies can vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the disclosed antibodies to elicit the desired response in the individual. Therapeutic effective amounts are also the amounts in which the beneficial therapeutic effect exceeds any toxic or harmful effects of the antibody or antibody fraction. The effective dose and dosing regimen of the disclosed antibodies depend on the disease or condition to be treated and can be determined by those skilled in the art. A physician with ordinary skill in the art can readily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician may begin the use of the disclosed antibody in the pharmaceutical composition at a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Typically, the appropriate dose of the disclosed composition will be such that the amount of the compound is the minimum dose that effectively produces a therapeutic effect according to a specific dosing regimen. This effective dose typically depends on the factors described above. For example, a therapeutically effective amount for therapeutic purposes can be measured by its ability to stabilize disease progression. Typically, the ability of a compound to treat autoimmune diseases is evaluated, for example, in animal model systems used to predict the efficacy of treatments for autoimmune diseases. Alternatively, this property of the composition can be assessed by examining the compound's ability to inhibit an induced immune response using in vitro assays known to a skilled physician. A therapeutically effective amount of the therapeutic compound can reduce an immune or inflammatory response, or alleviate symptoms in a subject. Those skilled in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen. Exemplary, non-limiting ranges for the therapeutically effective amount of the antibody disclosed herein are about 0.1-100 mg / kg, for example about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, for example about 0.1-10 mg / kg, for example about 0.5, about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. Exemplary, non-limiting ranges for the therapeutically effective amount of the antibody disclosed herein are 0.02-100 mg / kg, for example about 0.02-30 mg / kg, for example about 0.05-10 mg / kg, or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. It can be administered, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously, such as near the target site. The dosage regimen in the above-described treatment methods and uses is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased depending on the urgency of the treatment situation. In some embodiments, therapeutic efficacy is monitored during treatment, for example, at predefined time points.In some embodiments, efficacy can be monitored by visualization of the disease area or by other diagnostic methods further described herein, such as by performing one or more PET-CT scans using labeled antibodies, fragments derived from antibodies of this disclosure, or small antibodies. If desired, the effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more sub-dose (optionally, in unit dosage form) at appropriate intervals throughout the day. In some embodiments, the potent monoclonal antibody of this disclosure is administered by a slow, continuous infusion over a prolonged period, such as more than 24 hours, to minimize any undesirable side effects. Effective doses of the antibody of this disclosure can also be administered using dosing periods of weekly, bi-weekly, or bi-weekly. Dosing periods can be limited to, for example, 8 weeks, 12 weeks, or until clinical progress is established. As a non-limiting example, on at least one day of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 after the start of treatment, or on at least one week of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, every 24, 12 A single or divided dose, or any combination thereof, may be administered over a daily dose of the antibody disclosed herein at amounts of approximately 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg.

[0202] This disclosure also provides therapeutic applications in which the antibody of this disclosure is used in combination with at least one other therapeutic agent associated with the disease or condition to be treated as described above. This administration can be simultaneous, separate, or sequential. For simultaneous administration, the agents can be administered as a composition or as separate compositions as needed. Other therapeutic agents are typically associated with the condition to be treated. Exemplary therapeutic agents include other anticancer antibodies or ADCs, cytotoxic agents, immunotherapies, chemotherapeutic agents, antiangiogenic agents, anticancer immunogens, cell cycle regulators / apoptotic regulators, hormone regulators, and other agents listed below.

