Antibodies that bind to Nectin-4 and their use

Abstract

Antibodies and antigen-binding fragments that specifically bind to Nectin-4, as well as test kits containing the antibodies or antigen-binding fragments, are provided. Also provided are nucleic acids encoding the antibodies, host cells containing the nucleic acids, and methods for preparing the antibodies. Furthermore, the use of antibodies that specifically bind to Nectin-4 in diagnosis and prognosis prediction is also provided.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs. In particular, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-4, and a kit containing the antibody or the antigen-binding fragment. Furthermore, the present invention relates to a nucleic acid encoding the antibody, a host cell containing the nucleic acid, and a method for preparing the antibody. The present invention also relates to the use of an antibody that binds to Nectin-4 in the diagnosis and prognosis prediction.

Background Art

[0002] Nectin-4 (also known as "Poliovirus Receptor-Like Molecule 4 (PVRL4)") is a type I transmembrane glycoprotein with a molecular weight of approximately 66 KD. Nectin-4 belongs to the Nectin family members within the immunoglobulin superfamily. The extracellular domain of Nectin-4 consists of three Ig-like domains (VCC type) and, in cooperation with cadherin, is involved in the formation and maintenance of adherens junctions.

[0003] Nectin-4 is specifically expressed in embryos, placentas, and tumor cells, but is either not expressed at all or at very low levels in normal tissues and organs. Nectin-4 is a classic clinical tumor marker and serves as an ideal target for antibody-drug conjugates (ADCs). ADCs targeting Nectin-4 bind to Nectin-4 overexpressed on the surface of tumor cells, forming an ADC-receptor complex. Subsequently, the ADC undergoes target-mediated endocytosis, allowing it to enter the cell. Within lysosomes, the ADC is cleaved at its linker by cathepsin, releasing toxic small molecules that lead to the specific death of tumor cells. PADCEV® (also known as enfortumab vedotin-ejfv; Chinese name: enfortumab vedotin) is an ADC drug formed by conjugating enfortumab, a human IgG1 monoclonal antibody that targets the Nectin-4 protein, with the cytotoxic substance MMAE (monomethyl auristatin E, a microtubule disruptor) (Non-Patent Literature 1). PADCEV® is FDA approved for the treatment of locally advanced or metastatic urothelial carcinoma. However, PADCEV®, which targets Nectin-4, is intended for therapeutic use only and cannot be used for immunohistochemical (IHC) staining, thus lacking diagnostic utility.

[0004] In this field, there is a need for anti-Nectin-4 antibodies that specifically recognize Nectin-4 and can assay the presence and / or expression level of Nectin-4 in a sample with high sensitivity (e.g., by IHC assay), thereby achieving disease diagnosis or prognosis assessment. This is crucial for improving cancer patient survival rates, extending survival time, avoiding overtreatment with chemotherapy, and improving quality of life. Therefore, the use of highly specific and sensitive anti-Nectin-4 antibodies in diagnosis and prognosis prediction is of great practical value. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pia M. Challita-Eid et al. (2016) Cancer Res. 76(10):3003-13 [Overview of the project]

[0006] The present invention provides an anti-Nectin-4 antibody with high specificity and sensitivity that can reliably and sensitively detect Nectin-4. The antibody or antigen-binding fragment thereof of the present invention that specifically binds to Nectin-4 has one or more of the following characteristics. (a) When measured in an ELISA assay, it has the ability to specifically bind only to Nectin-4, and does not show specific binding to the Nectin family members Nectin-1, Nectin-2, Nectin-3, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5. (b) When measured in a flow cytometry assay, the ability to bind to the Nectin-4 molecule expressed on the cell, (c) When measured by Western blotting, Nectin-4 protein can be specifically detected, and when an excess of Nectin-4 protein is added, its binding to Nectin-4 protein on the membrane is inhibited, and / or (d) When measured by immunohistochemical staining, tissues expressing the Nectin-4 molecule are specifically stained, and for example, the tissues are selected from the group consisting of tonsils, salivary glands, brain, cerebellum, placenta, bladder, lungs, mammary glands, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureters, ovaries, fallopian tubes, cervix, endometrium, skin, kidneys, prostate, pancreas, thyroid, spleen, and liver.

[0007] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Nectin-4, comprising a heavy chain variable region and a light chain variable region. (a) The heavy chain variable region includes a variant of HCDR1 shown in SEQ ID NO: 5 having amino acid changes of HCDR1 or HCDR2 or less, a variant of HCDR2 shown in SEQ ID NO: 6 having amino acid changes of HCDR2 or less, and a variant of HCDR3 shown in SEQ ID NO: 7 having amino acid changes of HCDR3 or less, and the light chain variable region includes a variant of LCDR1 shown in SEQ ID NO: 8 having amino acid changes of LCDR1 or LCDR2 or less, a variant of LCDR2 shown in SEQ ID NO: 9 having amino acid changes of LCDR2 or less, and a variant of LCDR3 shown in SEQ ID NO: 10 having amino acid changes of LCDR3 or less, (b) The heavy chain variable region includes a variant of HCDR1 shown in SEQ ID NO: 15 having amino acid changes of HCDR1 or HCDR2 or less, a variant of HCDR2 shown in SEQ ID NO: 16 having amino acid changes of HCDR2 or less, and a variant of HCDR3 shown in SEQ ID NO: 17 having amino acid changes of HCDR3 or less, and the light chain variable region includes a variant of LCDR1 shown in SEQ ID NO: 18 having amino acid changes of LCDR1 or HCDR2 or less, a variant of LCDR2 shown in SEQ ID NO: 19 having amino acid changes of HCDR2 or less, and a variant of LCDR3 shown in SEQ ID NO: 20 having amino acid changes of HCDR3 or less, or (c) The heavy chain variable region includes the HCDR1 shown in SEQ ID NO: 25 or a variant of HCDR1 shown in SEQ ID NO: 25 having amino acid changes of 2 or less, the HCDR2 shown in SEQ ID NO: 26 or a variant of HCDR2 shown in SEQ ID NO: 26 having amino acid changes of 2 or less, and the HCDR3 shown in SEQ ID NO: 27 or a variant of HCDR3 shown in SEQ ID NO: 27 having amino acid changes of 2 or less; the light chain variable region includes the LCDR1 shown in SEQ ID NO: 28 or a variant of LCDR1 shown in SEQ ID NO: 28 having amino acid changes of 2 or less, the LCDR2 shown in SEQ ID NO: 29 or a variant of LCDR2 shown in SEQ ID NO: 29 having amino acid changes of 2 or less, and the LCDR3 shown in SEQ ID NO: 30 or a variant of LCDR3 shown in SEQ ID NO: 30 having amino acid changes of 2 or less.

