Antibody to Nectin-4 and use thereof

Abstract

The present invention provides an antibody molecule that binds to human Nectin-4 or a fragment thereof. The antibody is obtained by hybridoma screening and humanization techniques and can be used for the prevention or treatment of cancer and as a clinical lead drug molecule.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to Chinese Patent Application No. 202010320420.3, filed on April 21, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure belongs to the field of antibody drugs, and in particular relates to antibodies against human Nectin-4 and the use of the antibodies in the preparation of drugs. [Background technology]

[0003] Nectin-4 (PVRL4, also known as poliovirus receptor-like molecule 4) is a type I transmembrane glycoprotein with a molecular mass of 66 kDa and belongs to the nectin family of Ig superfamily proteins. Nectin-4 has an extracellular region containing three immunoglobulin-like (Ig-like) domains (VCC) and plays a role in the formation and maintenance of adherens junctions together with cadherins.

[0004] Nectin-4 is closely related to the generation and development of various tumor cells. Nectin-4 has been found to be expressed in many solid tumors, especially bladder cancer. As a tumor-associated antigen, Nectin-4 has tissue expression detection rates in breast cancer, ovarian cancer, and lung cancer, corresponding to 50% of breast cancers, 49% of ovarian cancers, and 86% of lung cancers, respectively, and plays an important role in the development, invasion, and metastasis of these epithelial malignant tumors. Therefore, Nectin-4 has become an important target for the diagnosis and treatment of multiple solid tumors.

[0005] Currently, the primary drug targeting Nectin-4 is enfortumab vedotin, an antibody-drug conjugate formed by conjugating an anti-Nectin-4 monoclonal antibody with the cytotoxic agent monomethyl auristatin E (MMAE). Enfortumab vedotin is primarily used to treat bladder cancer, particularly urothelial carcinoma, and received FDA Breakthrough Therapy Designation in March 2018. Additionally, other studies have shown that the adhesion factor Nectin-4 can be used not only as an effective prognostic factor in breast cancer but also as an effective therapeutic target for patients with triple-negative breast cancer (TNBC). Furthermore, in vitro and in vivo studies have demonstrated that anti-Nectin-4 antibody-drug conjugates (ADCs) have better curative effects on localized and metastatic TNBC. Summary of the Invention [Problem to be solved by the invention]

[0006] The technical problem that the present disclosure aims to solve is to obtain a high-affinity antibody that specifically binds to Nectin-4 through hybridoma screening and humanization, which requires obtaining a fully human antibody sequence through humanization engineering. [Means for solving the problem]

[0007] In response to the above-mentioned technical problems, an object of the present disclosure is to provide an antibody molecule or a fragment thereof that specifically binds to Nectin-4, particularly human Nectin-4, and to provide uses thereof. The "fragment" of an antibody described herein particularly encompasses various functional fragments of an antibody, such as its antigen-binding portion, for example, Fab, F(ab')2, or scFv fragments.

[0008] The present disclosure provides the following technical solutions:

[0009] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (1) CDR-H1 (GYTFTTY), CDR-H2 (YPGNVN), and CDR-H3 (GLYYFDY) shown in SEQ ID NO: 35, SEQ ID NO: 39, and SEQ ID NO: 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49; (2) CDR-H1 (GYTFTTYYIH), CDR-H2 (WIYPGNVNTK), and CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 36, 40, and 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49; (3) CDR-H1 (TYYIH), CDR-H2 (WIYPGNVNTKYNEKFKG), and CDR-H3 (GLYYFDY) shown in SEQ ID NO: 37, SEQ ID NO: 41, and SEQ ID NO: 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49; (4) CDR-H1 (TTYYIH), CDR-H2 (WIGWIYPGNVNTK), and CDR-H3 (ARGLYYFD) shown in SEQ ID NO: 38, SEQ ID NO: 42, and SEQ ID NO: 44; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRY), and CDR-L3 (QQDYSSPY) shown in SEQ ID NO: 46, SEQ ID NO: 48, and SEQ ID NO: 50; (5) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65; (6) CDR-H1 (GFSLIDYGVS), CDR-H2 (VIWGDGKIY), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 52, SEQ ID NO: 56, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65; (7) CDR-H1 (DYGVS), CDR-H2 (VIWGDGKIYYNSVLKS), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 53, SEQ ID NO: 57, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65; (8) CDR-H1 (IDYGVS), CDR-H2 (WLGVIWGDGKIY), and CDR-H3 (AKQGGLLFYAMD) shown in SEQ ID NO: 54, SEQ ID NO: 58, and SEQ ID NO: 60; and CDR-L1 (LNSYSQKNYLAWY), CDR-L2 (LLIYFASTRE), and CDR-L3 (QQHYNTPF) shown in SEQ ID NO: 62, SEQ ID NO: 64, and SEQ ID NO: 66; (9) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, SEQ ID NO: 55, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 67, SEQ ID NO: 63, and SEQ ID NO: 65; (10) CDR-H1 (GFSLIDY), CDR-H2 (WGDAK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, SEQ ID NO: 68, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 67, SEQ ID NO: 63, and SEQ ID NO: 65; (11) CDR-H1 (GFSLIDY), CDR-H2 (WGGGK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NO: 51, SEQ ID NO: 69, and SEQ ID NO: 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NO: 67, SEQ ID NO: 63, and SEQ ID NO: 65; (12) CDR-H1 (GYTFTSY), CDR-H2 (YPGNAN), and CDR-H3 (SVYYFDY) shown in SEQ ID NO: 70, SEQ ID NO: 74, and SEQ ID NO: 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NO: 45, SEQ ID NO: 80, and SEQ ID NO: 49; (13) CDR-H1 (GYTFTSYYIH), CDR-H2 (WIYPGNANNK), and CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 71, 75, and 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49; (14) CDR-H1 (SYYIH), CDR-H2 (WIYPGNANNKYNENFKG), and CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 72, 76, and 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49; (15) CDR-H1 (TSYYIH), CDR-H2 (WIGWIYPGNANNK), and CDR-H3 (ARSVYYFD) shown in SEQ ID NOs: 73, 77, and 79; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRN), and CDR-L3 (QQDYSSPY) shown in SEQ ID NOs: 46, 81, and 50; (16) CDR-H1 (GYSFTDY), CDR-H2 (NPNNGN), and CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 82, SEQ ID NO: 86, and SEQ ID NO: 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96; (17) CDR-H1 (GYSFTDYYMH), CDR-H2 (RVNPNNGNTL), and CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 83, SEQ ID NO: 87, and SEQ ID NO: 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96; (18) CDR-H1 (DYYMH), CDR-H2 (RVNPNNGNTLYNQKFRG), and CDR-H3 (EDRYAFAY) shown in SEQ ID NO: 84, SEQ ID NO: 88, and SEQ ID NO: 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96; and (19) CDR-H1 (TDYYMH), CDR-H2 (WIGRVNPNNGNTL), and CDR-H3 (AREDRYAFA) shown in SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 91; and CDR-L1 (STSSYTYMHWY), CDR-L2 (LLIKYASNLE), and CDR-L3 (QHTWEIPY) shown in SEQ ID NO: 93, SEQ ID NO: 95, and SEQ ID NO: 97, The present invention provides an antibody molecule or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL) comprising a combination of heavy chain CDRs and light chain CDRs selected from:

