Antibodies specific for nectin cell adhesion molecule 4 and uses thereof

Abstract

The present disclosure relates to modified antibodies or antigen-binding fragments thereof specific for Nectin cell adhesion molecule 4 (Nectin-4). The present disclosure also relates to methods for detecting or diagnosing whether a subject has or is at risk for developing a tumor, or for assessing the prognosis of a tumor, and to pharmaceutical compositions for use in the treatment, prophylactic treatment and / or prevention of a tumor in a subject suffering from a tumor.

Description

[Technical field]

[0001] Priority information This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 266,711, filed January 12, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .xml format and is incorporated herein by reference in its entirety. The .xml copy created on January 12, 2023 is named "G4590-15400PCT_SeqListing_20230112.xml" and is 45 kilobytes in size.

[0003] Field of the Disclosure The present disclosure relates to antibodies, or antigen-binding fragments thereof, specific for Nectin cell adhesion molecule 4 (Nectin-4) for detecting and / or treating tumors. [Background technology]

[0004] Cancer is one of the leading causes of death worldwide. Since cancer mortality can be reduced if cases are detected and treated early, there is a need to have viable screening options for the general population.

[0005] Immunotherapy is one of the most promising advances in cancer treatment. Cancer immunotherapy is utilized to generate and increase anti-tumor immune responses, for example, by treatment with antibodies specific for antigens on tumor cells, by fusion of antigen-presenting cells with tumor cells, or by activation of anti-tumor T cells. The ability to recruit immune cells (e.g., T cells) against tumor cells in patients provides a therapeutic modality to combat cancer types and metastases that were previously considered incurable.

[0006] When carcinoma is diagnosed early and the disease is limited to the organ of origin, the survival rate is dramatically improved. In fact, physical methods for screening, such as Pap smear for cervical cancer and mammography for breast cancer, have dramatically reduced the mortality rate. Therefore, research is encouraged to develop new early detection biomarkers using available body fluids, such as serum, urine or saliva. Although some known biomarkers can reliably detect the disease at advanced stages, they lack sufficient sensitivity and especially specificity for early cancer diagnosis, and therefore are mainly used for prognosis, staging, monitoring and therapy selection. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need to develop new approaches to the treatment and detection of cancer. [Means for solving the problem]

[0008] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to epitopes on an antigen or fragment thereof for the treatment and detection of cancer.

[0009] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof specific for an epitope on a nectin cell adhesion molecule 4 or a fragment thereof; comprising a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region, the CDR of the heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 regions, the CDR of the light chain variable region comprising CDRL1, CDRL2 and CDRL3 regions, and the CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6, or a substantially similar sequence having at least 90% sequence identity; the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, or a substantially similar sequence having at least 90% sequence identity; the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-18, or a substantially similar sequence having at least 90% sequence identity; and The present invention provides an antibody or antigen-binding fragment thereof, wherein the CDRL1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 24, or a substantially similar sequence having at least 90% sequence identity; the CDRL2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 30, or a substantially similar sequence having at least 90% sequence identity; and the CDRL3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 36, or a substantially similar sequence having at least 90% sequence identity.

[0010] In one embodiment of the present disclosure, the nectin-4 is human nectin-4.

[0011] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is specific for an epitope on the extracellular domain of Nectin-4 or a fragment thereof; examples of Nectin-4 fragments include, but are not limited to, amino acids 94-435, 94-732, or 94-957 of Nectin-4-Q96NY8 (PVRL4_human).

[0012] In one embodiment of the present disclosure, the antibody Nectin4-scFv-L1 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:2 or a substantially similar sequence having at least 90% sequence identity, a CDRH2 region comprising SEQ ID NO:8 or a substantially similar sequence having at least 90% sequence identity, a CDRH3 region comprising SEQ ID NO:14 or a substantially similar sequence having at least 90% sequence identity, a CDRL1 region comprising SEQ ID NO:20 or a substantially similar sequence having at least 90% sequence identity, a CDRL2 region comprising SEQ ID NO:26 or a substantially similar sequence having at least 90% sequence identity, and a CDRL3 region comprising SEQ ID NO:32 or a substantially similar sequence having at least 90% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37 or a substantially similar sequence having at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38 or a substantially similar sequence having at least 90% sequence identity.

[0013] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S2 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:3 or a substantially similar sequence having at least 90% sequence identity, a CDRH2 region comprising SEQ ID NO:9 or a substantially similar sequence having at least 90% sequence identity, a CDRH3 region comprising SEQ ID NO:15 or a substantially similar sequence having at least 90% sequence identity, a CDRL1 region comprising SEQ ID NO:21 or a substantially similar sequence having at least 90% sequence identity, a CDRL2 region comprising SEQ ID NO:27 or a substantially similar sequence having at least 90% sequence identity, and a CDRL3 region comprising SEQ ID NO:33 or a substantially similar sequence having at least 90% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39 or a substantially similar sequence thereof; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:40 or a substantially similar sequence thereof.

[0014] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S6 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO: 4 or a substantially similar sequence having at least 90% sequence identity, a CDRH2 region comprising SEQ ID NO: 10 or a substantially similar sequence having at least 90% sequence identity, a CDRH3 region comprising SEQ ID NO: 16 or a substantially similar sequence having at least 90% sequence identity, a CDRL1 region comprising SEQ ID NO: 22 or a substantially similar sequence having at least 90% sequence identity, a CDRL2 region comprising SEQ ID NO: 28 or a substantially similar sequence having at least 90% sequence identity, and a CDRL3 region comprising SEQ ID NO: 34 or a substantially similar sequence having at least 90% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 41 or a substantially similar sequence having at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 42 or a substantially similar sequence having at least 90% sequence identity.