[0008] In some embodiments, the changes in amino acids are conservative amino acid modifications.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 of the present invention is (a) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, (b) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 13, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 14, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or (c) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 24, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0010] In some embodiments, the antigen-binding fragment of the antibody that specifically binds to Nectin-4 of the present invention is Fab, Fab', F(ab')2, Fv, single-stranded Fv, single-stranded Fab, or diabody.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to Nectin-4 of the present invention is (a) The heavy chain sequence of SEQ ID NO: 1, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain sequence of SEQ ID NO: 2, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, for example, the heavy chain sequence shown in SEQ ID NO: 1 and the light chain sequence shown in SEQ ID NO: 2, (b) The heavy chain sequence of SEQ ID NO: 11, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain sequence of SEQ ID NO: 12, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, for example, the heavy chain sequence shown in SEQ ID NO: 11 and the light chain sequence shown in SEQ ID NO: 12, or (c) The heavy chain sequence of Sequence ID No. 21, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and the light chain sequence of Sequence ID No. 22, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, for example, the heavy chain sequence shown in Sequence ID No. 21 and the light chain sequence shown in Sequence ID No. 22.

[0012] In some embodiments, the antibody or antigen-binding fragment that specifically binds to Nectin-4 of the present invention is a mouse antibody or a rabbit antibody.

[0013] In some embodiments, the antibody or antigen-binding fragment that specifically binds to Nectin-4 in the present invention is an IgG class antibody.

[0014] In a second embodiment, the present invention provides a nucleic acid encoding the anti-Nectin-4 antibody or its antigen-binding fragment as described in the first embodiment, a nucleic acid-containing vector (preferably an expression vector), and a host cell containing the nucleic acid or vector. In some embodiments, the host cell is a prokaryotic or eukaryotic cell, selected from the group consisting of, for example, Escherichia coli (E. coli) cells, yeast cells, mammalian cells, or other cells suitable for the preparation of antibodies or their antigen-binding fragments. In some embodiments, the host cell is a HEK293 cell or a CHO cell.

[0015] In a third aspect, the present invention provides a method for producing an anti-Nectin-4 antibody or its antigen-binding fragment as described in the first aspect, comprising culturing a host cell as described in the second aspect under conditions suitable for expressing a nucleic acid encoding the anti-Nectin-4 antibody or its antigen-binding fragment as described in the first aspect, and recovering the anti-Nectin-4 antibody or its antigen-binding fragment expressed from the culture.

[0016] In a fourth aspect, the present invention provides a kit comprising the anti-Nectin-4 antibody or antigen-binding fragment described in the first aspect.

[0017] In a fifth aspect, the present invention provides the use of the anti-Nectin-4 antibody or an antigen-binding fragment thereof according to the first aspect for detecting the Nectin-4 level in a sample.

[0018] In a sixth aspect, the present invention provides a method for diagnosing cancer in a subject using the anti-Nectin-4 antibody or an antigen-binding fragment thereof according to the first aspect above.

[0019] In some embodiments, the present invention provides a method for determining the eligibility of a subject for treatment with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., an anti-Nectin-4 antibody or an antigen-binding fragment thereof having therapeutic efficacy) using the anti-Nectin-4 antibody or an antigen-binding fragment thereof according to the first aspect above.

[0020] In some embodiments, the present invention provides a method for predicting the prognosis of a subject having cancer using the anti-Nectin-4 antibody or an antigen-binding fragment thereof according to the first aspect above, the method comprising a subject having cancer being a subject treated with an anti-tumor agent (e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., an anti-Nectin-4 antibody or an antigen-binding fragment thereof having therapeutic efficacy)).

[0021] The preferred embodiments of the present invention, which will be described in detail below, are better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show the current preferred embodiments. However, it should be understood that the present invention is not limited to the exact configurations and means shown in the drawings.

Brief Description of the Drawings

[0022] [Figure 1]This figure shows an assay by ELISA for the binding of antibodies MM11, R012, and R036 to Nectin family proteins (Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, Nectin-like-5). [Figure 2] This figure shows the assays of binding of antibodies MM11, R012, and R036 to Nectin-4 expressing cells by flow cytometry (FACS). In the figure, "Blank" represents human prostate cancer cells (PC-3) treated only with the secondary antibody, without the addition of antibody MM11, antibody R012, or antibody R036 as the primary antibody; "NC" represents PC-3 cells treated only with blank buffer; and "PC-3-Nectin4" represents a PC-3 cell line expressing Nectin-4. [Figure 3] This figure shows the reactivity of antibodies MM11, R012, and R036 with Nectin-4 expressing cell lysates detected by Western blotting. In the figure, lane M: protein molecular weight marker, lane 1: PC-3 cell lysate, lane 2: PC-3-Nectin-4 cell lysate. The upper panel of Figure 3 shows that antibodies MM11, R012, and R036 each react with lysates derived from cells expressing Nectin-4, but not with lysates derived from cells that do not express Nectin-4. In the lower panel of Figure 3, "MM11 (inhibition)" and "R012 (inhibition)" show the results obtained by incubating membrane-transferred cell lysates with "MM11 antibody" or "R012 antibody," respectively, with the addition of an excess amount of recombinant Nectin-4 protein, while "Gapdh" shows the reaction in which membrane-transferred cell lysates were incubated with an anti-glyceraldehyde-3-phosphate dehydrogenase antibody (which functions as an internal reference in Western blotting). [Figure 4]This figure shows the results of immunohistochemical staining of representative normal human tissues using Nectin-4 rabbit monoclonal antibodies R012 and R036, and the positive control Nectin-4 rabbit monoclonal antibody EPR15613-68 (scale bar: 200 μm). [Figure 5] This figure shows the results of immunohistochemical staining of human bladder cancer tissue using Nectin-4 rabbit monoclonal antibodies R012 and R036, and the positive control Nectin-4 rabbit monoclonal antibody EPR15613-68 (scale bar: 200 μm). [Figure 6] This figure shows the results of immunohistochemical staining of human lung cancer tissue using Nectin-4 rabbit monoclonal antibodies R012 and R036, and the positive control Nectin-4 rabbit monoclonal antibody EPR15613-68 (scale bar: 200 μm). [Figure 7] This figure shows the results of immunohistochemical staining of human breast cancer tissue using Nectin-4 rabbit monoclonal antibodies R012 and R036, and the positive control Nectin-4 rabbit monoclonal antibody EPR15613-68 (scale bar: 200 μm). [Figure 8] This figure shows the results of immunohistochemical staining of representative normal human tissues using the Nectin-4 mouse monoclonal antibody MM11 and the positive control, the Nectin-4 mouse monoclonal antibody M22-321b41.1 (scale bar: 100 μm). [Figure 9] This figure shows the results of immunohistochemical staining of human prostate cancer tissue using the Nectin-4 mouse monoclonal antibody MM11 and the positive control, the Nectin-4 mouse monoclonal antibody M22-321b41.1 (scale bar: 50 μm). [Figure 10] This figure shows the results of immunohistochemical staining of human lung cancer tissue using the Nectin-4 mouse monoclonal antibody MM11 and the positive control, the Nectin-4 mouse monoclonal antibody M22-321b41.1 (scale bar: 50 μm). [Figure 11]This figure shows the results of immunohistochemical staining of human triple-negative breast cancer tissue using the Nectin-4 mouse monoclonal antibody MM11 and the positive control, the Nectin-4 mouse monoclonal antibody M22-321b41.1 (scale bar: 50 μm). [Modes for carrying out the invention]

[0023] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions described herein, and these methods and conditions may be modified.