[0010] The heavy chain variable region or light chain variable region in the antibody molecule or fragment thereof of the present disclosure comprises the above domain components arranged as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (where FR is a framework region), in accordance with the domain configuration of heavy chain variable regions or light chain variable regions in antibodies generally known in the art.

[0011] Preferably, in the antibody molecule or fragment thereof provided by the present disclosure, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:12, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or an amino acid sequence with at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 25, or an amino acid sequence with at least 75% identity to the amino acid sequence set forth, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:30, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28, SEQ ID NO:31 or SEQ ID NO:32, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, or The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth.

[0012] According to certain embodiments of the present disclosure, the heavy and light chain variable regions of the antibody molecule or fragment thereof are selected from the following combinations of amino acid sequences: (1) an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; (2) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; (3) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; (4) the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; (5) the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; (6) the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; (7) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 22; (8) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 23; (9) the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (10) the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 19; and the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (11) The amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; and the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (12) The amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; and the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (13) the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; and the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (14) An amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 30; and an amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; or (15) The amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; and the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 34.

[0013] According to the above-mentioned specific amino acid sequences of the heavy chain variable region or light chain variable region provided by the present disclosure, the amino acid sequences of the heavy chain CDRs and light chain CDRs contained therein can be routinely determined by those skilled in the art, and the resulting heavy chain CDRs and light chain CDRs, as well as combinations thereof, determined by other methods known in the art are also within the scope of the present disclosure.

[0014] The antibody molecules or fragments thereof provided by the present disclosure bind to poliovirus receptor-like molecule 4 (Nectin-4), preferably mammalian Nectin-4, more preferably primate Nectin-4, even more preferably human or cynomolgus monkey Nectin-4, and in particular human Nectin-4.

[0015] Preferably, the antibody molecule is a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody, and the fragment is any fragment of an antibody molecule capable of specifically binding to Nectin-4, such as a single-chain variable fragment (scFv), a disulfide-stabilized Fv fragment (dsFv), a (disulfide-stabilized Fv fragment)2 (dsFv)2, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a variable fragment (Fv).

[0016] Preferably, the antibody molecule is a monoclonal antibody or a single chain antibody.

[0017] Preferably, the antibody molecule or fragment thereof further comprises a human or mouse constant region, preferably a mouse or human heavy chain constant region (CH) and / or a light chain constant region (CL), and preferably the antibody molecule or fragment thereof comprises a heavy chain and a light chain, for example two heavy chains and two light chains. More preferably, the antibody molecule or fragment thereof comprises an IgG-, IgA-, IgM-, IgD- or IgE-type heavy chain constant region and / or a kappa- or lambda-type light chain constant region.

[0018] According to certain embodiments of the present disclosure, the antibody molecule provided by the present disclosure is a monoclonal antibody, preferably a humanized monoclonal antibody, and preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 type and the light chain constant region is of the kappa type. For example, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0019] In the context of the present disclosure, "at least 75% identity" refers to any percent identity between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0020] In another aspect, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region, a light chain variable region, a heavy chain, or a light chain comprised in an antibody molecule or fragment thereof according to the present disclosure.

[0021] The nucleic acid molecules of the present disclosure can be cloned into a vector, which then transfects or transforms a host cell. Thus, in yet another aspect, the present disclosure provides a vector comprising a nucleic acid molecule of the present disclosure. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0022] The vectors or nucleic acid molecules of the present disclosure can be used in any manner to transform or transfect a host cell, or to enter a host cell, such as for storage or expression of an antibody. Thus, in a further aspect, the present disclosure provides a host cell comprising or transformed or transfected with a nucleic acid molecule and / or vector according to the present disclosure. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, or animal cell.

[0023] Antibody molecules according to the present disclosure can be obtained using any conventional technique known in the art. For example, the heavy and / or light chain variable regions of the antibody molecule, or the heavy and / or light chains of the antibody molecule, can be obtained from nucleic acid molecules provided by the present disclosure, and then antibody molecules can be obtained by assembling them with any other domains of the antibody molecule, or host cells provided by the present disclosure can be cultured under conditions that allow the host cells to express the heavy and / or light chain variable regions of the antibody molecule, or the heavy and / or light chains of the antibody molecule, and assemble them into antibodies. Optionally, this method can further comprise the step of recovering the produced antibody molecules.

[0024] The antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, or fusion protein provided by the present disclosure may be contained in a composition, more particularly, a pharmaceutical preparation, and may be used for various purposes as needed. Thus, in a further aspect, the present disclosure also provides a composition comprising the antibody molecule or fragment thereof, nucleic acid molecule, vector, and / or host cell according to the present disclosure. The composition is preferably a pharmaceutical composition optionally containing a pharmaceutically acceptable carrier, adjuvant, or excipient.

[0025] In yet another aspect, the present disclosure also provides the use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the manufacture of an agent for the detection or diagnosis of a disease or disorder.

[0026] Accordingly, the present disclosure further provides methods for detecting or diagnosing a disease or disorder, comprising contacting the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell and / or composition with a sample derived from a subject, wherein the subject is a mammal, preferably a primate, more preferably a human.

[0027] In a further aspect, the present disclosure also provides the use of an antibody molecule or a fragment thereof in the preparation of an antibody drug conjugate.

[0028] Thus, the present disclosure provides an antibody drug conjugate formed by conjugating an antibody molecule or fragment thereof according to the present disclosure to a cytotoxic moiety.

[0029] Preferably, the cytotoxic moiety is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder. More preferably, the tubulin inhibitor is selected from the group consisting of maytansinoids, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl dolastatin 10, tubulysin and its derivatives, cryptophycin and its derivatives, and taltubulin. Preferably, the topoisomerase inhibitor is selected from the group consisting of PNU-159682 and its derivatives, which are metabolites of doxorubicin, and SN38 and its derivatives, which are metabolites of irinotecan (CPT-11). Preferably, the DNA binder is selected from the group consisting of PBD and its derivatives, and duocarmycin and its derivatives.