[0015] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S8 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:5 or a substantially similar sequence having at least 90% sequence identity, a CDRH2 region comprising SEQ ID NO:11 or a substantially similar sequence having at least 90% sequence identity, a CDRH3 region comprising SEQ ID NO:17 or a substantially similar sequence having at least 90% sequence identity, a CDRL1 region comprising SEQ ID NO:23 or a substantially similar sequence having at least 90% sequence identity, a CDRL2 region comprising SEQ ID NO:29 or a substantially similar sequence having at least 90% sequence identity, and a CDRL3 region comprising SEQ ID NO:35 or a substantially similar sequence having at least 90% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:43 or a substantially similar sequence having at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:44 or a substantially similar sequence having at least 90% sequence identity.

[0016] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S21 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:6 or a substantially similar sequence having at least 90% sequence identity, a CDRH2 region comprising SEQ ID NO:12 or a substantially similar sequence having at least 90% sequence identity, a CDRH3 region comprising SEQ ID NO:18 or a substantially similar sequence having at least 90% sequence identity, a CDRL1 region comprising SEQ ID NO:24 or a substantially similar sequence having at least 90% sequence identity, a CDRL2 region comprising SEQ ID NO:30 or a substantially similar sequence having at least 90% sequence identity, and a CDRL3 region comprising SEQ ID NO:36 or a substantially similar sequence having at least 90% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:45 or a substantially similar sequence thereof; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:46 or a substantially similar sequence thereof.

[0017] In some embodiments of the disclosure, the antibody or fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an scFv antibody or fragment thereof.

[0018] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent, examples of which include, but are not limited to, antimetabolites, alkylating agents, alkylating-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes.

[0019] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is expressed on the surface of a cell. The cell can be an immune cell, a cancer stem cell or a stem cell. In one embodiment of the present disclosure, the immune cell is a T cell.

[0020] The present disclosure also provides vectors encoding the antibodies or antigen-binding fragments thereof.

[0021] The disclosure also provides engineered cells that express the antibodies or antigen-binding fragments thereof or contain the vectors.

[0022] The present disclosure also provides a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof, or the genetically engineered cell.

[0023] In another aspect, the disclosure provides a method of detecting or diagnosing whether a subject has or is at risk for developing a tumor, or assessing the prognosis of a tumor, comprising contacting a sample from the subject with an antibody or antigen-binding fragment thereof.

[0024] The present disclosure provides a method for detecting Nectin-4 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof.

[0025] The present disclosure also provides a kit for detecting Nectin-4 in a sample, comprising an antibody or an antigen-binding fragment thereof.

[0026] In another aspect, the disclosure provides a method of treating, prophylactically treating and / or preventing a tumor in a subject suffering from a tumor comprising administering to the subject a pharmaceutical composition. Alternatively, the disclosure provides a pharmaceutical composition for use in treating, prophylactically treating and / or preventing a tumor in a subject suffering from a tumor comprising an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, or a genetically engineered cell. Examples of tumors include, but are not limited to, squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, head and neck cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia. In particular, the tumor is triple-negative breast cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, head / neck cancer, or esophageal cancer.

[0027] The present disclosure is described in detail in the following sections. Other features, objects, and advantages of the present disclosure can be found in the detailed description and claims. [Brief description of the drawings]

[0028] [Figure 1] Figure 1 shows the expression of PVRL4 fragments in Example 1. Lane 1: supernatant after Ni+ Sepharose binding; Lane 2: collected wash buffer; Lane 3: collected elution buffer (imidazole); Lane 4: Ni+ Sepharose after elution. Expected sizes: r342p: 15 kDa; r639p: 48 kDa; r864p: 34 kDa. [Figure 2A]Figure 2A shows an scFv sequence alignment of the light chain variable region of chicken immunoglobulins, and Figure 2B shows an scFv sequence alignment of the heavy chain variable region of chicken immunoglobulins. [Figure 2B] FIG. 2B shows an scFv sequence alignment of the heavy chain variable regions of chicken immunoglobulins. [Diagram 3] Figure 3A shows the affinity assay of anti-nectin-4 M13 phage with nectin-4 expressed or not on BML01 cells, and Figure 3B shows the affinity fold (BML01-nectin-4 OD450 / BML01 OD450) for anti-nectin-4 M13 phage. [Figure 4-1] FIG. 4 shows the results of detecting the binding activity of candidate phage clones using an anti-HA antibody. [Figure 4-2] FIG. 4 shows the results of detecting the binding activity of candidate phage clones using an anti-HA antibody. [Figure 4-3] FIG. 4 shows the results of detecting the binding activity of candidate phage clones using an anti-HA antibody. [Diagram 5] FIG. 5 shows protein expression of scFv. [Figure 6] FIG. 6 shows the binding ability of anti-PVRL4 scFv to PVRL4 864 by Western blotting. [Figure 7] FIG. 7 shows the results of binding assays of L1 / S6 / S21 / L4 to r864p by competitive ELISA or ELISA. [Figure 8] FIG. 8 shows the binding ability of monoclonal anti-PVRL4 scFv on MCF-7 cells in Western blot analysis. [Figure 9A] Figures 9A and 9B show the results of binding assays of anti-PVRL4 scFv to breast cancer cell lines by immunofluorescence: Figure 9A: MCF7; Figure 9B: MCF10A. [Figure 9B] Figures 9A and 9B show the results of binding assays of anti-PVRL4 scFv to breast cancer cell lines by immunofluorescence: Figure 9A: MCF7; Figure 9B: MCF10A. [Figure 10] FIG. 10 shows the results of immunofluorescence staining of S21 on breast cancer cell line MCF7 by confocal microscopy. [Figure 11] FIG. 11 shows the results of characterization of monoclonal anti-PVRL4 scFv(S21) in a staining assay. [Figure 12] FIG. 12 shows the binding ability of monoclonal anti-PVRL4 scFv on MCF-7 cells using ELISA (data shown as mean±SD, N=2). [Figure 13] FIG. 13 shows the results of MCF7 cell proliferation after treatment with anti-PVRL4 scFv S21. [Figure 14] FIG. 14 shows the results of epitope mapping of anti-PVRL4 scFv S21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In the description that follows, certain terms will be used and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms not expressly defined herein will be used in accordance with their plain and ordinary meaning.