[0024] I. Definition Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0025] The term "approximately" used in relation to a number is intended to encompass a range of numbers from a lower limit of 5% below the specified number to an upper limit of 5% above the specified number.

[0026] As used herein, the term "and / or" refers to one of the options or two or more of the options.

[0027] As used herein, the terms “comprise” or “include” also include elements, elements, or processes that describe the subject unless otherwise specified. For example, when referring to an antibody variable region that “contains” a particular sequence, it is also intended to include the antibody variable region consisting of that particular sequence.

[0028] As used herein, the term “antibody” refers to an immunoglobulin molecule that binds to an antigen. Embodiments of an antibody include monoclonal antibodies, polyclonal antibodies, or chimeric antibodies. Antibodies may belong to any class (e.g., IgG, IgE, IgM, IgD, IgA).

[0029] The exemplary antibodies of this disclosure are immunoglobulin G (IgG) class antibodies, comprising four polypeptide chains, namely two heavy chains (HC) and two light chains (LC), cross-linked by interchain disulfide bonds. The amino-terminus of each of the four polypeptide chains contains a variable region comprising approximately 100 to 125 or more amino acids, primarily involved in antigen recognition. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region.

[0030] The VH and VL regions can be further subdivided into structures in which highly variable regions known as complementarity-determining regions (CDRs) and more conserved regions known as framework regions (FRs) are alternately arranged. The CDRs are exposed on the surface of the protein and constitute important regions responsible for the antigen-binding specificity of the antibody. Each VH and VL contains three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the heavy chain are called "HCDR1, HCDR2, and HCDR3," while the three CDRs of the light chain are called "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues responsible for forming antigen-specific interactions.The amino acid residues are from Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard Conformations for the Canonical Structures of Immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (International, available from www.imgt.org). CDRs can be assigned according to an established numbering scheme, including those described in the ImMunoGeneTics database (see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212).

[0031] However, it should be noted that the CDR boundaries in the variable region of an antibody, as determined by different numbering schemes, may differ. That is, the CDR sequences in the variable region of the same antibody, as defined based on different numbering schemes, may differ. Therefore, when defining an antibody having a specific CDR sequence in the present invention, the range of antibodies may include antibodies that contain a specific CDR sequence in their variable region sequence, but whose boundaries are assigned using different numbering schemes (e.g., different numbering scheme rules or combinations thereof), and thus may have different CDR boundaries than the specific CDR boundary defined in the present invention.

[0032] The CDR boundary of the antibody of the present invention may be artificially determined by any numbering scheme or combination thereof within the art. Unless otherwise specified, the terms “CDR” or “CDR sequence” in the present invention include the CDR sequence determined by any of the above schemes.

[0033] An "antigen-binding fragment" refers to a complete antibody or a portion or segment of a complete antibody or the entire antibody that contains fewer amino acid residues than the entire antibody, and that retains the ability to bind to an antigen or to compete with the complete antibody (i.e., the complete antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Examples of antigen-binding fragments, but not limited to, include Fab, Fab', F(ab')2, Fv, single-stranded Fv (scFv), single-stranded Fab, diabodies, single-domain antibodies (sdAb, nanobodies), camelid Ig, Ig NAR, F(ab')3 fragments, bis-scFv, (scFv)2, minibodies, diabodies, tribodies, tetrabodies, and disulfide-stabilized Fv proteins ("dsFv"). This term also encompasses genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies), hybrid antibodies (e.g., bispecific antibodies), and their antigen-binding fragments. For further details, see also: Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL), Kuby, Journal of Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0034] The terms "Nectin family proteins" and "Nectin family" are used interchangeably to refer to cell adhesion molecules that form physical connections between adjacent cells, enabling intercellular communication, migration, and other important cellular processes. Nectin family proteins include at least Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5. Of these, the expression of Nectin-4 protein in healthy humans is primarily limited to placental and embryonic tissues. Compared to healthy adult tissues, many tumor cell types exhibit high expression of Nectin-4 protein. Nectin-4 protein is a tumor-associated antigen.

[0035] In the context of antigens and antibodies, the terms “binding” or “specific binding” mean that the binding is selective to the antigen and can be distinguished from undesirable or nonspecific interactions. The ability of an antibody to bind to a particular antigen can be determined using enzyme-linked immunosorbent assays (ELISA), SPR, biolayer interference, or other conventional binding assays known in the art.

[0036] To determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps can be introduced in either or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, and 100% of the reference sequence length. Then, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0037] Sequence comparison and percentage identity calculation between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlemane-Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm, incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percentage identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using an NWSgapdna.CMP matrix, gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and one of the parameter sets that should be used unless otherwise specified) is the Blossum 62 scoring matrix, a gap penalty of 12, a gap expansion penalty of 4, and a gap frame shift penalty of 5.

[0038] The percentage identity between two amino acid or nucleotide sequences can also be determined using the ALIGN program (version 2.0), which incorporates the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), using a PAM120 weighted remainder table with gap length penalties 12 and 4.

[0039] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used, for example, as “query sequences” for performing searches against public databases to identify sequences of other family members or related sequences.

[0040] With respect to polypeptide sequences, "conservative modification" or "conservative amino acid modification" refers to substitutions, deletions, or additions to the polypeptide sequence that result in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Mutants having such conservative modifications are additional to, and not excluded from, the polymorphic mutants, interspecific homologs, and alleles of the present invention. The following eight groups contain amino acids that are conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative amino acid modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing an amino acid sequence.

[0041] As used herein, “vector” refers to a construct capable of delivering one or more target genes or sequences into a host cell, and preferably capable of expressing such genes or sequences within the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phagemids, DNA or RNA expression vectors associated with cationic flocculants, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells such as producer cells.

[0042] In this invention, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which foreign nucleic acids have been introduced (including progeny cells derived from such cells). Host cells encompass “transformed organisms” and “transformed cells,” which include primary transformed cells and progeny cells derived from such cells, regardless of the number of passages. Progeny cells may not be identical to parent cells in terms of nucleic acid content and may have mutations. This disclosure includes mutant progeny cells that exhibit the same function or biological activity as cells screened or selected from initially transformed cells.

[0043] As used herein, “subject,” “individual,” or “subject” refers to an animal, preferably a mammal, more preferably a human, that requires the alleviation, prevention, treatment, and / or diagnosis of a disease or disorder. Mammals include, but are not limited to, livestock, athletic animals, pets, primates, horses, dogs, cats, mice, and rats. This term refers to a subject that has or is at risk of having a disease.