[0030] In a further aspect, the present disclosure also provides the use of the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition and / or antibody drug conjugate in the manufacture of a medicament for the prevention or treatment of a disease or disorder.

[0031] In another aspect, the present disclosure also provides a method for preventing or treating a disease or disorder, comprising administering an antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition and / or antibody drug conjugate according to the present disclosure to a subject in need thereof. The subject is a mammal, more preferably a human.

[0032] Thus, in a further aspect, the present disclosure provides a kit comprising an antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition and / or antibody drug conjugate according to the present disclosure, which kit can be used for therapeutic, detection or diagnostic purposes, such as treating, detecting or diagnosing a disease or disorder.

[0033] According to various embodiments provided by the present disclosure, the antibody molecule or fragment thereof, nucleic acid molecule, vector, host cell, composition, and / or antibody-drug conjugate can be used for the prevention, treatment, detection, or diagnosis of a disease or disorder associated with high expression of Nectin-4. Preferably, the disease or disorder is a tumor or cancer, particularly a solid tumor, in which Nectin-4 is highly expressed. For example, the disease or disorder is bladder cancer, pancreatic cancer, breast cancer (including triple-negative and basal subtypes), non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, etc., particularly bladder cancer, breast cancer, ovarian cancer, or lung cancer.

[0034] Compared with the prior art, in the present disclosure, high-affinity antibodies that specifically bind to Nectin-4 are obtained through hybridoma screening and humanization, and fully human antibody sequences are obtained through humanization engineering. Furthermore, studies on the physicochemical properties and cellular activity of the provided molecules confirm that the sequences of clinically effective lead drug molecules can be obtained.

[0035] Embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0036] [Figure 1A] Figure 1 shows the results of identifying antigen-expressing cell lines by flow cytometry. Panel 1A: HT-1376 bladder cancer cells [Figure 1B] Panel 1B shows the results of identifying antigen-expressing cell lines by flow cytometry. Panel 1B: CHO-huNectin4 S8 cells. [Figure 2A] Panel 2A shows the results of detecting the binding activity of hybridoma cell culture supernatants by FACS assay. Panel 2A: First screening [Figure 2B] Panel 2B shows the results of detecting the binding activity of hybridoma cell culture supernatants by FACS assay. Panel 2B: Second screening [Figure 2C] FIG. 1 shows the results of detecting the binding activity of hybridoma cell culture supernatants by FACS assay. [Figure 3A] FIG. 1 shows experimental results of antibody binding to BT474 cells by FACS assay. [Figure 3B] FIG. 1 shows experimental results of antibody binding to BT474 cells by FACS assay. [Figure 3C] FIG. 1 shows experimental results of antibody binding to BT474 cells by FACS assay. [Figure 3D] FIG. 1 shows experimental results of antibody binding to BT474 cells by FACS assay. [Figure 4A] FIG. 1 shows experimental results of the endocytic activity of antibodies in BT474 cells. [Figure 4B] FIG. 1 shows experimental results of the endocytic activity of antibodies in BT474 cells. [Figure 4C] FIG. 1 shows experimental results of the endocytic activity of antibodies in BT474 cells. [Figure 4D] FIG. 1 shows experimental results of the endocytic activity of antibodies in BT474 cells. [Figure 5A] FIG. 1 shows experimental results of antibody binding to cells expressing huNectin4, muNectin4, and cynoNectin4 by FACS assay. [Figure 5B] FIG. 1 shows experimental results of antibody binding to cells expressing huNectin4, muNectin4, and cynoNectin4 by FACS assay. [Figure 5C] FIG. 1 shows experimental results of antibody binding to cells expressing huNectin4, muNectin4, and cynoNectin4 by FACS assay. [Figure 6A] Figure 6 shows experimental results of antibody binding to members of the nectin family. Panel 6A: Nectin-1 [Figure 6B] Figure 6B shows experimental results of antibody binding to members of the nectin family. Panel 6B: Nectin-2 [Figure 6C]Figure 6 shows experimental results of antibody binding to members of the nectin family. Panel 6C: Nectin-3 [Figure 6D] Figure 6D shows experimental results of antibody binding to members of the nectin family. Panel 6D: Nectin-4 [Figure 7A] Figure 7 shows experimental results of antibody stability in cynomolgus monkey serum. Panel 7A: Control antibody Enfortumab [Figure 7B] Figure 7B shows the experimental results of antibody stability in cynomolgus monkey serum. Panel 7B: 42D20 hz10. [Figure 8A] Figure 8 shows the in vivo pharmacokinetics of antibodies in mice. Panel 8A: Control antibody Enfortumab [Figure 8B] In vivo pharmacokinetics of antibodies in mice. Panel 8B: 42D20 hz10 DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure in any way.

[0038] All experimental procedures in the following examples are conventional unless otherwise specified. All raw materials and reagents used in the following examples are commercially available unless otherwise specified.

[0039] The heavy and light chain amino acid sequences of the control antibody enfortumab are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0040] The sequences of the antibodies provided by this disclosure are shown in Appendices I-IV.

[0041] The antigen, i.e., recombinant NECTIN4 protein (accession number: NP_002178.2, 32aa to 349aa), is shown in SEQ ID NO:3. [Example]

[0042] Example 1 Synthesis and Expression of Control Antibodies Completely synthetic genes encoding the heavy and light chain variable regions of enfortumab were cloned upstream of the genes encoding the human kappa light chain constant region and human IgG1 heavy chain constant region of the eukaryotic expression vector pCDNA3.1, respectively, to obtain the light and heavy chain expression vectors for enfortumab. These two vectors were then transformed into Escherichia coli for propagation, and large amounts of vectors carrying the enfortumab antibody light chain (SEQ ID NO: 2) and heavy chain (SEQ ID NO: 1) genes were isolated. HEK293 cells were co-transfected with the two vectors mixed with polyethyleneimine (PEI), and the culture supernatant was collected 5 to 6 days after cell transfection. The antibody enfortumab was obtained by purifying the expression supernatant using a Mabselect affinity chromatography column.