[0030] Unless otherwise specified, "a" or "an" means "one or more."

[0031] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0032] The term "antibody" as used herein means any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., Nectin-4). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or VVR). HThe heavy chain constant region is made up of three domains: H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L1 ) is included. V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-Nectin-4 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0033] As used herein, the term "specific" means that the antibody does not cross-react to any significant extent with other epitopes.

[0034] As used herein, the term "complementarity determining region" (CDR) refers to the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definitions include overlapping or subsets of amino acid residues when compared to each other.

[0035] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, for example by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains, cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0036] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies are obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means known or available in the art.

[0037] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0038] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0039] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0040] As used in this disclosure, the term "therapeutic agent" means any compound, substance, drug, prodrug or active ingredient having a therapeutic or pharmacological effect suitable for administration to a mammal, e.g., a human.

[0041] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0042] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0043] The term "genetically engineered" cells or "genetic engineering" of cells refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels in the cell. The genetic material can be in the form of DNA or RNA. Genetic material can be introduced into cells by various means including viral transduction and non-viral transfection. After being genetically engineered, the expression levels of certain genes in the cells can be altered permanently or temporarily.

[0044] As used in this disclosure, the term "pharmaceutical composition" means a mixture containing a therapeutic agent that is administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or pathological condition from which the mammal suffers.

[0045] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.

[0046] As used herein, the terms "treatment," "treating," and the like, encompass any treatment of disease in a mammal, particularly a human, including: (a) preventing the disease from developing in a subject who may have a predisposition to the disease, but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0047] The terms "preventing" or "prevention" are art-recognized and when used in reference to a condition, it includes administering an agent prior to the onset of the condition that reduces the frequency or severity of symptoms of, or delays the onset of, a medical condition in a subject compared to a subject who does not receive the agent.

[0048] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0049] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of humans, a physician, nurse, clinical nurse, or individual; in the case of animals, including non-human mammals, a veterinarian) that a subject needs or will benefit from treatment. This judgment is made based on a variety of factors that are within the caregiver's area of ​​expertise, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the compounds of the present disclosure.

[0050] "Cancer," "tumor," and similar terms include precancerous, neoplastic, transformed, and cancerous cells and may refer to solid tumors or nonsolid cancers (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign and malignant neoplasms (abnormal growth). "Transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell proliferation, reduced contact inhibition and colonization, and / or malignant tumors (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Transformation can result from infection with a transforming virus and integration of new genomic DNA, or uptake of exogenous DNA, but it can also occur spontaneously or following exposure to carcinogens.

[0051] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient that can be used in a diagnostic or monitoring assay. This definition includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny.

[0052] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope on Nectin-4 or a fragment thereof.

[0053] In one embodiment, the antibody or antigen-binding fragment thereof (anti-Nectin-4) comprises a complementarity determining region (CDR) of a heavy chain variable region and a complementarity determining region of a light chain variable region, the complementarity determining region of the heavy chain variable region comprises CDRH1, CDRH2 and CDRH3 regions, the complementarity determining region of the light chain variable region comprises CDRL1, CDRL2 and CDRL3 regions, and the CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-18, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and The CDRL1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-24, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; the CDRL2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-30, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the CDRL3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-36, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0054] Nectin cell adhesion molecule 4 (nectin-4), also known as poliovirus receptor-related 4 (PVRL4), is a type I transmembrane protein and a member of the nectin family of related immunoglobulin-like adhesion molecules. Nectin-4 is known to be located on the cell surface and can bind to other cells or the extracellular matrix (ECM). In humans, nectin-4 is expressed primarily during development at epithelial adherens junctions in the trachea, lung, and skin (JBC 2001, 276(46):43205-43215). Its expression is reduced in adults, while expression of other nectins continues in adult tissues. However, nectin-4 is re-expressed as a tumor-associated antigen with carcinogenic properties in various carcinomas, including breast cancer. In tumors, nectin-4 and the active form of ADAM17 / TACE metalloprotease are overexpressed, and cleaved nectin-4 (soluble form) is observed in the cytoplasm (JBC 2005, 280(20):19543-50; BMC Cancer 2007, 7:73). Specifically, the nectin-4 disclosed herein is human nectin-4.

[0055] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is specific for an epitope on the extracellular domain of Nectin-4 or a fragment thereof; examples of Nectin-4 fragments include, but are not limited to, amino acids 94-435, 94-732, or 94-957 of Nectin-4-Q96NY8 (PVRL4_human, NCBI Reference Sequence: NM_030916.3, UniProtKB / Swiss-Prot(Q96NY8.1)).