[0203] Typically, the antibodies of this disclosure are administered to a subject in the form of pharmaceutical compositions comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. For administration to a patient, the compositions are formulated for administration to a patient. The compositions of this disclosure can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable cartridge. The techniques used herein include subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion. The sterile injectable form of the compositions disclosed herein can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, such as 1,3-butanediol solutions. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are commonly used as solvents or suspension media. For this purpose, any mild fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acid and its glycerol derivatives, can be used to prepare injectable formulations, similar to naturally occurring pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, commonly used in the preparation of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of this disclosure can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of oral tablets, common carriers include lactose and corn starch. Typically, lubricants, such as magnesium stearate, are also added. For oral administration in capsule form, useful diluents include, for example, lactose. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may also be added. Alternatively, the compositions of this disclosure can be administered in the form of suppositories for rectal administration.These can be prepared by mixing the reagent with a suitable, non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thus melting in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of this disclosure can also be applied topically, particularly when the therapeutic target includes areas or organs easily accessible for topical application, including eye, skin, or lower intestinal diseases. Suitable topical formulations are readily prepared for each of these areas or organs. For topical application, the compositions can be formulated into suitable ointments containing an active ingredient suspended or dissolved in one or more carriers. Carriers for topical application of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the compositions can be formulated into suitable lotions or creams containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water. Topical application to the lower intestine can be performed using rectal suppositories (see above) or suitable enema formulations. Patches may also be used. The compositions disclosed herein can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulations and can be prepared as saline solutions using benzyl alcohol or other suitable preservatives, bioavailability enhancers, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0204] For example, the antibody present in the pharmaceutical composition of this disclosure can be provided at a concentration of 10 mg / mL in a single-use 100 mg (10 mL) or 500 mg (50 mL) vial. The product is formulated for intravenous administration: 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An exemplary suitable dose range for the antibody in the pharmaceutical composition of this disclosure is about 1 mg / mL. 2 -500mg / m 2 However, it should be understood that these details are exemplary and that optimal details and protocols may be tailored to take into account the affinity and tolerability of a particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of this disclosure for injection (e.g., intramuscular or intravenous injection) can be prepared containing sterile buffered water (e.g., 1 ml for intramuscular injection) and about 1 ng to about 100 mg, such as about 50 ng to about 30 mg, or more preferably about 5 mg to about 25 mg of the anti-Nectin-4 antibody of this disclosure.

[0205] Diagnostic and prognostic methods

[0206] In one embodiment, this disclosure relates to a method for detecting the presence or expression level of Nectin-4 in a biological sample from a subject, said method comprising: contacting the biological sample with an anti-Nectin-4 antibody disclosed herein and detecting the presence of the bound antibody.

[0207] Typically, detection is performed via immunohistochemistry.

[0208] Biological samples are typically as described above, especially fixed tissues, and more particularly FFPE tissues.

[0209] The subjects are typically as described above.

[0210] As used herein, the term "detection" includes both quantitative and qualitative detection. Therefore, this disclosure includes detecting the presence and / or expression level of Nectin-4 in a sample. Selective binding of antibodies to Nectin-4 is established, in particular, by the presence of membrane staining, since Nectin-4 is a membrane protein.

[0211] In some implementations, the anti-Nectin-4 antibody provided herein can be used to detect the presence of Nectin-4 in biological samples.

[0212] In one embodiment, a Nectin-4 antibody disclosed herein is provided for use in diagnostic or detection methods. In another instance, this disclosure provides the use of an anti-Nectin-4 antibody as previously described in the preparation of reagents for diagnostic or detection methods.

[0213] In one embodiment, for example, a method for detecting the presence of Nectin-4 in a biological sample as described below is provided. In some embodiments, the method includes contacting a biological sample with the Nectin-4 antibody described herein under conditions that allow the anti-Nectin-4 antibody to bind to Nectin-4, and detecting whether a complex is formed between the anti-Nectin-4 antibody and Nectin-4. This method can be an in vitro or in vivo method. The anti-Nectin-4 antibody disclosed herein can be used, for example, in immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), Western blotting (e.g., Western blotting), flow cytometry (e.g., FACS). TM ) and enzyme-linked immunosorbent assay (ELISA). More specifically, the immunoassay is IHC, and the biological sample is fixed tissue, especially FFPE.

[0214] In one embodiment, the anti-Nectin-4 antibody is used to select subjects suitable for treatment with an anti-Nectin-4 antibody (particularly the same anti-Nectin-4 antibody), typically where Nectin-4 is a biomarker used for patient selection. This disclosure also provides the use of the anti-Nectin-4 antibody in a method for diagnosing subjects with a condition (e.g., cancer), the method comprising: determining the presence or expression level of Nectin-4 in the sample by contacting a sample obtained from the subject with the anti-Nectin-4 antibody described above herein and detecting the presence of the bound antibody.

[0215] For example, the method provides the use of anti-Nectin-4 antibodies in diagnosing a subject with cancer, the method comprising: determining the presence or expression level of Nectin-4 in a sample obtained from the subject by contacting the sample with the anti-Nectin-4 antibody described earlier herein and detecting the presence of the bound antibody. In some embodiments, the sample is selected from the group consisting of tissue samples, whole blood samples, serum samples, and plasma samples. More specifically, the sample is a tissue sample, particularly a fixed tissue sample. In some cases, the tissue sample is a tumor sample.