[0044] A “biological sample” from a subject refers to a collection of cells, tissues, or bodily fluids obtained from an individual or subject. Sources of tissue or cell samples may include solid tissues such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or fine-needle aspiration samples; blood or any blood component; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid (ascites), or interstitial fluid; or cells derived from a subject at any stage of pregnancy or development. Tissue samples may contain compounds not naturally present in tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics. Examples of tumor specimens used herein, but not limited to, include tumor biopsy specimens, fine-needle aspiration specimens, bronchoalveolar lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from or exhibiting tumor-like properties, and preserved tumor specimens such as formalin-fixed, paraffin-embedded tumor tissue sections or frozen tumor specimens.

[0045] The terms “reference sample,” “reference cells,” “reference tissue,” “control sample,” “control cells,” or “control tissue” refer to a sample, cell, tissue, or standard level used for comparison purposes. In one embodiment, the reference sample, reference cells, reference tissue, control sample, control cells, or control tissue are obtained from healthy and / or non-disease sites of the same subject or individual body and are healthy and / or non-disease tissues or cells. In yet another embodiment, the reference sample, reference cells, reference tissue, control sample, control cells, or control tissue are obtained from healthy tissues or cells of an individual that is not a subject. The presence or expression level of Nectin-4 in the “reference sample,” “reference cells,” “reference tissue,” “control sample,” “control cells,” or “control tissue” is referred to as the “reference presence or expression level.”

[0046] The term "primary antibody" refers to an antibody that specifically binds to Nectin-4 in a biological sample. The term "secondary antibody" refers to an antibody that specifically binds to the primary antibody, thereby forming a crosslink between the primary antibody and (if necessary) a subsequent reagent.

[0047] Immunofluorescence (IF) staining is one of the earliest techniques developed among labeled immunoassay methods. Based on the principle of antigen-antibody reactions, a known antigen or antibody is first labeled with a fluorescent group, and then the corresponding antigen (or antibody) in cells or tissues is detected using the fluorescent antibody (or antigen) as a probe. The antigen-antibody complex formed in tissue or cells contains the labeled fluorophore. When the specimen is observed under a fluorescence microscope, the fluorophore emits bright fluorescence (yellow-green or reddish-orange) when exposed to external excitation light. This allows for the visualization of tissue cells containing fluorescence, the determination of the properties and localization of the antigen or antibody, and the measurement of its content using quantitative methods.

[0048] Immunoprecipitation (IP) is a method for purifying and enriching specific proteins using antigen-antibody specificity reactions, and its main steps include capture and fixation.

[0049] Co-immunoprecipitation (Co-IP) is an extension based on immunoprecipitation. Co-IP is a method developed by utilizing the specific binding between antigen proteins and antibodies, and the phenomenon in which bacterial proteins, known as "protein A / G," specifically bind to the Fc fragment (immunoglobulin) of antibodies. Typically, protein A / G is pre-bound to agarose beads. After reacting the antigen protein (e.g., cell lysate) and bait protein X with a solution containing an antibody targeting them, protein A / G on the agarose beads adsorbs bait protein X via the antibody. Subsequently, bait protein X interacts with target protein Y, thereby separating target protein Y from the cell lysate, and the objective of co-immunoprecipitation is achieved.

[0050] II. The antibody that specifically binds to Nectin-4 according to the present invention The present invention provides antibodies that specifically bind to Nectin-4. In some embodiments, the antibodies of the present invention are monoclonal antibodies derived, for example, from any eukaryotic clone, prokaryotic clone, or phage clone. Monoclonal antibodies can be produced by hybridoma technology, recombination technology, phage display technology, B cell clone sorting technology, synthesis technology (e.g., CDR grafting), or a combination of other technologies known in the art.

[0051] Methods for antibody production and purification are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 5-8 and 15, ISBN 0-87969-314-2. For example, mice, rabbits, or other animals can be immunized with human Nectin-4, and the produced antibodies can be recovered and purified, and their amino acid sequences can be determined using conventional methods well known in the art. Similarly, phage libraries can be screened by evaluating the interaction between thousands of Fab fragments and human Nectin-4. The resulting interacting factors can then be recovered and purified, and their amino acid sequences can be determined using conventional methods well known in the art.

[0052] The terms “antibody that binds to Nectin-4 protein,” “antibody that binds to Nectin-4,” “antibody against Nectin-4 protein,” “anti-Nectin-4 antibody,” “isolated antibody that binds to Nectin-4 protein,” “Nectin-4 antibody,” and “Nectin-4 protein antibody” are used herein without distinction and refer to such antibodies of the present invention that can specifically bind to the Nectin-4 protein, thereby enabling the antibodies to be used as diagnostic and / or detection agents targeting the Nectin-4 protein.

[0053] The isolated anti-Nectin-4 antibody and antigen-binding fragment of the present invention specifically bind to the Nectin-4 protein and include a heavy chain variable region and a light chain variable region. (a) The heavy chain variable region includes a variant of HCDR1 shown in SEQ ID NO: 5 having amino acid changes of HCDR1 or HCDR2 or less, a variant of HCDR2 shown in SEQ ID NO: 6 having amino acid changes of HCDR2 or less, and a variant of HCDR3 shown in SEQ ID NO: 7 having amino acid changes of HCDR3 or less, and the light chain variable region includes a variant of LCDR1 shown in SEQ ID NO: 8 having amino acid changes of LCDR1 or LCDR2 or less, a variant of LCDR2 shown in SEQ ID NO: 9 having amino acid changes of LCDR2 or less, and a variant of LCDR3 shown in SEQ ID NO: 10 having amino acid changes of LCDR3 or less, (b) The heavy chain variable region includes a variant of HCDR1 shown in SEQ ID NO: 15 having amino acid changes of HCDR1 or HCDR2 or less, a variant of HCDR2 shown in SEQ ID NO: 16 having amino acid changes of HCDR2 or less, and a variant of HCDR3 shown in SEQ ID NO: 17 having amino acid changes of HCDR3 or less, and the light chain variable region includes a variant of LCDR1 shown in SEQ ID NO: 18 having amino acid changes of LCDR1 or HCDR2 or less, a variant of LCDR2 shown in SEQ ID NO: 19 having amino acid changes of HCDR2 or less, and a variant of LCDR3 shown in SEQ ID NO: 20 having amino acid changes of HCDR3 or less, or (c) The heavy chain variable region includes the HCDR1 shown in SEQ ID NO: 25 or a variant of HCDR1 shown in SEQ ID NO: 25 having amino acid changes of 2 or less, the HCDR2 shown in SEQ ID NO: 26 or a variant of HCDR2 shown in SEQ ID NO: 26 having amino acid changes of 2 or less, and the HCDR3 shown in SEQ ID NO: 27 or a variant of HCDR3 shown in SEQ ID NO: 27 having amino acid changes of 2 or less; the light chain variable region includes the LCDR1 shown in SEQ ID NO: 28 or a variant of LCDR1 shown in SEQ ID NO: 28 having amino acid changes of 2 or less, the LCDR2 shown in SEQ ID NO: 29 or a variant of LCDR2 shown in SEQ ID NO: 29 having amino acid changes of 2 or less, and the LCDR3 shown in SEQ ID NO: 30 or a variant of LCDR3 shown in SEQ ID NO: 30 having amino acid changes of 2 or less.