[0056] In one embodiment of the present disclosure, the antibody Nectin4-scFv-L1 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO: 2 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH2 region comprising SEQ ID NO: 8 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising SEQ ID NO: 14 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising an amino acid sequence similar to SEQ ID NO:20, a CDRL1 region comprising SEQ ID NO:20 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, a CDRL2 region comprising SEQ ID NO:26 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a CDRL3 region comprising SEQ ID NO:32 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0057] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S2 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:3 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH2 region comprising SEQ ID NO:9 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising SEQ ID NO:15 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising the sequence SEQ ID NO:21, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, a CDRL2 region comprising SEQ ID NO:27, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a CDRL3 region comprising SEQ ID NO:33, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:40, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0058] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S6 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO: 4 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH2 region comprising SEQ ID NO: 10 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising SEQ ID NO: 16 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising an analogous sequence, a CDRL1 region comprising SEQ ID NO:22 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, a CDRL2 region comprising SEQ ID NO:28 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a CDRL3 region comprising SEQ ID NO:34 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:41 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:42 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0059] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S8 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO:5 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH2 region comprising SEQ ID NO:11 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising SEQ ID NO:17 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising an amino acid sequence of SEQ ID NO:23 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, a CDRL2 region comprising SEQ ID NO:29 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a CDRL3 region comprising SEQ ID NO:35 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:43 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:44 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0060] In one embodiment of the present disclosure, the antibody Nectin4-scFv-S21 or an antigen-binding fragment thereof comprises a CDRH1 region comprising SEQ ID NO: 6 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH2 region comprising SEQ ID NO: 12 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising SEQ ID NO: 18 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDRH3 region comprising an analogous sequence, a CDRL1 region comprising SEQ ID NO:24 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, a CDRL2 region comprising SEQ ID NO:30 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a CDRL3 region comprising SEQ ID NO:36 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:45 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:46 or a substantially similar sequence with at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0061] The sequence listing is shown in Table 1.

[0062] [Table 1] TIFF2025502198000003.tif205170

[0063] Antibodies according to the present disclosure may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments), and may be modified as needed to affect functionality.

[0064] Various techniques known to those skilled in the art can be used to determine whether an antibody "specifically binds to one or more amino acids" in a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide to which an antibody specifically binds is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0065] Whether an antibody specifically binds to or competes for binding with the same epitope as a reference anti-Nectin-4 antibody can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Nectin-4 antibody of the present disclosure, the reference antibody is bound to Nectin-4 protein. The ability of the test antibody to bind to a Nectin-4 molecule is then evaluated. If the test antibody can bind to Nectin-4 after saturation binding with the reference anti-Nectin-4 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Nectin-4 antibody. On the other hand, if the test antibody cannot bind to a Nectin-4 molecule after saturation binding with the reference anti-Nectin-4 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-Nectin-4 antibody of the present disclosure. Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antibodies bind to the same (or overlapping) epitope if, for example, a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay. Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only the subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0066] Antibodies also include antigen-binding fragments of complete antibody molecules. Antigen-binding fragments of antibodies may be obtained, for example, from complete antibody molecules using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0067] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody, or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0068] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated with V H In an antigen-binding fragment having a domain,H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer and has a V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V H Or V L It may contain domains.

[0069] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H1 (ii) V H -C H2 (iii) V H -C H3 ;(iv)V H -C H1 -C H2 ;(v)V H -C H1 -C H2 -C H3 ;(vi)V H -C H2 -C H3 ;(vii)V H -C L ;(viii)V L -C H1 ;(ix)V L -C H2 ;(x)V L -C H3 ;(xi)V L -C H1 -C H2 ;(xii)V L -C H1 -C H2 -C H3 ;(xiii)V L -C H2 -C H3 and (xiv) V L -CL In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V domains (e.g., by disulfide bond(s)). H Or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association.

[0070] The anti-Nectin-4 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in the germline sequence of another mammal, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H Domain and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0071] The present disclosure also provides a method for the preparation of a nucleotide sequence comprising administering to a subject a polypeptide having one or more conservative substitutions as disclosed herein. H , V L , and / or any variant of the CDR amino acid sequence. H , V L , and / or any of the CDR amino acid sequences, e.g., 10 or less, 8 or less, 6 or less, 4 or less conservative amino acid substitutions, H , V L and / or an anti-Nectin-4 antibody having the CDR amino acid sequence.

[0072] In some embodiments of the present disclosure, the antibody according to the present disclosure is a humanized antibody. To improve the binding affinity of the humanized antibody according to the present disclosure, some amino acid residues in the human framework region are replaced by the corresponding amino acid residues in the CDR of a species such as a rodent.

[0073] The antibodies of the present disclosure may be monospecific, bispecific, or multispecific. A multispecific antibody may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. The anti-Nectin-4 antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof may be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent bonding, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody with a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the immunoglobulin is specific for Nectin-4 or a fragment thereof and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.

[0074] In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent.