[0043] Example 2 Preparation of a cell line expressing the antigen Nectin-4 The Nectin-4 gene reading frame was cloned by PCR from a vector containing Nectin-4 cDNA (Sino Biological, Inc., catalog number: HG19771-UT) and then cloned into a stable expression vector containing the glutamine synthetase (GS) gene for screening by enzymatic digestion. Suspension-cultured CHO-K1 cells were electrotransfected (Nucleofector IIb, Lonza). The transfected cells were transferred to CD CHO AGT™ medium (Gibco, catalog number: 12490-025) containing 50 μM MSX (Sigma, catalog number: M5379) and seeded into 96-well cell culture plates. After 2–3 weeks at 37°C and 5% CO2, nine wells containing cells were obtained by pre-screening with MSX pressure. The cells were then expanded in 24-well cell culture plates, and clone S8, which showed high antigen expression, was finally selected by flow cytometry (FACS) analysis. The clones were scaled up and cryopreserved.

[0044] The highly expressing clone S8 was named CHO-huNectin4 S8. The results of cell identification compared with HT-1376 bladder cancer cells that endogenously express Nectin-4 are shown in Figure 1.

[0045] Example 3 Screening and identification of hybridoma cells 1. Immunization of Mice Ten 8-week-old Balb / c mice were divided into two groups and immunized by a conventional immunization procedure using the modified CHO-huNectin4 S8 cell line as the immunizing agent, and a rapid immunization procedure using recombinant human Nectin4 protein (purchased from Novoprotein Scientific, Inc., catalog number: CJ19) as the immunizing agent.

[0046] Blood collected from mice before immunization was used as a negative control. Two immunizing agents were intraperitoneally injected, and the second and third immunizations were performed with a two-week interval. Blood was collected one week after the third immunization, and the serum titer was tested. Mice with high titers were selected and given a final boost three days before fusion.

[0047] 2. Fusion and Screening SP20 myeloma cells were harvested and grown to a certain size. One day before fusion, the cells were fed with fresh medium to ensure good cell growth conditions for fusion. On the day of fusion, the myeloma cells were harvested, centrifuged, suspended in basal medium, counted, and used.

[0048] Spleens and lymph nodes were aseptically removed from mice, pulverized to prepare cell suspensions, and then filtered through a cell filter. Erythrocyte lysis was performed, and the resulting suspensions were pooled and counted. B cells and SP20 myeloma cells were mixed at a 1:2 ratio, centrifuged, washed twice with electrofusion buffer, and resuspended to approximately 1 × 10 cells. 7 pieces / ml~2×10 7The cell density was adjusted to 100 cells / ml. The cell suspension was placed in an electric shock cup for fusion, added to complete culture medium, and placed in an incubator at 37°C with 8% CO2 for 30 to 240 minutes to recover. The cells were then added to HAT culture medium and cultured in a 384-well plate. On day 5, the cells were replenished with HAT culture medium, and on day 7, the HAT culture medium was replaced with HT culture medium. On days 8 to 10, screening for positive hybridomas was performed as follows.

[0049] Culture supernatants of hybridoma cells were collected and analyzed by FACS assay. Positive wells were screened for binding to CHO-huNectin4 S8 cells, which stably express the human Nectin4 antigen on their cell surface, but not to blank CHOK1 cells. Single cells from the screened positive wells were isolated by limiting dilution, and the subcloning procedure was terminated when 100% of the cells obtained after two consecutive subclonings were detected to be 100% positive. Each of the resulting hybridoma cell clones secreted only one antibody.

[0050] The results of detecting the binding activity of the hybridoma cell culture supernatant by FACS assay are shown in FIG. 2 and Tables 1 to 3.

[0051] [Table 1]

[0052] [Table 2]

[0053] [Table 3]

[0054] The resulting mouse monoclonal antibodies were named after the IDs of the hybridoma cell lines.

[0055] Example 4 Identification of variable region sequences of mouse monoclonal antibodies Monoclonal hybridoma cells secreting anti-human Nectin-4 antibodies were grown in culture, and total cellular RNA was extracted using the RNAfast200 kit (Shanghai Flytech Biotechnology Co., Ltd.) according to the instructions provided with the kit. The resulting hybridoma total RNA was reverse transcribed into cDNA using 5x PrimeScript RT Master Mix (Takara). The antibody light chain variable region (IgVL(κ)) and heavy chain variable region (VH) sequences were amplified using degenerate primers (Anke Krebber, 1997) and Extaq PCR reagent (Takara). The PCR amplified products were purified using a PCR Cleanup Gel Extraction Kit (Macherey-Nagel GmbH & Co.), ligated into a T-vector using the pClone007 Simple Vector Kit (Tsingke Biotechnology Co., Ltd.) according to the instructions provided with the kit, and transformed into competent E. coli cells. The variable region sequences of the monoclonal antibodies were obtained by DNA sequencing after strain amplification and plasmid extraction.

[0056] Example 5 Preparation of chimeric antibodies The heavy chain variable region sequence of each mouse anti-human Nectin-4 monoclonal antibody was spliced ​​together with the heavy chain constant region sequence of a published human monoclonal antibody of the IgG1 subclass (SEQ ID NO: 4) to construct a mammalian cell expression vector. The light chain variable region sequence of each mouse anti-human Nectin-4 monoclonal antibody was spliced ​​together with the light chain constant region sequence of a published human monoclonal antibody of the kappa subclass (SEQ ID NO: 5) to construct a mammalian cell expression vector. The heavy and light chain vectors of the constructed anti-human Nectin-4 chimeric antibody were mixed in pairs, and the vectors were transfected into HEK293 cells using polyethyleneimine (PEI). After approximately 7 days, the cell supernatant was collected, and the anti-human Nectin-4 chimeric antibody protein was obtained using MabSelect.

[0057] The resulting chimeric antibodies were named according to the format "mouse antibody abbreviation-xiIgG".

[0058] Example 6 Humanization of mouse antibodies and preparation of humanized antibodies Based on a comprehensive analysis of the antibody coding scheme, the amino acid sequence regions of the six complementarity-determining regions (CDRs) of the heavy and light chains of each mouse antibody, as well as the framework regions supporting the conserved three-dimensional structure, were determined. Subsequently, the heavy chain variable region sequences of human antibodies similar to the mouse antibodies, primarily IGHV1|IGHJ4*01, were searched against known human antibody sequences. The framework region sequences in the sequences were then selected as templates, and the heavy chain CDRs of the mouse antibody were combined with the framework regions of the human antibody to finally generate a humanized heavy chain variable region sequence. Similarly, a humanized light chain variable region sequence was generated.