[0216] This disclosure also provides the use of a Nectin-4 antibody in a reagent for preparing a method for diagnosing a subject with a condition (e.g., cancer), said method comprising: determining the presence or expression level of Nectin-4 in a sample obtained from a subject by contacting a sample with the anti-Nectin-4 antibody disclosed herein and detecting the presence of the bound antibody. In some embodiments, the sample is selected from the group consisting of tissue samples, whole blood samples, serum samples, and plasma samples. More specifically, the sample is a tissue sample, particularly a fixed tissue sample. In some cases, the tissue sample is a tumor sample.

[0217] In another embodiment, this disclosure provides a method for identifying a subject suffering from a condition (e.g., cancer) that may respond to treatment, the method comprising: determining the presence or expression level of Nectin-4 in a sample obtained from the subject by contacting a sample with an anti-Nectin-4 antibody disclosed herein and detecting the presence of the bound antibody, wherein the presence or expression level of Nectin-4 in the sample indicates that the subject may respond to treatment.

[0218] In one embodiment, this disclosure also provides a method for predicting the responsiveness of an individual with cancer to treatment with an anticancer therapy. Typically, the anticancer therapy comprises an anti-Nectin-4 antibody as described herein (e.g., conjugated to a cytotoxic portion) or a chimeric antigen receptor (CAR) as previously defined herein. The method includes determining the presence or expression level of Nectin-4 in a sample obtained from a subject by contacting a sample with the anti-Nectin-4 antibody disclosed herein and detecting the presence of the bound antibody, wherein the presence or expression level of Nectin-4 in the sample indicates that the subject is more likely to respond to the anticancer therapy. In some embodiments, the sample is selected from the group consisting of tissue samples, whole blood samples, serum samples, and plasma samples. More specifically, the sample is a tissue sample, particularly a fixed tissue sample. In some cases, the tissue sample is a tumor sample.

[0219] This disclosure also covers methods for monitoring the efficacy of treatment in patients receiving anti-Nectin-4 antibodies or CARs as defined herein. The methods include measuring the expression level (or concentration) of Nectin-4 protein in a patient's biological sample at two or more time points. A measurement of a higher Nectin-4 protein expression level (typically assessed by measuring the concentration of Nectin-4 in the biological sample) at a later time point compared to a value obtained in the patient's biological sample at an earlier time point (and thus typically used as a reference value) indicates that the patient is unresponsive to treatment. A measurement of a lower Nectin-4 protein expression level (or Nectin-4 concentration) indicates that the patient is responsive to treatment. Measuring equal Nectin-4 protein levels (or Nectin-4 concentrations) at two or more time points indicates a Nectin-4-related disease, typically cancer, that is not developing in the patient (i.e., stable).

[0220] More specifically, in the embodiments of this disclosure as described above, the anti-Nectin-4 antibody of this disclosure can be used, along with IHC and staining protocols, to determine the presence and / or expression level / amount of Nectin-4 in a sample. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence of proteins in a sample. In one embodiment, the expression level of Nectin-4 is determined using a method comprising: (a) performing IHC analysis on a sample (e.g., a tumor sample obtained from a subject) with the anti-Nectin-4 antibody disclosed herein; and (b) determining the presence and / or expression level of Nectin-4 in the sample. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line staining sample, a tissue sample from a non-cancerous patient, or a reference sample known to have a predetermined Nectin-4 expression level).

[0221] IHC can be performed in combination with other techniques, such as morphological staining and / or in situ hybridization (e.g., FISH). Two general methods of IHC are available: direct and indirect assays. According to the first assay, the binding of an antibody to a target antigen is directly measured. This direct assay uses labeled reagents, such as fluorescently labeled or enzyme-labeled primary antibodies, which can be visualized without further antibody-antibody interactions. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is conjugated with an enzyme label, a chromogenic or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

[0222] Primary and / or secondary antibodies used for IHC typically have detectable labeling. Many labels are available, generally falling into the following categories: (a) radioactive isotopes, such as... 35 S, 14 C 125 1. 3 H and 131 1; (b) colloidal gold particles; (c) fluorescent labels, including but not limited to rare earth chelates (europium chelates), Texas red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercially available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and / or any one or more derivatives thereof; (d) various enzyme-substrate labels are available, and a review of some of them is provided in U.S. Patent No. 4,275,149. Examples of enzyme-labeled enzymes include luciferases (e.g., firefly luciferase and bacterial luciferase; see, for example, U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with catalase peroxidase as a substrate; alkaline phosphatase (AP) with p-nitrophenyl ester as a chromogenic substrate; and β-D-galactosidase (β-D-gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorescent substrate (e.g., 4-methylumbellatus-D-galactosidase). For a review of these, see, for example, U.S. Patents 4,275,149 and 4,318,980.