[0075] In some embodiments of the present disclosure, the therapeutic agent represents a cytostatic or cytotoxic agent, or an isotope chelator with a corresponding radioisotope. Examples of cytostatic or cytotoxic agents include, but are not limited to, antimetabolites (e.g., fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase, gemcitabine, capecitibine, azathioprine, cytosine methotrexate, trimethoprim, pyrimethamine, or pemetrexed); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorethamine, uramustine, dibromomannitol, tetranitrate, procarbazine, altretamine, mitozolomide, or temozolomide); alkylating agents (e.g., cisplatin, carboplatin, nedaplatin, oxaliplatin, satrap, platin, or triplatin; DNA minor groove alkylating agents (e.g., duocarmycins, such as CC-1065, and any analogs or derivatives thereof; pyrrolobenzodiazepines, or any analogs or derivatives thereof); anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin); antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, mitomycin (e.g., for example, mitomycin C; calicheamicin; antimitotics (e.g., maytansinoids (such as DM1, DM3, and DM4), auristatins (including, for example, monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF)), dolastatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel, or the new taxanes), tubulysins, and colchicine);topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine, or mitoxantrone); HDAC inhibitors (e.g., vorinostat, romidepsin, chidamide, panobinostat, or belinostat); proteasome inhibitors (e.g., peptidyl boronic acid); and radioisotopes, e.g., At; 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or 213 , P 32 , and Lu 177 Examples of isotope chelators include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N",N"-pentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate (DOTA), 1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (THP), triethylenetetraamine-N,N,N',N",N"',N"'-hexaacetate (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetrakis(methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

[0076] In one embodiment of the present disclosure, the antibody or its antigen-binding fragment is expressed on the surface of the cell. In particular, the cell is a T cell or a stem cell, such as an iPSC. Induced pluripotent stem cells can be reprogrammed by inducing Yamanaka factors into somatic cells. Similar to embryonic stem cells, iPSCs have the ability to differentiate into cells of the three germ layers without ethical concerns. This characteristic makes iPSCs promising for clinical use.

[0077] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0078] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct that includes at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in a CAR polypeptide construct are in the same polypeptide chain, e.g., constituting a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not adjacent to each other, e.g., in different polypeptide chains.

[0079] The antibody or antigen-binding fragment thereof may be encoded by a vector encoding the antibody or antigen-binding fragment thereof. An exemplary vector is a lentiviral vector. "Lentivirus" refers to a genus of retroviruses that can infect dividing and non-dividing cells. Some examples of lentiviruses include HIV (including human immunodeficiency viruses: HIV types 1 and 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0080] In another aspect, the disclosure provides a genetically engineered cell that expresses an antibody or antigen-binding fragment thereof or contains a vector. The genetically engineered cell can be an immune cell or a stem cell. The disclosure also provides an immune cell differentiated from the genetically engineered cell.

[0081] The present disclosure provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof, genetically engineered cells or immune cells of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with suitable diluents, carriers, excipients, and other agents that provide improved translocation, delivery, tolerability, and the like. The compositions may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition used, as well as the excipients, diluents, and / or carriers, will depend on the intended use of the antibody and, in the case of therapeutic use, the mode of administration. Many suitable formulations can be found in a formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0082] The dose of the antibody administered to the patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, etc. The preferred dose is typically calculated according to body weight or body surface area. When the antibody of the present disclosure is used to treat a condition or disease associated with EPHA10 in an adult patient, it may be advantageous to administer the antibody of the present disclosure intravenously. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the antibody may be determined empirically; for example, the progress of the patient can be monitored by periodic evaluation and the dosage adjusted accordingly. Furthermore, inter-species adjustment of the dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0083] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, encapsulation in receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as, for example, oral mucosa, rectal and intestinal mucosa, etc.), or together with other biologically active agents. Administration can be systemic or local.

[0084] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device can be easily applied to deliver the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen delivery device does not have a replaceable cartridge. Rather, a disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is removed and the reservoir is emptied, the entire device is discarded.

[0085] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, controlled release systems can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0086] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium customarily used for injections. Examples of aqueous media for injections include isotonic solutions containing physiological saline, glucose and other auxiliary agents, and the like, which may be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil, soybean oil, and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The injections thus prepared are preferably filled into suitable ampoules.

[0087] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses adapted to the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0088] In another aspect, the disclosure provides a method of detecting or diagnosing whether a subject has or is at risk for developing a tumor, or assessing the prognosis of a tumor, comprising contacting a sample from the subject with an antibody or antigen-binding fragment thereof.

[0089] The present disclosure provides a method for detecting Nectin-4 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof.

[0090] The present disclosure also provides a kit for detecting Nectin-4 in a sample, comprising an antibody or an antigen-binding fragment thereof.

[0091] In another aspect, the disclosure provides a method of treating, prophylactically treating and / or preventing a tumor in a subject suffering from a tumor comprising administering to the subject a pharmaceutical composition. Alternatively, the disclosure provides a pharmaceutical composition for use in treating, prophylactically treating and / or preventing a tumor in a subject suffering from a tumor comprising an effective amount of an antibody or antigen-binding fragment thereof disclosed herein, or a genetically engineered cell.