[0059] Antibodies in which mouse CDRs are directly grafted onto human framework regions often exhibit a dramatic decrease in binding activity, necessitating the conversion of individual amino acids within the framework regions from human to mouse. To determine which positions need to be reverted to the original mouse residues, the designed humanized antibody sequence must be compared with the original mouse antibody sequence to identify amino acid differences and determine whether these different amino acids are important for supporting antibody structure or binding to the antigen. The sequence obtained by humanization design must also be checked for potential post-translational modification sites, such as N-glycosylation sites, N-deamidation sites, and D-aspartic acid isomerization sites.

[0060] The humanized heavy and light chain variable regions were combined to obtain a humanized antibody by referring to the procedure for preparing a chimeric antibody described in Example 5. The humanized antibody was named according to the format "mouse antibody abbreviation-hzmn", where the numbers of the modified humanized sequences of VH and VL of the mouse antibody (VH_hz and VL_hz) are m and n, respectively.

[0061] Example 7 Preparation of Antibody Drug Conjugates (ADCs) The antibody was reduced with 2.0 to 2.6 equivalents of TECP in PBS (pH 7.4) for 2 hours, and a solution of DMA containing vcMMAE was added to the TECP-reduced antibody solution (at a molar ratio of vcMMAE to antibody of 6:1). After stirring at 2 to 8°C for 1 hour, DMA and small molecule residues were removed by ultrafiltration. The absorbance of the conjugate was measured at 248 to 280 nm using a UV spectrophotometer, and the conjugate concentration was calculated. The resulting conjugate was subpackaged into cryotubes for storage at -80°C, and the DAR value of the conjugate (4.0 ± 1) was determined by HPLC-HIC.

[0062] ADCs were named by adding the suffix "E" to the name of the corresponding antibody.

[0063] Example 8 In vitro cell binding assay The anti-human Nectin-4 control antibody Enfortumab and the antibodies or ADCs of the present disclosure were diluted two-fold from an initial concentration of 100 nM to obtain solutions of each antibody or ADC at 16 concentrations. 10 μL of each solution at different concentrations was added to each well of a 384-well plate.

[0064] BT474 cells (breast cancer cells) expressing Nectin-4 on the cell surface were collected by centrifugation at 100 g for 5 minutes at room temperature, washed once with PBS containing 0.5% BSA, and centrifuged at 100 g for 5 minutes at room temperature. Approximately 2 × 10 cells were collected. 6 The cells were resuspended at a density of 1000 cells / ml and 10 μl was added to each well of a 384-well plate containing antibody or ADC. After 1 hour of incubation at 4°C, a fluorescently labeled goat anti-human IgG secondary antibody was added. After 1 hour of continued incubation at 4°C, the mean fluorescence reading of the cell population was analyzed by flow cytometry.

[0065] Experimental results of binding of murine antibody molecules of the present disclosure to BT474 cells by FACS assay are shown in FIG. 3, panel 3A and Tables 4 and 5.

[0066] [Table 4]

[0067] [Table 5]

[0068] Experimental results of binding of modified humanized molecules of the present disclosure to BT474 cells by FACS assay are shown in Figure 3, panels 3B and 3C and in Tables 6 and 7.

[0069] [Table 6]

[0070] [Table 7]

[0071] Experimental results of binding of ADCs of the disclosure to BT474 cells by FACS assay are shown in Figure 3, panel 3D and Table 8.

[0072] [Table 8]

[0073] Example 9 In vitro cytodiagnostic assay of antibodies 9.1 Endocytosis assay in BT474 cells 1. BT474 cells were collected by centrifugation at 1200 rpm for 8 minutes and washed twice with DPBS (Gibco, Catalog No.: 14190-136). 2. 1E5 cells were seeded per well, and each antibody or ADC was diluted 2-fold from an initial concentration of 10 μg / ml to obtain a total of 7 concentrations of each antibody or ADC solution, with the last concentration actually used as a blank well. The antibody and ADC solutions were added to the cells, and the resulting mixture was incubated on ice for 1 hour. 3. The cells were washed twice with ice-cold PBS and centrifuged at 1200 rpm for 8 minutes. Then, the cells were resuspended in RPMI 1640 medium supplemented with L-glutamine and HEPES and divided into three equal portions, one of which was incubated at 37°C for different times, one of which was kept on ice at all times and used as a zero-point control with no endocytosis, and one of which was not added with antibody or ADC and used as a NC control. 4. The cells were washed with citric acid (pH 2.7) for 3.5 minutes, neutralized with 1 M Tris-HCl solution (pH 9.5), washed twice with PBS, resuspended in an appropriate amount of 1% BSA-PBS, and then detected using an IQplus instrument. 5. Data were analyzed and processed using GraphPad Prism software.

[0074] The experimental results of the endocytic activity of the murine antibody molecules of the present disclosure in BT474 cells are shown in FIG. 4, panel 4A and Table 9.

[0075] [Table 9]

[0076] The experimental results of the endocytic activity of modified humanized molecules of the present disclosure in BT474 cells are shown in Figure 4, panels 4B and 4C, and Tables 10 and 11.

[0077] [Table 10]

[0078] [Table 11]

[0079] The experimental results of the endocytic activity of the ADCs of the disclosure in BT474 cells are shown in Figure 4, panel 4D and Table 12.

[0080] [Table 12]

[0081] 9.2 Growth inhibition assay in BT474 cells Nectin4-expressing breast cancer BT474 cells were cultured, harvested by trypsinization, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Cells were then plated at a density of 4,000 cells per well and incubated at 37°C and 5% CO2 for 24 hours. The antibodies or ADCs to be tested were dissolved in medium containing 1% BSA and diluted three-fold from an initial concentration of 200 μg / ml, resulting in a total of nine concentrations of each antibody or ADC solution, including zero. 100 μl of diluted antibody or ADC solution was added per well to a 96-well plate, and 100 μl of cell culture was added to each well containing the antibody or ADC solution, mixed, and incubated at 37°C and 5% CO2 for 120 hours. CCK-8 was prepared at a concentration of 5 μM in medium containing 1% BSA. 20 μl of the CCK-8 solution was added to each well of the 96-well plate and incubated at 37°C and 5% CO2 for 4 hours. Finally, the plate was incubated at room temperature for 15 minutes and then thoroughly mixed. The plate was read using a detection wavelength of 450 nm. Using SoftMax Pro, the working concentration (ng / ml) of naked antibody or ADC was plotted on the X-axis against the measured absorbance value on the Y-axis, and a four-parameter fit was performed to obtain the EC50 values ​​for the naked antibody and ADC.

[0082] The results are shown in Tables 13, 14 and 15.