[0054] Here, amino acid changes refer to the addition, deletion, or substitution of amino acids, such as conservative amino acid substitutions. CDRs are defined according to the Kabat numbering scheme.

[0055] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention is (a) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, (b) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 13, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 14, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or (c) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 24, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0056] In some embodiments, the antibody of the present invention binds to mammalian Nectin-4, such as human Nectin-4. In some embodiments, the Nectin-4 antibody of the present invention binds to one or more extracellular domains of Nectin-4.

[0057] In some embodiments, the antibody of the present invention has one or more of the following properties: (a) When measured in an ELISA assay, it has the ability to specifically bind only to Nectin-4, and does not show specific binding to the Nectin family members Nectin-1, Nectin-2, Nectin-3, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5. (b) When measured in a flow cytometry assay, the ability to bind to the Nectin-4 molecule expressed on the cell, (c) When measured by Western blotting, Nectin-4 protein can be specifically detected, and when an excess of Nectin-4 protein is added, its binding to Nectin-4 protein on the membrane is inhibited, and / or (d) When measured by immunohistochemical staining, tissues expressing the Nectin-4 molecule are specifically stained, and for example, the tissues are selected from the group consisting of tonsils, salivary glands, brain, cerebellum, placenta, bladder, lungs, mammary glands, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureters, ovaries, fallopian tubes, cervix, endometrium, skin, kidneys, prostate, pancreas, thyroid, spleen, and liver.

[0058] III. Nucleic acids of the present invention and host cells containing the same In one embodiment, the present invention provides nucleic acids encoding any of the aforementioned Nectin-4 antibodies, their antigen-binding fragments, or their individual chains. In one embodiment, a vector comprising nucleic acids is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising nucleic acids or a vector is provided. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for the preparation of antibodies or antigen-binding fragments. In another embodiment, the host cell is a prokaryotic cell.

[0059] The present invention further includes nucleic acids that hybridize with the above nucleic acids under stringent conditions, or nucleic acids that encode polypeptide sequences having one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions relative to the above nucleic acids.

[0060] In one embodiment, one or more vectors containing the above-mentioned nucleic acids are provided. In one embodiment, the vector is an expression vector such as a eukaryotic expression vector. Examples of vectors, but not limited to, include viral vectors, plasmids, cosmids, λ phagemids, or yeast artificial chromosomes (YACs).

[0061] Once the expression vector or expression DNA sequence is prepared, the expression vector can be transfected or introduced into suitable host cells. For this purpose, several techniques can be employed, including protoplast fusion, calcium phosphate precipitation, electroporation, retroviral introduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are cultured in a culture medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells, as well as methods and conditions for recovering the produced antibody molecules, are known to those skilled in the art and may be modified or optimized based on the specific expression vector and mammalian host cells used, according to methods known herein and in the prior art.

[0062] IV. Production and Purification of the Antibody of the Present Invention In one embodiment, the present invention provides a method for preparing a Nectin-4 antibody, the method comprising culturing host cells containing a nucleic acid encoding the Nectin-4 antibody or an expression vector containing said nucleic acid under conditions suitable for expressing the nucleic acid encoding the Nectin-4 antibody, and optionally isolating the Nectin-4 antibody. In one embodiment, the method further comprises recovering the Nectin-4 antibody from the host cells or host cell culture.

[0063] To recombinantly produce the antibody of the present invention, first, the nucleic acid encoding the Nectin-4 antibody of the present invention is isolated and inserted into a vector for further cloning and / or expression in host cells. Such nucleic acid can be easily isolated and sequenced using conventional procedures, for example, by using an oligonucleotide probe that can specifically bind to the nucleic acid encoding the Nectin-4 antibody of the present invention.

[0064] The antibodies of the present invention, prepared as described herein, can be purified using known prior art techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions for purifying specific proteins also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibodies of the present invention can be determined by any of the various well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0065] V. Activity assay of the antibody of the present invention The Nectin-4 antibodies provided herein can be identified, screened, or characterized for their physicochemical properties and / or biological activity using various assays known in the art.

[0066] In some embodiments, the antigen-binding activity of the antibody of the present invention is assayed using known methods such as ELISA, Western blotting, and FACS.

[0067] The cells used in any of the aforementioned in vitro assays include cells that spontaneously express Nectin-4, or cell lines that have been engineered to express Nectin-4. Engineered cell lines that express Nectin-4 are cell lines that do not normally express Nectin-4 but express Nectin-4 after transfection with DNA encoding Nectin-4.

[0068] VI. Methods and kits for diagnosis and detection In some embodiments, any of the Nectin-4 antibodies provided herein may be used to assay the presence and / or level of Nectin-4 in a biological sample.

[0069] As used herein, the term “assay” includes “assay quantitatively or qualitatively.” Exemplary assays may include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody-conjugated magnetic bead assay, or ELISA assay. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from a hyperplastic or neoplastic lesion.

[0070] In one embodiment, a Nectin-4 antibody is provided for use in diagnostic or detection methods for the purpose of diagnosis or prognosis evaluation, for example, to detect and / or evaluate the presence and / or level of Nectin-4, to determine the effectiveness of a given treatment regimen.

[0071] In one embodiment, a method is provided for assaying the presence and / or levels of Nectin-4 in a biological sample. In some embodiments, the method includes assaying the presence and / or levels of Nectin-4 protein in a biological sample.

[0072] In some embodiments, Nectin-4 is human Nectin-4. In certain embodiments, the method includes contacting a biological sample with the Nectin-4 antibody described herein under conditions that enable the Nectin-4 antibody to bind to Nectin-4, and assaying whether a complex is formed between the Nectin-4 antibody and Nectin-4. This complex formation indicates the presence of Nectin-4. The method may be in vitro or in vivo. In one embodiment, the Nectin-4 antibody is used to select subjects suitable for treatment with a therapeutic Nectin-4 antibody, for example, Nectin-4 functions as a biomarker for subject selection.

[0073] In one embodiment, the antibody of the present invention can be used to diagnose cancer or tumors expressing the Nectin-4 protein, for example, to evaluate therapeutic efficacy in a subject, or to diagnose solid tumors expressing the Nectin-4 protein (e.g., sarcomas and carcinomas affecting multiple organ systems, e.g., those invading the esophagus, lungs, breasts, ovaries, lymphoid tissue, gastrointestinal tract (e.g., colon), anus, genitals, and urogenital tract (e.g., kidneys, bladder epithelium, bladder cells, prostate), pharynx, central nervous system (e.g., brain, nerve cells, or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma), and pancreas; as well as adenocarcinomas (including malignant tumors); blood It can be used to evaluate the progression, diagnosis, and / or staging of cancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma). In some embodiments, the cancer diagnosed using the anti-Nectin-4 antibody may be early, intermediate, or advanced, or may be metastatic cancer, and may include, for example, carcinoma in situ or metastatic cancer expressing the Nectin-4 protein, but are not limited to these. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, rectal cancer, colon cancer, esophageal cancer, skin cancer, urothelial carcinoma, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, and prostate cancer.