[0092] An example of a cancer is a solid tumor. Another example of a cancer is a liquid tumor, such as lymphoma, leukemia, and hematological malignancies. Further examples of cancers of the present disclosure include triple negative breast cancer, breast cancer, breast cancer metastasis, metastasis of any cancer described herein, colon cancer, colon cancer metastasis, sarcoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, such as Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, pediatric astrocytoma, astrocytoma, pediatric atypical teratoid / rhabdoid tumor, CNS atypical teratoid / rhabdoid tumor, atypical teratoid / rhabdoid tumor, basal cell carcinoma, skin cancer, bile duct cancer, bladder. , bone cancer, Ewing's sarcoma family of cancers, osteosarcoma, chondroma, chondrosarcoma, primary and metastatic bone cancer, malignant fibrous histiocytoma, childhood brain stem glioma, brain stem glioma, brain tumor, brain and spinal cord tumor, central nervous system embryonal tumor, childhood central nervous system embryonal tumor, central nervous system germ cell tumor, childhood central nervous system germ cell tumor, craniopharyngioma, childhood craniopharyngioma, ependymoma, childhood ependymoma, breast cancer, bronchial tumor, childhood bronchial tumor, Burkitt's lymphoma, carcinoid tumor, gastrointestinal cancer, carcinoma of unknown primary, cardiac tumor, childhood cardiac tumor, primary lymphoma, cervical cancer, bile duct cell alveolar cancer, chordoma, childhood chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse midline glioma, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, childhood esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, pediatric extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, ovarian cancer, testicular cancer , gestational trophoblastic disease, glioma, glioblastoma multiforme (GBM), low-grade glioma (LGG), cerebral gliomatosis, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Langerhans cell histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, melanoma, melanoma metastasis, islet cell tumor, pancreatic neuroendocrine tumor, kidney cancer, renal cell tumor, Wilms' tumor, pediatric kidney tumor, lip and oral cavity cancer, liver cancer, lung cancer, medulloblastoma, non-Hodgkin's lymphoma, macroglobulinemia, Waldenström's macroglobulinemia, male breast cancer, Merkel cell carcinoma,Metastatic squamous cell neck cancer of unknown primary site, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, childhood multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, multiple myeloma, myeloproliferative neoplasm, chronic myeloproliferative neoplasm, mucinous papillary ependymoma, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oligoastrocytoma, oropharyngeal cancer, ovarian cancer, low malignant potential tumors, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis, childhood papillomatosis, paraganglioma, paranasal sinus and These include nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pharyngeal cancer, pilocytic astrocytoma, pituitary tumor, pleomorphic xanthoastrocytoma (PXA), pleuropulmonary blastoma, childhood pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, pregnancy-related cancer, rhabdomyosarcoma, childhood rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the kidney, renal pelvis, and ureter, uterine cancer, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vascular tumors, and vulvar cancer.

[0093] Nectin-4 is expressed in 61% of ductal carcinomas and 6% in lobular type. Without wishing to be limited by theory, it is believed that the CEA / CA15.3 / Nectin-4 association allows for monitoring 74% of breast cancers compared to 67% with the CEA / CA15.3 association. Nectin-4 also upregulates EMT, metastasis and the WNT / β-catenin pathway via the Pi3k / Akt axis. Clinicopathological data show Nectin-4 induced in breast tumor metastasis to axillary lymph nodes. In another aspect, Nectin-4 is a potential angiogenic biomarker in breast cancer stem cells. The Nectin-4 ectodomain physically interacts with integrin-β4 and activates angiogenic pathways. The interaction of nectin-4 with integrin-β4 promotes angiogenesis via the Src, PI3K, AKT, and iNOS pathways, but not via the Phospho-Erk or NF-κβ pathways. EXAMPLES

[0094] The following examples are provided to aid those of ordinary skill in the art in practicing the present disclosure.

[0095] Working Example Example 1: Expression and purification of recombinant PVRL4 fragment proteins and anti-PVRL4 scFv Briefly, the nectin-4-Q95NY8 (PVRL4_human) gene fragments of PVRL4_342 (94-435, 342bp, r342p), PVRL4_639 (94-732, 639bp, r639p), and PVRL4_864 (94-957, 864bp, r864p) were constructed in pET-21a(+) plasmid, and the resulting vector was transformed into Escherichia coli (E. coli). Bacterial cultures from single colonies were grown overnight at 37°C in 5 ml of LB medium containing ampicillin (50 μg / ml), diluted 100-fold into the same LB medium, and the OD 600 The cells were further grown until the β-terminal tail reached between 0.4 and 0.8. To induce PVRL4 protein expression, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1.0 mM. The cell pellet was resuspended in His-binding buffer containing 6 M urea and lysed by sonication. After centrifugation, the resulting cell lysate was diluted with Ni 2+ Recombinant PVRL4 fusion protein was purified by incubation with -Sepharose (GE™ Healthcare Life Science, Pittsburgh, PA, USA) according to the manufacturer's instructions.

[0096] Protein products were analyzed by SDS-PAGE as shown in FIG.

[0097] Immunization of animals. First, female White Leghorn (Gallus domesticus) chickens were immunized intramuscularly with 50 μg of purified his-PVRL4_342 or 20 μg of purified his-PVRL4_864 in 0.5 ml of Freund's complete adjuvant (Sigma™, USA). Three booster immunizations with 50 μg of his-PVRL4_342 at 7 or 8 day intervals were performed, followed by one booster immunization with 30 μg of his-PVRL4_342 one month later. Similarly, a second immunization with 25 μg of his-PVRL4_863, a third immunization with 20 μg of his-PVRL4_863, and a fourth immunization with 15 μg of his-PVRL4_863 at 7 day intervals were performed. After each immunization, polyclonal IgY antibodies in the egg yolk were partially purified and titrated by enzyme-linked immunosorbent assay (ELISA) to determine the presence of a humoral anti-PVRL4 immune response. IgY antibodies were purified from egg yolk using 10% dextran sulfate. Purified IgY antibodies were dissolved in 5 ml of TBS containing 0.05% sodium azide and stored at -20°C.