[0083] [Table 13]

[0084] [Table 14]

[0085] [Table 15]

[0086] Example 10 Analysis of Antibody Cross-Binding Activity to Antigen - Facs cell: CHO-huNectin4 S8; HEK293 cells expressing mouse Nectin4 (NP_082169): HEK-muNectin4; and HEK293 cells expressing Cyno Nectin4 (SEQ ID NO: 6): HEK-cynoNectin4.

[0087] This experiment was carried out with reference to the procedure described in Example 8. The results are shown in Panels 5A, 5B, and 5C of Figure 5 and Tables 16, 17, and 18.

[0088] [Table 16]

[0089] [Table 17]

[0090] [Table 18]

[0091] Example 11. Antibody in vitro binding affinity and kinetics assays The interaction between the antibody and antigen was measured using a GE BIAcore S200 instrument. Referring to the instructions provided with the GE Healthcare biotin capture kit, the analytical and control sample channels on the CAP sensor chip were first bound to the antigen, i.e., His-tagged human NECTIN4. Then, antibody-containing samples (each diluted 3-fold from an initial concentration of 20 nM to obtain a total of eight antibody solutions, with a repeat concentration of 0.741 nM) were flowed through both the analytical and sample channels, and the optical response upon antibody-antigen binding was measured. The association constant (Kon), dissociation constant (Koff), and affinity constant (KD) of each antibody were finally obtained by instrument software fitting analysis (1:1 binding mode).

[0092] The results are shown in Table 19.

[0093] [Table 19]

[0094] Example 12: Verification of cross-binding activity of antibodies against antigenic proteins belonging to the same family antigen: Human Nectin-1 (C-6His), Novoprotein catalog number C492; Human Nectin-2 (C-6His), Novoprotein catalog number C440; Human Nectin-3 (C-6His), Novoprotein catalog number C630; Human Nectin-4 (C-6His), Novoprotein catalog number CJ19. Antibody (primary antibody): An antibody of the present disclosure. Control antibody: CD111 / Nectin-1 / PVRL1 antibody, rabbit Fab, 80244-RP01-100, Sino biologics; Anti-Nectin 2 antibody (ab233085), rabbit antibody, Abcam; Anti-Nectin 3 antibody (ab137961), rabbit antibody, Abcam. Secondary antibody: Goat anti-rabbit IgG-Fc secondary antibody (HRP) catalog number SSA003, Jackson Immuno.

[0095] Plates were coated with 1 μg / ml of antigen and incubated overnight at 4°C. Serially diluted antibodies were then added to the plates. Finally, HRP-labeled secondary antibodies were added, and absorbance at 450 nm was detected. The results are shown in panels 6A, 6B, 6C, and 6D of Figure 6.

[0096] These results demonstrate that the antibodies of the present disclosure exhibited properties consistent with the control antibodies, all specifically recognized the antigen Nectin-4, exhibited dose-dependent binding effects, and had no cross-binding activity against other proteins in the same family as Nectin-4.

[0097] Example 13 In vitro stability study of antibodies in cynomolgus monkey serum Experimental materials: antibody to be tested, FBS, antigen to which the antibody binds, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML) and HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098). Experimental equipment: 37°C incubator and microplate reader.

[0098] Testing Procedure: Sample preparation: 1) A solution of the antibody to be tested was adjusted to a concentration of 20 μg / ml, filtered for sterilization, and aliquoted into 250 μl / tube for use. 2) An equal volume of cynomolgus monkey serum was added to each aliquot of the antibody solution to obtain a sample containing serum at a final concentration of 50% and antibody at a final concentration of 10 μg / ml. 3) A total of seven such samples were prepared, sealed with sealing film, and placed at 37°C to keep the samples sterile throughout the process. 4) Samples were taken on days 0, 3, 7, 10, 14 and 21, respectively, and kept at 4°C for testing, and the sample taken on day 21 was kept at 4°C for at least one day.

[0099] Test Method: 1) Two 96-well ELISA plates were coated overnight at 4°C with 0.2 μg / ml of antigen in PBS and 100 μl per well of anti-IgG Fab monoclonal antibody in PBS. 2) Prepare the following required reagents: Blocking buffer: 5% BSA + PBS Antibody dilution buffer 5%BSA+PBS+50%FBS ELISA plate washing buffer 0.1% Tween + PBS 3) The two coated ELISA plates were washed three times with 300 μl / well of PBS to wash away uncoated antigen. 4) 200 μl of blocking buffer was added per well, and the plate was blocked at 37° C. for 1 hour. 5) The antibody to be tested in each sample was diluted to 2 μg / ml with antibody dilution buffer, and then further diluted 3-fold in a gradient fashion to obtain a total of 8 concentrations of each antibody solution. 6) The blocking buffer in the two coated ELISA plates was discarded, and 100 μl of the diluted antibody solution was added to each well of the plate, followed by incubation at 37° C. for 1 hour. 7) The plate was washed three times with PBST. 8) 100 μl of secondary antibody diluted 1:5000 was added per well to the washed ELISA plate and then incubated at RT for 40 minutes. 9) The plate was washed three times with PBST. 10) 100 μl of TMB substrate was added per well, and the color was developed in the dark for 10 minutes. 11) The reaction was stopped by adding 50 μl of 2 M HCl, and the absorbance was read at 450 nm.

[0100] Data Processing: The changes in the antibody binding curves over different time periods were observed, and the binding curves were plotted using ELISA values ​​to evaluate the stability of the antibody binding activity. The results are shown in panels 7A and 7B of Figure 7.

[0101] The results demonstrate that the effective antibody content of the antibodies of the present disclosure did not change after incubation at 37°C for 21 days, i.e., the antibodies can be stably stored at 37°C for more than 21 days.

[0102] Example 14 Analysis of antibody pharmacokinetics in mice Experimental materials: the antibody under test, serum collected from mice at different time points, the human Nectin-4 antigen to which the antibody binds, an anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), and an HRP-conjugated goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0103] Testing Procedure: Serum collection: 1) Female Balb / C mice were divided into groups of 3 mice, and 200 μg of antibody was administered to each group via the tail vein or intraperitoneally. 2) Blood was collected from the tail vein at different time points according to the experimental design, and the collected blood samples were left at room temperature for at least 30 minutes. After that, the serum was separated by centrifugation at 4000 rpm for 15 minutes and stored at -20°C. Assuming that the final serum sample may evaporate during frozen storage, it is recommended to collect a final serum sample of more than 20 μl. 3) The final serum sample collected should be frozen and stored at -20°C for at least 24 hours.