[0074] In some embodiments, labeled Nectin-4 antibodies are provided. Labels include, but are not limited to, directly detectable labels (e.g., fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radioactive labels) and indirectly detectable labels, such as those detected via enzymatic reactions or molecular interactions, e.g., enzymes or ligands. Examples of labels include, but are not limited to, 32 P, 14 C, 125 I, 3 H, and 131Examples include radioactive isotopes such as I, rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, fluorophores such as dansyl and umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that oxidize pigment precursors using hydrogen peroxide (e.g., HR, lactoperoxidase, or microperoxidase), biotin / avidin, spin labeling, bacteriophage labeling, and stable free radicals.

[0075] In some embodiments of any invention provided herein, the sample is obtained before treatment with Nectin-4 antibody. In some embodiments, the sample is obtained after cancer has metastasized. In some embodiments, the sample is a formalin-fixed and paraffin-embedded (FFPE) sample. In some embodiments, the sample is a biopsy sample (e.g., a core biopsy sample), a surgical specimen (e.g., derived from surgical excision), or a fine-needle aspiration.

[0076] In some embodiments, Nectin-4 is assayed before treatment, for example, before the initiation of treatment or before subsequent treatments administered at intervals.

[0077] In some embodiments, the present invention is a method for diagnosing Nectin-4 related diseases such as cancer in a subject, 1) Contacting a biological sample from a subject with the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment as a capture agent, wherein the biological sample is a tissue sample, cell sample, or body fluid sample from a subject, for example, a tissue sample from tumor tissue or healthy tissue, a blood sample, a serum sample, a urine sample, a saliva sample, or a tissue fluid sample, for example, a tissue section (e.g., paraffin section or frozen section) sample from tumor tissue or healthy tissue; 2) Assaying the binding of the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment to a biological sample from a subject, and optionally, 3) Determining the presence or expression level of Nectin-4 in a biological sample from the subject, and optionally comparing the presence or expression level of Nectin-4 in the biological sample from the subject with a reference Nectin-4 presence or expression level. Includes, The detection of Nectin-4 in the sample and / or a Nectin-4 expression level significantly higher than the reference level indicates a Nectin-4 related disease such as cancer in the subject. The present invention provides a method for optionally assaying the presence or expression level of Nectin-4 using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

[0078] In some embodiments, the present invention relates to a method for determining the eligibility of a subject for treatment with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (for example, the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment), 1) Contacting a biological sample from a subject with the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment as a capture agent, wherein the biological sample is a tissue sample, cell sample, or body fluid sample from a subject, for example, a tissue sample from tumor tissue or healthy tissue, a blood sample, a serum sample, a urine sample, a saliva sample, or a tissue fluid sample, for example, a tissue section (e.g., paraffin section or frozen section) sample from tumor tissue or healthy tissue; 2) Assaying the binding of the antibody of the present invention or its antigen-binding fragment to a biological sample from a subject, and optionally, 3) Determining the presence or expression level of Nectin-4 in a biological sample from the subject, and optionally comparing the presence or expression level of Nectin-4 in the biological sample from the subject with a reference Nectin-4 presence or expression level. Includes, Detection of Nectin-4 in a sample and / or detection of Nectin-4 expression levels higher than the reference level indicates that the subject is treatable with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment). The present invention provides a method for optionally assaying the presence or expression level of Nectin-4 using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

[0079] In some embodiments, the present invention relates to a method for treating cancer in a subject requiring treatment for cancer, comprising a pre-step of diagnosing cancer or determining eligibility for treatment with an anti-Nectin-4 antibody, wherein the pre-step includes performing an immunohistochemical (IHC) assay, an enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sort (FACS) assay, a Western blot assay, an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay on a biological sample from the subject using an anti-Nectin-4 antibody as disclosed herein, for example, the pre-step is: 1) Contacting a biological sample with the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment as a capture agent, wherein the biological sample is a tissue sample, cell sample, or body fluid sample from a subject, for example, a tissue sample, blood sample, serum sample, urine sample, saliva sample, or tissue fluid sample derived from tumor tissue or healthy tissue, for example, a tissue section (e.g., paraffin section or frozen section) sample derived from tumor tissue or healthy tissue; 2) Assaying the binding of antibodies or their antigen-binding fragments to biological samples, and optionally, 3) Determine the expression of Nectin-4 in the biological sample and compare the expression level of Nectin-4 in the biological sample with the expression level of reference Nectin-4, Includes, The present invention provides a method for optionally assaying the expression level of Nectin-4 using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

[0080] In some embodiments, the present invention provides a method for predicting the prognosis of a subject with cancer, wherein the subject with cancer has been treated with an antitumor agent, and the antitumor agent is not limited to, but includes metabolic inhibitors, antibiotic anticancer agents, plant alkaloid anticancer agents, topoisomerase inhibitors, antitumor alkylating agents, monoclonal antibodies, ADCs, etc. This method is 1) Contacting a biological sample from a subject treated with an antitumor agent (e.g., anti-Nectin-4 antibody or anti-Nectin-4 antibody-drug conjugate (e.g., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment)) with the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention as a capture agent, where the biological sample is a tissue sample, cell sample, or body fluid sample from the subject, for example, a tissue sample, blood sample, serum sample, urine sample, saliva sample, or tissue fluid sample derived from tumor tissue or healthy tissue, for example, a tissue section sample (e.g., paraffin section or frozen section) derived from tumor tissue or healthy tissue; 2) Assaying the binding of the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment to a biological sample from a subject, 3) To determine the presence or expression level of Nectin-4 in biological samples from the subject, Includes, Detection of Nectin-4 in a sample and / or detection of Nectin-4 expression levels higher than the reference level indicates a poor prognosis, while the absence of Nectin-4 in a sample, or the presence of a reference level or detection of Nectin-4 levels lower than the reference level, indicates a good prognosis. The present invention provides a method for optionally assaying the presence or expression level of Nectin-4 using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

[0081] In some embodiments, the present invention further provides a kit comprising the anti-Nectin-4 antibody of the present invention and optionally an accompanying document instructing on how to use the kit.

[0082] In some embodiments, a method for treating a tumor is provided, comprising: testing a subject (e.g., a sample from a subject) (e.g., a subject sample containing cancer cells) for the presence of Nectin-4 to determine a Nectin-4 value; comparing the Nectin-4 value to a control value (e.g., a Nectin-4 value in a sample from a healthy individual); and, if the Nectin-4 value from the subject sample is higher than the control value, administering a therapeutically effective amount of anti-Nectin-4 antibody (e.g., a therapeutic anti-Nectin-4 antibody) to the subject, optionally in combination with one or more other treatments, thereby treating the tumor.