[0223] As previously mentioned, the detection of soluble Nectin-4 in the serum of cancer patients, particularly those with breast cancer, is associated with poor prognosis. Furthermore, Nectin-4 levels have been shown to increase with metastasis and decrease after treatment (Reference 1). Therefore, in some embodiments, this disclosure covers methods for assessing the clinical outcomes of subjects with cancer. These methods include determining the presence or expression level of Nectin-4 in a sample by contacting a sample obtained from the subject with the anti-Nectin-4 antibody described earlier herein and detecting the presence of the bound antibody. Typically, the detection of Nectin-4 and / or the detection of Nectin-4 expression levels in the sample above a reference level indicates a poor prognosis. Alternatively, the absence of Nectin-4 in the sample or the detection of Nectin-4 levels below a reference value indicates a favorable prognosis.

[0224] This disclosure also includes a method for treating cancer, comprising (i) a diagnostic step as defined above, and (ii) administering an antibody as defined above, particularly an antibody conjugated to a cytotoxic portion, if Nectin-4 is detected and / or if the level of Nectin-4 is greater than or equal to a reference value, and / or administering a chimeric antigen receptor as defined above or a composition comprising thereto.

[0225] In particular, this disclosure includes methods for treating patients who have or are suspected of having cancer, comprising:

[0226] Step 1), which assesses Nectin-4 expression in tissue samples from subjects suspected of having cancer, or assesses the responsiveness of said cancer patients to anticancer treatments, includes:

[0227] (1a) Contact the tissue sample with the antibody or its antigen-binding fragment disclosed herein;

[0228] (1b) Detecting the binding of the antibody or its antigen-binding fragment to the tissue sample; and

[0229] (1c) Determining the expression of Nectin-4 in the tissue sample, wherein the expression level of Nectin-4 in the tissue sample is compared with a reference expression level of Nectin-4; and

[0230] Step 2), that is, when an increased Nectin-4 expression level is observed compared to the reference, the anticancer therapeutic agent is administered to the patient;

[0231] Optionally, the expression level of Nectin-4 was detected using immunohistochemistry (IHC), Western blotting, fluorescence-activated cell sorting (FACS), or enzyme-linked immunosorbent assay (ELISA).

[0232] Optionally, the anticancer therapeutic agent comprises an anti-Nectin-4 antibody or an antibody-drug conjugate of an anti-Nectin-4 antibody, typically an antibody or a variant thereof according to this disclosure.

[0233] In the methods described above, reference values ​​can be selected as defined above. The samples defined herein can also be obtained as previously stated. The term cancer in this document means as previously defined, particularly as defined in this section.

[0234] This disclosure will be further illustrated by the following drawings and embodiments. However, these embodiments and drawings should not be construed in any way as limiting the scope of this disclosure.

[0235] Table 1: Sequences of this disclosure:

[0236] The CDRs in the table below are numbered according to the IMGT nomenclature.

[0237]

[0238] Attached Figure Description

[0239] Figure 1 : Competitive determination of 5A12.2 binding to nectin-4.

[0240] A: The binding of 5A12.2-HRP mAb to recombinant nectin-4 protein was measured by ELISA in the presence of other nectin-4 mAbs at different concentrations. No cross-inhibition was detected.

[0241] B: A table summarizing the competition between anti-nectin-4 mAbs. Black: mAb competition. Gray: No mAb competition. The 5A12.2 tablet differs from N41 mAb, 14A5.2, and Ha22.2.

[0242] Figure 2 Epigenetic affinity binding constant (K) D Determination of the value:

[0243] The binding of MAb to nectin-4 expressed on the cell surface of SUM190 tumor cells was detected. The epigenetic affinity binding constant (Kb) was calculated using Graph PadPrism software. D The F-Max value corresponds to the maximum fluorescence intensity (au) measured by FACS analysis.

[0244] Figure 3 Cell cytotoxicity analysis:

[0245] The ADC activity against Nectin-4 mAb was evaluated on the SUM190 tumor cell line using the mab-ZAP kit from ATS-bio. IC50 was calculated using Graph Pad Prism software.