[0104] Test Method: 1) Two 96-well ELISA plates were coated overnight at 4°C with 0.2 μg / ml of antigen in PBS and 100 μl per well of anti-IgG Fab monoclonal antibody in PBS. 2) Prepare the following required reagents: Blocking buffer: 5% BSA + PBS Antibody dilution buffer: 5% BSA + PBS + 50% blank mouse serum ELISA plate washing buffer 0.1% Tween + PBS 3) The two coated ELISA plates were washed three times with 300 μl / well of PBS. 4) 200 μl of blocking buffer was added per well, and the plate was blocked at 37° C. for 1 hour. 5) Each serum sample was first diluted to an appropriate concentration with blocking buffer, and then diluted with antibody dilution buffer to obtain serial dilutions across the appropriate concentration range. The specific dilution ratios used to achieve the concentrations were determined by preliminary testing, provided that the dilution ratios allowed the final measurement values ​​for serum sample color development to fall within the range of the standard measurement values. 6) Antibody standards were diluted with antibody dilution buffer. The standard dilution method was also determined by adjustment in a preliminary test so that a linear curve could be fitted using the standards (or a sigmoidal curve could be fitted if appropriate software was available). 7) The blocking buffer in the two coated ELISA plates was discarded, and 100 μl of diluted antibody standards and serum samples to be tested were added to the plates per well, followed by incubation at 37° C. for 1 hour. 8) The plate was washed three times with PBST. 9) 100 μl of secondary antibody diluted 1:5000 was added per well to the washed ELISA plate, followed by incubation at 37° C. for 40 minutes. 10) The plate was washed three times with PBST. 11) 100 μl of TMB substrate was added per well and allowed to develop in the dark for 10 minutes. 12) The reaction was stopped by adding 50 μl of 2 M HCl, and the absorbance was read at 450 nm.

[0105] The results are shown in panels 8A and 8B of FIG. 8 and in Table 20.

[0106] [Table 20]

[0107] The above description of the embodiments of the present disclosure is not intended to limit the present disclosure, and those skilled in the art can make various changes and modifications to the present disclosure without departing from the spirit of the present disclosure, which should be included in the scope of the appended claims.

[0108] [Table 21]

[0109] [Table 22]

[0110] [Table 23]

[0111] [Table 24]

[0112] [Table 25]

[0113] [Table 26]

[0114] [Table 27]

[0115] [Table 28]

[0116] Table 29

[0117] Table 30

Claims

1. (1) CDR-H1 (GYTFTTY), CDR-H2 (YPGNVN), and CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 35, 39, and 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49; (2) CDR-H1 (GYTFTTYYIH), CDR-H2 (WIYPGNVNTK), and CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 36, 40, and 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49; (3) CDR-H1 (TYYIH), CDR-H2 (WIYPGNVNTKYNEKFKG), and CDR-H3 (GLYYFDY) shown in SEQ ID NOs: 37, 41, and 43; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRYT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 47, and 49; (4) CDR-H1 (TTYYIH), CDR-H2 (WIGWIYPGNVNTK), and CDR-H3 (ARGLYYFD) shown in SEQ ID NOs: 38, 42, and 44; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRY), and CDR-L3 (QQDYSSPY) shown in SEQ ID NOs: 46, 48, and 50; (5) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK) and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 55 and 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES) and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63 and 65; (6) CDR-H1 (GFSLIDYGVS), CDR-H2 (VIWGDGKIY) and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 52, 56 and 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES) and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63 and 65; (7) CDR-H1 (DYGVS), CDR-H2 (VIWGDGKIYYNSVLKS) and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 53, 57 and 59; and CDR-L1 (KSSQSLLNSYSQKNYLA), CDR-L2 (FASTRES) and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 61, 63 and 65; (8) CDR-H1 (IDYGVS), CDR-H2 (WLGVIWGDGKIY), and CDR-H3 (AKQGGLLFYAMD) shown in SEQ ID NOs: 54, 58, and 60; and CDR-L1 (LNSYSQKNYLAWY), CDR-L2 (LLIYFASTRE), and CDR-L3 (QQHYNTPF) shown in SEQ ID NOs: 62, 64, and 66; (9) CDR-H1 (GFSLIDY), CDR-H2 (WGDGK) and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 55 and 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES) and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63 and 65; (10) CDR-H1 (GFSLIDY), CDR-H2 (WGDAK), and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 68, and 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES), and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63, and 65; (11) CDR-H1 (GFSLIDY), CDR-H2 (WGGGK) and CDR-H3 (QGGLLFYAMDY) shown in SEQ ID NOs: 51, 69 and 59; and CDR-L1 (KSSQSLLNTYSQKNYLA), CDR-L2 (FASTRES) and CDR-L3 (QQHYNTPFT) shown in SEQ ID NOs: 67, 63 and 65; (12) CDR-H1 (GYTFTSY), CDR-H2 (YPGNAN), and CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 70, 74, and 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49; (13) CDR-H1 (GYTFTSYYIH), CDR-H2 (WIYPGNANNK) and CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 71, 75 and 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT) and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80 and 49; (14) CDR-H1 (SYYIH), CDR-H2 (WIYPGNANNKYNENFKG), and CDR-H3 (SVYYFDY) shown in SEQ ID NOs: 72, 76, and 78; and CDR-L1 (KASQSVSNDVA), CDR-L2 (YASNRNT), and CDR-L3 (QQDYSSPYT) shown in SEQ ID NOs: 45, 80, and 49; (15) CDR-H1 (TSYYIH), CDR-H2 (WIGWIYPGNANNNK), and CDR-H3 (ARSVYYFD) shown in SEQ ID NOs: 73, 77, and 79; and CDR-L1 (SNDVAWY), CDR-L2 (LLIYYASNRN), and CDR-L3 (QQDYSSPY) shown in SEQ ID NOs: 46, 81, and 50; (16) CDR-H1 (GYSFTDY), CDR-H2 (NPNNGN), and CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 82, 86, and 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96; (17) CDR-H1 (GYSFTDYYMH), CDR-H2 (RVNPNNGNTL), and CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 83, 87, and 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES), and CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96; (18) CDR-H1 (DYYMH), CDR-H2 (RVNPNNGNTLYNQKFRG) and CDR-H3 (EDRYAFAY) shown in SEQ ID NOs: 84, 88, and 90; and CDR-L1 (RASQSVSTSSYTYMH), CDR-L2 (YASNLES) and CDR-L3 (QHTWEIPYT) shown in SEQ ID NOs: 92, 94, and 96; and (19) CDR-H1 (TDYYMH), CDR-H2 (WIGRVNPNNGNTL) and CDR-H3 (AREDRYAFA) shown in SEQ ID NOs: 85, 89 and 91; and CDR-L1 (STSSYTYMHWY), CDR-L2 (LLIKYASNLE) and CDR-L3 (QHTWEIPY) shown in SEQ ID NOs: 93, 95 and 97; An antibody molecule or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL) comprising a combination of heavy chain CDRs and light chain CDRs selected from the group consisting of:

2. the antibody molecule or antigen-binding fragment thereof, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:10, or an amino acid sequence with at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:12, or an amino acid sequence with at least 75% identity to the amino acid sequence set forth; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or an amino acid sequence with at least 75% identity to said amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25, or an amino acid sequence with at least 75% identity to said amino acid sequence set forth, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:30, or an amino acid sequence having at least 75% identity to said amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28, SEQ ID NO:31, or SEQ ID NO:32, or an amino acid sequence having at least 75% identity to said amino acid sequence set forth, or 2. The antibody molecule or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 75% identity to the amino acid sequence set forth.