[0083] The following embodiments are provided to aid in understanding the present invention. These embodiments are not intended to limit, and should not be construed to limit, the scope of protection of the present invention in any way. [Examples]

[0084] Example 1. Preparation and sequencing of Nectin-4 antibody The Nectin-4 antibody of the present invention may be prepared by the method described below. However, the method for preparing the Nectin-4 antibody of the present invention is not limited to the method described in these examples. The Nectin-4 antibody of the present invention may also be prepared by other methods known in the art.

[0085] Fragments derived from human Nectin-4, such as a fragment containing the extracellular domain of human Nectin-4 (SEQ ID NO: 31), were used as immunization antigens. Primary immunization was performed using the antigen with complete Freund's adjuvant (CFA). Booster immunization was performed using the antigen with incomplete Freund's adjuvant (IFA). Emulsions containing the antigen were administered subcutaneously.

[0086] 1.1. Preparation by hybridoma method: Balb / c mice were immunized with recombinant Nectin-4 extracellular protein (UniProt database accession number Q96NY8, positions 32-349 (SEQ ID NO: 32), prepared using a mammalian cell expression system). When serum from immunized mice showed high titer anti-Nectin-4 antibodies, the mouse spleen was sterile excised and a splenocyte suspension was prepared. The splenocytes in the suspension were fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and divided into 96-well cell culture plates. The plates were cultured in a 37°C 5% CO2 incubator to establish hybridoma cell lines. The optimal Nectin-4 mouse monoclonal antibody (named MM11) was screened using immunohistochemical assays.

[0087] The sequence information for antibody MM11 was as follows: The heavy chain constant region corresponds to the mouse IgG1 antibody sequence, and the heavy chain variable region and light chain variable region are shown in italics and underline, respectively. The complementarity-determining region (CDR) sequence, defined according to KABAT nomenclature, is further highlighted in bold.

[0088] Heavy chain amino acid sequence TIFF2026520735000001.tif46170

[0089] Light chain amino acid sequence TIFF2026520735000002.tif23170

[0090] 1.2. Preparation using phage display technology: Large-eared Japanese white rabbits were immunized with recombinant Nectin-4 extracellular protein (see UniProt database accession number Q96NY8, positions 32-349). Mononuclear cells were isolated from the blood of the immunized rabbits. Total RNA was then extracted from the mononuclear cells to create a cDNA gene library for Nectin-4 rabbit antibodies. A phage library was constructed from the cDNA gene library of Nectin-4 rabbit antibodies. After biopanning of the phage library, Nectin-4 rabbit monoclonal antibodies with favorable immunohistochemical results were screened and named antibody R012 and antibody R036, respectively.

[0091] The sequence information for antibodies R012 and R036 was as follows: The heavy chain constant region is the rabbit IgG antibody sequence, and the heavy chain variable region and light chain variable region are shown in italics and underline. The complementarity-determining region (CDR) sequence, defined according to KABAT nomenclature, is further highlighted in bold.

[0092] Antibody R012 Heavy chain amino acid sequence TIFF2026520735000003.tif46170

[0093] Light chain amino acid sequence TIFF2026520735000004.tif23170

[0094] Antibody R036 Heavy chain amino acid sequence TIFF2026520735000005.tif47170

[0095] Light chain amino acid sequence TIFF2026520735000006.tif23170

[0096] Example 2. Detection of antibody binding ability to Nectin-4 and its family proteins by ELISA. To verify the antigen-binding specificity of the Nectin-4 antibody obtained in Example 1, cross-reactivity between the antibody and other Nectin family proteins was detected using an enzyme-linked immunosorbent assay (ELISA).

[0097] The Nectin family of proteins includes the following nine proteins: Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5. Specific details are as follows.

[0098] [Table 1]

[0099] The ELISA assay was performed as follows.

[0100] Coating: Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5 were each diluted to 5 μg / ml in coating buffer (carbonate buffer, pH 9.6). A 96-well plate was coated with 100 μl of the liquid per well and incubated overnight at 2°C to 8°C.

[0101] Blocking: Remove the plate, tap it gently to dry, wash it twice with 200 μl / well of PBST, and then tap it gently to dry. Add 200 μl / well of 3% BSA / PBST and incubate at room temperature for 1.5 hours to block.

[0102] Sample Loading: Remove the plate, tap it gently to dry, wash it twice with 200 μl / well PBST, and then tap it gently to dry. The Nectin-4 antibodies prepared in Example 1 (antibody MM11, antibody R012, and antibody R036) were diluted with 1% BSA / PBST to concentrations of 10 μg / ml, 1 μg / ml, 100 ng / ml, and 10 ng / ml, respectively. The 1% BSA / PBST solutions used for dilution were set as a blank control. 100 μl per well was added to a 96-well plate and incubated at room temperature for 2 hours.

[0103] Secondary antibody addition: Remove the plate, tap it gently to dry, wash it six times with 200 μl / well PBST, then wipe it gently to dry. Dilute goat anti-mouse IgG-HRP (Jackson, catalog no. 115-035-003) at a ratio of 1:20000, or dilute goat anti-rabbit IgG(H+L) antibody-HRP (ThermoFisher, catalog no. 31460) at a ratio of 1:10000. Add 100 μl / well and incubate at room temperature for 1 hour.

[0104] Color development: Remove the plate, tap it gently to dry, wash it six times with 200 μl / well PBST, then wipe it gently to dry. Add 100 μl / well TMB substrate and incubate at room temperature for approximately 5 minutes.

[0105] Reaction completion and plate reading: The reaction was completed by adding 100 μl / well of 1 mol / L H3PO4, and the plate was read. Using 650 nm as the reference wavelength, the absorbance at 450 nm in each well of the plate was read and the OD (Optical Dioxide) was determined. 450nm-650nm It was recorded as such.

[0106] Results of the ELISA assay: The results of the ELISA assay are shown in Table 1 and Figure 1. Mouse antibody MM11, rabbit antibody R012, and rabbit antibody R036 were able to specifically bind to recombinant human Nectin-4 protein and did not cross-react with other family proteins.

[0107] [Table 2]

[0108] Example 3. Flow cytometry analysis of antibodies that bind to Nectin-4 expressing cells To verify the binding specificity of the Nectin-4 antibody obtained in Example 1 to Nectin-4 expressing cells, flow cytometry (FACS) was used to detect the binding of the antibody to Nectin-4 expressing cells.

[0109] Flow cytometry was performed as follows.

[0110] Cell processing: Human prostate cancer cells (PC-3) purchased from ATCC did not express Nectin-4. Human prostate cancer cells (PC-3) were used as a negative control.

[0111] PC-3-Nectin-4 cells are a genetically engineered cell line constructed by Jiangsu Mabwell Health Pharmaceutical R&D Co., Ltd., and are a PC-3 cell line expressing Nectin-4. Specifically, the full-length human Nectin-4 gene (NCBI Gene ID: 81607, 1533 bp) was knocked into PC-3 cells. A stable transfected cell line with high Nectin-4 expression was obtained through monoclonal cell screening. High Nectin-4 expression on the cell surface was confirmed by flow cytometry, and this cell line was named PC-3-Nectin-4 cells.