[0246] Figure 4 IHC staining of nectin-4 in samples from patients with primary triple-negative breast cancer:

[0247] FFPE tissues were stained with 5A12.2 mAb as described in Materials and Methods. Nectin-4 expression was scored (Quick Score, QS) by multiplying the percentage of positive cells (P) by the intensity (I). Formula: QS = P x I. Maximum score was 300. Left; T1: Patient tumor 1, T2: Patient tumor 2. Right: Membrane expression of Nectin-4 as expected (arrow). Detailed Implementation

[0248] Materials and methods

[0249] 5A12.2 Selection of Hybridoma

[0250] Recombinant human Nectin-4-Fc protein was used as an immunogen to immunize Balb / C mice. This protein consists of the distal IgV domain of the extracellular region of Nectin-4. Immunized spleen cells were fused with mouse X63Ag8.653 myeloma cells. Hybridoma supernatants (~2000) were screened by FACS on Nectin-4-transfected and untransfected cells. The affinity and fluorescence intensity of hybridomas producing Nectin-4 antibodies were further screened and compared with previous Nectin-4 antibodies. 5A12.2 was selected as an antibody with improved properties.

[0251] Competition Measurement

[0252] Mab competition assay was performed by ELISA. A 96-well tray was coated overnight at +4 °C with 0.125 μg / ml recombinant nectin4VCC-Fc protein. Binding of peroxidase-conjugated 5A12.2 mAb (0.55 μg / ml 5A12.2-HRP) was measured in the presence of four different anti-nectin-4 mAbs (5A12, N41, 14A5, and Ha22-2 mab) at variable concentrations.

[0253] Flow cytometry:

[0254] FACS analysis was performed on the SUM190 breast cancer cell line using a specified concentration of nectin-4 monoclonal antibody. The cells were then stained with phycoerythrin-conjugated goat anti-mouse antibody (Beckman-Coulter).

[0255] Epigenetic affinity binding constant (K D ) Value and maximum fluorescence intensity determination :

[0256] K was determined by FACS analysis using serially diluted monoclonal antibodies. D Values. Graph Pad Prism is used for precise measurements. Maximum fluorescence intensity is measured via FACS analysis.

[0257] ADC-based in vitro nectin-4 targeting

[0258] The ability of the anti-nectin-4 monoclonal antibody to induce ADC-based cytotoxicity in SUM190 tumor cells was tested. Experiments were performed by incubating 3000 cells / well with serially diluted antibody and a goat anti-mouse monoclonal antibody conjugated with saponin in triplicate, according to the manufacturer's (mab-ZAP kit, ATS-bio) recommended method. For analysis of the effects, cell growth was measured using the alamarBlue staining procedure recommended by the manufacturer (Biosource, CA, USA). This assay incorporates a fluorescent redox indicator. Fluorescence intensity is proportional to the reduction in cellular metabolism. AlamarBlue was measured on day 5 by incubating 1 / 10 volume of alamarBlue solution at 37°C for 2 hours and reading at 595 nm (FLUOstar Optima, BMG Labtech).

[0259] IHC staining on FFPE breast cancer samples

[0260] Samples were fixed in 4% formaldehyde for 24 to 48 hours. Antigen retrieval was performed for 12 min at 95°C and pH 8.0 using the Discovery HQ procedure. 5A12 mAb was then incubated at 37°C for 3 h at 1 μg / ml. Experiments were conducted using the DiscoveryXT Ventana from Roche. Staining was detected using Discovery Anti-Mouse HQ, Discovery Anti-HQ, and ChromomapDAB according to the manufacturer's recommendations (Roche Diagnostics).

[0261] result

[0262] The results of this invention disclose a novel anti-nectin-4 mAb with improved properties. This mAb, named 5A12.2, recognizes the IgG-like distal domain in the extracellular region of Nectin-4. Its binding properties are compared with other patented mAbs in terms of epitope recognition (PCT / EP2018 / 055109 and PCT / EP2106 / 071076).

[0263] like Figure 1 As shown in A and B, 5A12.2 mAb identifies epitopes different from those of the three other tested mAbs, namely N41, 14A5.2, and Ha22.2. FACS analysis shows that 5A12.2 mAb exhibits better apparent affinity compared to N41 and 14A5 mAb. Figure 2 (Top). Interestingly, the maximum binding affinity of 5A12.2 mAb is higher than that of the other two mAbs (top). Figure 2 ,bottom).