[0098] Construction of scFv antibody library. Briefly, spleens collected from chickens after the final immunization were immediately placed in Trizol for homogenization. 20 μg of total RNA was reverse transcribed into first strand cDNA using SuperScript RT kit (INVITROGEN™, USA). After amplification using chicken-specific primers, PCR products of heavy and light chain variable (VH and VL) regions were subjected to a second round of PCR to form full-length scFv fragments with short linkers (e.g., SEQ ID NO: 47) or long linkers (SEQ ID NO: 48 or 49), which were further cloned into pComb3X vector. Recombinant phage DNA was transformed into E. coli strain ER2738 by electroporation. Recombinant phage generation was initiated by the addition of wild-type VCS-M13 helper phage, which was subsequently precipitated with 4% polyethylene glycol 8000 and 3% NaCl (w / v), and finally resuspended in 1× phosphate-buffered saline.

[0099] Cell-based phage display panning. 50 μl of the phage library (10 12 ~10 13 ) and 150 μl of 1% BSA in PBS for 1 hour at 4° C. For negative selection, 100 μl of 10 6 BML01 cells and 80 μl of phages were placed at room temperature for 30 min twice. For positive selection, 10 6 BML01-Nectin4 cells were added and placed at 4°C for 1 h. The mixture was washed 5-8 times with PBS. Phages were eluted by incubating the cells with 100 μl of 0.2 M glycine-HCl (pH 2.2) for 10 min at room temperature. The supernatant was collected and neutralized with 15 μl of 1 M Tris-HCl, pH 9.1.

[0100] The phage titers were determined and amplified using E. coli ER2738. The phage titers and amplification results are shown in Table 2.

[0101] [Table 2]

[0102] The ScFv sequence alignment with chicken immunoglobulin heavy and light chain variable region genes is shown in Figures 2A and 2B.

[0103] Cell-based ELISA. BML01 cells or nectin4-overexpressing BML01 cells were cultured at 1 × 10 6The cells were suspended at 100 μl / ml. 100 μl of cells were seeded in a 96-well round-bottom plate and 50 μl of candidate phage culture was added to each well. The mixture was incubated at room temperature for 2 hours with shaking at 150 rpm, then washed three times with PBS. 100 μl of anti-M13-HRP (diluted 1:5000 with 1% BSA in PBS) was added to the mixture, incubated at room temperature for 2 hours with shaking at 150 rpm, then washed six times with PBS. 100 μl of TMB was added to the mixture, and the reaction was stopped by adding 50 μl of 2N H2SO4. The results are shown in Figures 3A and 3B.

[0104] Anti-HA antibody was used to detect the binding activity of candidate phage clones. 5 To the cells, 20 μl of candidate phage clones were added, incubated at room temperature for 30 min, and washed twice. Anti-HA antibody was then added, incubated at room temperature for 30 min, and washed twice. The mixture was subjected to flow cytometry analysis and is shown in FIG. 4.

[0105] Protein expression of the scFv is shown in Figure 5. The binding ability of the anti-PVRL4 scFv to PVRL4 864 was assayed by Western blotting and is shown in Figure 6.

[0106] The binding ability of serial concentrations of anti-PVRL4 scFv to PVRL4 864 and BSA was assayed by ELISA. The concentration of anti-PVRL4 scFv at an OD450 nm value of 1 based on titration is shown in Table 3. This indicates that S21 has the strongest affinity to PVRL4 864.

[0107] [Table 3]

[0108] The results of binding assay of L1 / S6 / S21 / L4 to r864p by competitive ELISA or ELISA are shown in Figure 7. The primary antibody is purified scFv; the secondary antibody is goat anti-chicken light chain (1:3000); the tertiary antibody is HRC-conjugated donkey anti-goat IgG antibody (1:5000). The K d The values ​​are shown in Table 4.

[0109] [Table 4]

[0110] The present inventors have successfully produced recombinant PVRL4 fragment proteins and anti-PVRL4_864 scFv.

[0111] Example 2: Characterization of anti-PVRL4 scFv The binding ability of monoclonal anti-PVRL4 scFv to MCF-7 cells in Western blot analysis is shown in Figure 8. MDA-MB-231 cells served as a negative control. Primary antibodies were purified scFv S2, S21 and L4 (10 μg / ml); secondary antibody was goat anti-chicken light chain (1:3000); tertiary antibody was HRP-conjugated donkey anti-goat antibody (1:5000).

[0112] The results of binding assay of anti-PVRL4 scFv to breast cancer cell lines by immunofluorescence are shown in Figures 9A and 9B. The results of immunofluorescence staining of S21 to breast cancer cell line MCF7 by confocal microscopy are shown in Figure 10. The primary antibody was purified scFv S21 (300 μg / ml); the secondary antibody was goat anti-chicken light chain (1:400); the tertiary antibody was TRIC-conjugated rabbit anti-goat IgG antibody (1:400); the blank was the secondary and tertiary antibody without the primary antibody.

[0113] The results of characterization of monoclonal anti-PVRL4 scFv in staining assays are shown in Figure 11. S21 was used for immunofluorescence staining (300 μg / ml) on MCF-7 cells and visualized with TRITC-conjugated antibody (400x magnification; representative data).

[0114] The results of binding assay of anti-PVRL4 scFv to breast cancer cell line MCF7 by cell ELISA are shown in Figure 12. Primary antibodies were serially diluted purified scFv S21 and L4; secondary antibody was goat anti-chicken light chain (1:2000); tertiary antibody was HRC-conjugated donkey anti-goat IgG antibody (1:4000); blank was secondary and tertiary antibody without primary antibody; negative control (NC) was anti-RT scFv S3 (RTS3).

[0115] The results of MCF7 cell proliferation after treatment with anti-PVRL4 scFv S21 are shown in FIG.