3. The antibody molecule or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region and the light chain variable region of the antibody molecule or antigen-binding fragment thereof are selected from the following combinations of amino acid sequences: (1) an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 7; and an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 8; (2) an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; (3) an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 9; and an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; (4) an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 11; (5) an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 10; and an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 12; (6) An amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 13; and an amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 14; (7) The amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 16; and the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 22; (8) An amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and an amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 23; (9) An amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 16; and an amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (10) The amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 19; and the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (11) An amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 21; and an amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 25; (12) The amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 27; and the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 28; (13) An amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 29; and an amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; (14) An amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 30; and an amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 32; Or, (15) The amino acid sequence set forth in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO: 33; and the amino acid sequence set forth in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity to the amino acid sequence set forth in SEQ ID NO:

34.

4. The antibody molecule or antigen-binding fragment thereof of claim 1, which binds to mammalian Nectin-4.

5. The antibody molecule or antigen-binding fragment thereof of claim 1, which binds to primate Nectin-4.

6. The antibody molecule or antigen-binding fragment thereof according to claim 1, which binds to human or cynomolgus monkey Nectin-4.

7. The antibody molecule is a murine antibody, a chimeric antibody, or a fully or partially humanized antibody, and the antigen-binding fragment is a single-chain variable fragment (scFv), a disulfide-stabilized Fv fragment (dsFv), a disulfide-stabilized Fv fragment (dsFv), or a disulfide-stabilized Fv fragment (dsFv) of the antibody molecule. ２ (dsFv) ２ , Fab fragment, Fab' fragment, F(ab') ２ An antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a fragment or fragment variable (Fv).

8. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule is a monoclonal antibody or a single-chain antibody.

9. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule or antigen-binding fragment thereof comprises a constant region.

10. 10. The antibody molecule or antigen-binding fragment thereof of claim 9, wherein the constant region comprises a mouse or human heavy chain constant region (CH) and / or a light chain constant region (CL).

11. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule or antigen-binding fragment thereof comprises a heavy chain and a light chain.

12. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE, and / or a kappa or lambda type light chain constant region.

13. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule is a monoclonal antibody.

14. The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody molecule is a humanized monoclonal antibody.

15. 15. The antibody molecule or antigen-binding fragment thereof of claim 14, wherein the monoclonal antibody comprises a heavy chain constant region of the IgG1 type and a light chain constant region of the kappa type.

16. A nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region and a light chain variable region contained in the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 15, or a nucleotide sequence encoding the heavy chain and the light chain contained in the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 15.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the nucleic acid molecule of claim 16 and / or the vector of claim 17, or transformed or transfected with the nucleic acid molecule of claim 16 and / or the vector of claim 17.

19. A composition comprising the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16, the vector according to claim 17 or the host cell according to claim 18.

20. 20. The composition of claim 19, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

21. Use of the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16, the vector according to claim 17, the host cell according to claim 18 or the composition according to claim 19 or 20 in the manufacture of an agent for the detection or diagnosis of a disease or disorder.

22. Use of the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 15 in the preparation of an antibody drug conjugate.

23. 16. An antibody drug conjugate formed by conjugating the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 15 to a cytotoxic moiety.

24. 24. The antibody drug conjugate of claim 23, wherein the cytotoxic moiety is a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder.

25. 25. The antibody-drug conjugate of claim 24, wherein the tubulin inhibitor is selected from the group consisting of maytansinoids, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl dolastatin 10, tubulysin and its derivatives, cryptophycin and its derivatives, and taltubulin.

26. 25. The antibody drug conjugate of claim 24, wherein the topoisomerase inhibitor is selected from the group consisting of PNU-159682, a metabolite of doxorubicin, and its derivatives, and SN38, a metabolite of irinotecan (CPT-11), and its derivatives.

27. 25. The antibody drug conjugate of claim 24, wherein the DNA binding agent is selected from the group consisting of PBD and its derivatives, and duocarmycin and its derivatives.

28. 28. Use of the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17, the host cell of claim 18, the composition of claim 19 or 20, or the antibody drug conjugate of any one of claims 23 to 27 in the manufacture of a medicament for the prevention or treatment of a disease or disorder.

29. 28. A kit comprising the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 15, the nucleic acid molecule of claim 16, the vector of claim 17, the host cell of claim 18, the composition of claim 19 or 20, or the antibody drug conjugate of any one of claims 23 to 27.

30. 30. The kit of claim 29 for use in the treatment, detection or diagnosis of a disease or disorder.

31. The use according to claim 21 or 28, wherein the disease or disorder is a tumor or cancer in which Nectin-4 is highly expressed.

32. 30. The use of claim 21 or 28, wherein the disease or disorder is a solid tumor.

33. 29. The use of claim 21 or 28, wherein the disease or disorder is bladder cancer, pancreatic cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, or ovarian cancer.

34. 34. The use of claim 33, wherein the breast cancer comprises a triple negative subtype and a basal subtype.

35. 33. The use according to claim 31 or 32, wherein the disease or disorder is bladder cancer, breast cancer, ovarian cancer or lung cancer.

36. The kit described in claim 30, wherein the disease or disorder is a tumor or cancer in which Nectin-4 is highly expressed.

37. The kit of claim 30, wherein the disease or disorder is a solid tumor.

38. The kit described in claim 30, wherein the disease or disorder is bladder cancer, pancreatic cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, or ovarian cancer.

39. The kit described in claim 38, wherein the breast cancer includes a triple-negative subtype and a basal subtype.

40. The kit described in claim 36 or 37, wherein the disease or disorder is bladder cancer, breast cancer, ovarian cancer or lung cancer.

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