[0264] These properties led the inventors to compare the ADC properties of the mab. For example... Figure 3 As shown at the top, 5A12.2 mAb exhibits cytotoxicity when conjugated with the toxin saponin (a highly potent inhibitor of ribosome function). Its cytotoxicity is higher than that of N41 and 14A5 mAb. Figure 3 ,bottom).

[0265] Finally, IHC experiments were performed to evaluate the properties of 5A12.2 mAb in recognizing and staining paraffin-embedded tissues. Figure 4 As shown, Nectin-4 expression in breast cancer tissue is readily detected as expected by membrane staining. To the best of the inventors' knowledge, this is the first anti-Nectin-4 mAb to act on FFPE tissue. Therefore, 5A12.2 mAb represents a new option for the diagnosis, prognosis, and treatment of Nectin-4 positive tumors.

[0266] References:

[0267] 1. Fabre-Lafay S et al. Nectin-4 is a new histological and serological tumor associated marker for breast cancer. BMC Cancer 2007;7:73.

[0268] 2.Takano A et al.Identification of nectin-4 oncoprotein as adiagnostic and therapeutic target for lung cancer.Cancer Res 2009;69:6694-6703.

[0269] 3.Derycke MS et al.Nectin 4 overexpression in ovarian cancer tissuesand serum:potential role as a serum biomarker.Am J ClinPathol 2010;134:835-845.

[0270] 4.Nishiwada S et al.Nectin-4expression contributes to tumorproliferation,angiogenesis and patient prognosis in human pancreatic cancer.JExpClin Cancer Res 2015;34:30.

[0271] 5.Challita-Eid PM et al.EnfortumabVedotin Antibody-Drug ConjugateTargeting Nectin-4 Is a Highly Potent Therapeutic Agent in MultiplePreclinical Cancer Models.Cancer Res 2016;76:3003-3013.

[0272] 6.M-Rabet et al.Nectin-4:a new prognostic biomarker for efficienttherapeutic targeting of primary and metastatic triple-negative breastcancer.Ann Oncol.2017Apr 1;28(4):769-776

[0273] 7.Fabre S et al.,Prominent role of the Ig-like V domain in trans-interactions of nectins.Nectin-3 and nectin-4bind to the predicted C-C'-C"-Dbeta-strands of the nectin-1 V domain.J Biol Chem.2002 Jul 26;277(30):27006-13.

Claims

1. An antibody specific for human Nectin-4, comprising: a HCDR1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3, a HCDR3 of SEQ ID NO: 4, a LCDR1 of SEQ ID NO: 5, a LCDR2 of SEQ ID NO: 6 and a LCDR3 of SEQ ID NO:

7.

2. The anti-Nectin-4 antibody of claim 1, which competes for binding to Nectin-4 with the reference murine antibody mAb 5A12.2, obtainable from the hybridoma deposited at the CNCM under accession number CNCM 1-5407.

3. The anti-Nectin-4 antibody of claim 1, comprising a variable heavy chain (VH) domain and a variable light chain domain having at least 90% identity to the heavy chain and light chain of SEQ ID NO: 8 and 9, respectively; or the variable heavy chain (VH) domain and the variable light chain domain of SEQ ID NO: 8 and 9, respectively.

4. The anti-Nectin-4 antibody of claim 1, which is a chimeric antibody or a humanized antibody.

5. The anti-Nectin-4 antibody of any one of claims 1-4, characterized in that the antibody has at least one of the following functions: i. which has a K D Human Nectin-4 binding to SEQ ID NO: 1; ii. it selectively binds Nectin-4 in a biological sample in an immunohistochemistry (IHC) assay.

6. A nucleic acid molecule encoding the heavy and light chain of the anti-Nectin-4 antibody of any one of claims 1-5.

7. A host cell comprising the nucleic acid of claim 6.

8. A pharmaceutical composition comprising the anti-Nectin-4 antibody of any one of claims 1-5 and at least one pharmaceutically acceptable carrier.

9. Use of the anti-Nectin-4 antibody of any one of claims 1-5 for the manufacture of a reagent for the diagnosis of a cancer characterized by expression of Nectin-4 in a subject, wherein the diagnosis comprises detecting the presence or expression level of Nectin-4 in a biological sample, wherein the detection is performed by using the anti-Nectin-4 antibody of any one of claims 1-5 in an immunohistochemistry (IHC) assay.

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