[0116] The present inventors found that S21 could recognize PVRL4 on MCF7 cell line and inhibit its proliferation.

[0117] Example 3: Epitope mapping of PVRL4 using anti-PVRL4 scFv The results of epitope mapping of anti-PVRL4 scFv S21 are shown in Figure 14. The primary antibody is purified scFv S21 (20 μg / ml) or anti-r864 4th immunization IgY (1:5000) as a positive control; the secondary antibody is goat anti-chicken light chain (1:3000) or HRP-conjugated donkey anti-chicken IgY (1:5000); the tertiary antibody is HRC-conjugated donkey anti-goat IgG antibody (1:5000). The binding region of S21 is SEQ ID NO:50.

[0118] While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are deemed to be within the scope of the present disclosure.

Claims

1. An antibody or antigen-binding fragment thereof specific for an epitope on Nectin cell adhesion molecule 4 (Nectin-4) or a fragment thereof; comprising a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region, the CDR of the heavy chain variable region comprising CDRH1, CDRH2 and CDRH3 regions, the CDR of the light chain variable region comprising CDRL1, CDRL2 and CDRL3 regions, and the CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2-6; the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:8-12; and the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:14-18; and An antibody or antigen-binding fragment thereof, wherein the CDRL1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-24; the CDRL2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-30; and the CDRL3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-36.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the nectin-4 is human nectin-4.

3. The antibody or antigen-binding fragment thereof of claim 1, which is specific to an epitope on the extracellular domain of Nectin-4 or a fragment thereof.

4. The antibody or antigen-binding fragment thereof according to claim 1, which is specific to amino acids 94 to 435, 94 to 732, or 94 to 957 of Nectin-4-Q96NY8 (PVRL4_human).

5. the CDRH1 region comprises SEQ ID NO:2, the CDRH2 region comprises SEQ ID NO:8, the CDRH3 region comprises SEQ ID NO:14, the CDRL1 region comprises SEQ ID NO:20, the CDRL2 region comprises SEQ ID NO:26, and the CDRL3 region comprises SEQ ID NO:32; the CDRH1 region comprises SEQ ID NO:3, the CDRH2 region comprises SEQ ID NO:9, the CDRH3 region comprises SEQ ID NO:15, the CDRL1 region comprises SEQ ID NO:21, the CDRL2 region comprises SEQ ID NO:27, and the CDRL3 region comprises SEQ ID NO:33; the CDRH1 region comprises SEQ ID NO:4, the CDRH2 region comprises SEQ ID NO:10, the CDRH3 region comprises SEQ ID NO:16, the CDRL1 region comprises SEQ ID NO:22, the CDRL2 region comprises SEQ ID NO:28, and the CDRL3 region comprises SEQ ID NO:34; the CDRH1 region comprises SEQ ID NO:5, the CDRH2 region comprises SEQ ID NO:11, the CDRH3 region comprises SEQ ID NO:17, the CDRL1 region comprises SEQ ID NO:23, the CDRL2 region comprises SEQ ID NO:29, and the CDRL3 region comprises SEQ ID NO:35; or The antibody or antigen-binding fragment thereof of claim 1, wherein the CDRH1 region comprises SEQ ID NO: 6, the CDRH2 region comprises SEQ ID NO: 12, the CDRH3 region comprises SEQ ID NO: 18, the CDRL1 region comprises SEQ ID NO: 24, the CDRL2 region comprises SEQ ID NO: 30, and the CDRL3 region comprises SEQ ID NO:

36.

6. the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37, or a substantially similar sequence with at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:38, or a substantially similar sequence with at least 90% sequence identity; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39, or a substantially similar sequence with at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:40, or a substantially similar sequence with at least 90% sequence identity; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:41, or a substantially similar sequence with at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:42, or a substantially similar sequence with at least 90% sequence identity; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:43, or a substantially similar sequence with at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:44, or a substantially similar sequence with at least 90% sequence identity; or The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45 or a substantially similar sequence having at least 90% sequence identity; and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 46 or a substantially similar sequence having at least 90% sequence identity.

7. The antibody or antigen-binding fragment thereof according to claim 1 , which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or an scFv antibody or a fragment thereof.

8. The antibody or antigen-binding fragment thereof of claim 1 , conjugated to a therapeutic agent.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the therapeutic agent is selected from the group consisting of antimetabolites, alkylating agents, alkylation-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioactive isotopes.

10. The antibody or antigen-binding fragment thereof of claim 1 , which is expressed on the surface of a cell.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the cell is an immune cell or a stem cell.

12. A vector encoding the antibody or antigen-binding fragment thereof of claim 1.

13. A genetically engineered cell expressing the antibody or antigen-binding fragment thereof of claim 1.

14. A pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a genetically engineered cell according to claim 13.

15. 12. A method for detecting or diagnosing whether a subject has or is at risk of developing a tumor, or for assessing the prognosis of a tumor, comprising the step of contacting a sample derived from the subject with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

16. A method for detecting Nectin-4 in a sample, comprising the step of contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

17. A kit for detecting Nectin-4 in a sample, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11.

18. A pharmaceutical composition for use in the treatment, prophylactic treatment and / or prevention of a tumor in a subject suffering from a tumor, comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 or a genetically engineered cell according to claim 13.

19. 19. The pharmaceutical composition of claim 18, wherein the tumor is selected from the group consisting of squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, head and neck cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

20. 20. The pharmaceutical composition of claim 19, wherein the tumor is triple-negative breast cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, head / neck cancer or esophageal cancer.

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