[0112] PC-3-Nectin-4 cells and PC-3 cells (negative control) cultured to the logarithmic growth phase were digested with trypsin, centrifuged at 200g for 5 minutes, and collected to obtain approximately 5 × 10⁶ cells. 5 The cells were resuspended to a density of cells / ml. 1 ml of each cell suspension was transferred to a separate flow cytometry tube, and 5 × 10⁶ cells were extracted. 5Cells / flow cytometer tubes were obtained. The flow cytometer tubes were centrifuged at 350 g at 4°C for 5 minutes, and the supernatant was discarded. The flow cytometer tubes were washed twice with pre-cooled PBS for later use.

[0113] Dilution and incubation of antibody samples: Antibodies MM11, R012, and R036 were each diluted to 5 μg / ml in PBS. 200 μl of each antibody dilution buffer was taken and the respective cell types were resuspended. The mixtures were gently mixed and incubated on ice in the dark for 1 hour.

[0114] Addition of fluorescent secondary antibody: Flow cytometer tubes were centrifuged at 4°C and 350g for 5 minutes. The supernatant was discarded. Cells were washed twice with 1 ml of pre-cooled PBS added to each tube. Then, 500 μl of secondary antibody was added. For antibody MM11, the secondary antibody was goat anti-mouse IgG Fc-FITC (1:200) (Abcam, catalog number ab150113), and for antibodies R012 and R036, the secondary antibody was goat anti-rabbit IgG H&L-Alexa Flour™ 488 (1:2000) (Abcam, catalog number ab150077). Cells were resuspended and incubated on ice for 60 minutes, then centrifuged at 4°C and 350g for 5 minutes. The supernatant was discarded. Cells were washed twice with 1 ml of pre-cooled PBS added to each tube, and then resuspended in 500 μl of PBS.

[0115] detection: Each flow cytometry tube was detected using a flow cytometer according to the instrument's relevant operating procedures. The results were entered into the corresponding save path.

[0116] FACS detection results: Figure 2 shows the FACS detection results. When detected using antibodies MM11, R012, and R036, PC-3-Nectin-4 cells produced a higher fluorescence signal, but no significant fluorescence signal was observed in PC-3 cells. This result indicates that the anti-Nectin-4 antibodies MM11, R012, and R036 can specifically recognize and bind to Nectin-4 expressed on PC-3-Nectin-4 cells without recognizing other proteins on the cell.

[0117] Example 4. Western blot analysis of antibody reactivity with Nectin-4 expressing cell lysates. Western blotting was used to determine whether the Nectin-4 antibody obtained in Example 1 specifically recognized the Nectin-4 protein. Furthermore, to further confirm the specificity of the Nectin-4 antibody obtained in Example 1, inhibition experiments were performed by adding an excess amount of Nectin-4 protein in the Western blotting.

[0118] The Western blot assay was performed as follows.

[0119] Human prostate cancer cells (PC-3) were purchased from ATCC. Flow cytometry assays confirmed that PC-3 cells did not express Nectin-4.

[0120] PC-3-Nectin-4 cells are a genetically engineered cell line constructed by Jiangsu Mabwell Health Pharmaceutical R&D Co., Ltd., and are a PC-3 cell line expressing Nectin-4. Specifically, the full-length human Nectin-4 gene (NCBI Gene ID: 81607, 1533 bp) was knocked into PC-3 cells. A stable transfected cell line with high Nectin-4 expression was obtained through monoclonal cell screening. High Nectin-4 expression on the cell surface was confirmed by flow cytometry, and this cell line was named PC-3-Nectin-4 cells.

[0121] Protein Extraction: PC-3-Nectin-4 cells and PC-3 cells were thawed and cultured in cell culture flasks in an incubator until appropriate densities were reached. The culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Cells were lysed by adding RIPA lysis solution (radioimmunoprecipitation buffer) containing a protease inhibitor (Thermo, catalog number 89901). Cells were scraped off with a cell scraper, lysed on ice for 30 minutes, and then centrifuged at 12000 rpm at 4°C for 10 minutes. After centrifugation, the supernatant was transferred to a new centrifuge tube. Protein concentration was measured using the BCA method and adjusted to the appropriate concentration. Five times the amount of loading buffer was added and thoroughly mixed, and the protein was heated in a metal bath at 97°C for 10 minutes to completely denaturate it. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference for Western blotting.

[0122] Electrophoresis: 4.6 μg of cell lysate from each cell line was loaded. Electrophoresis conditions: 120 V, 80 minutes.

[0123] Membrane transfer: Wet transfer was performed. Wet transfer conditions: 0.35A, 90 minutes.

[0124] Blocking: The membrane was incubated at room temperature with 5% BSA / TBST for 1 hour with gentle shaking.

[0125] Addition of primary antibody: (1) The Nectin-4 antibodies prepared in Example 1 (antibody MM11, antibody R012, and antibody R036, respectively) were diluted to 20 ng / ml with 5% BSA / TBST and added as primary antibodies. The membrane was incubated overnight at 2°C to 8°C with gentle shaking, or (2) The Nectin-4 antibodies prepared in Example 1 (antibody MM11 and antibody R012, respectively) were diluted to 20 ng / ml with 5% BSA / TBST. The Nectin-4 antibody was added as the primary antibody, and 200 μg / ml of recombinant human Nectin-4 (Jiangsu Mabwell Health Pharmaceutical R&D Co., Ltd., lot: 20210101) was added and pre-incubated for 1 hour. The membrane was then incubated overnight at 2°C to 8°C with gentle shaking.

[0126] Secondary antibody addition: After re-warming for 30 minutes, the membrane was washed three times with TBST for 5 minutes each time. Goat anti-mouse IgG-HRP (Jackson, catalog number: 115-035-003) was diluted with 5% BSA / TBST at a ratio of 1:50000 and added as the secondary antibody, and incubated at room temperature for 1 hour with gentle shaking. Alternatively, goat anti-rabbit IgG(H+L) antibody-HRP (ThermoFisher, catalog number: 31460) was diluted with 5% BSA / TBST at a ratio of 1:25000 and added as the secondary antibody, and incubated at room temperature for 1 hour with gentle shaking.

[0127] Chromogenicity: After secondary antibody incubation was complete, the membrane was washed three times with TBST for 5 minutes each time. ECL luminescence solution, prepared in a 1:1 ratio for chromogenicity, was added, and the luminescence signal was captured.

[0128] Western blot results: The results of the Western blot are shown in Figure 3. The detection band for GAPDH (used as an internal reference) appeared at approximately 36 kDa, ensuring the accuracy and reliability of the experimental results. Specific bands binding to antibodies MM11, R012, and R036 were detected in Nectin-4 positive cells, but no specific bands were observed in Nectin-4 negative cells.

[0129] The specific bands on Nectin-4-positive cells bound to antibodies MM11 and R012 disappeared after the addition of excess recombinant Nectin-4 protein, demonstrating that anti-Nectin-4 antibodies specifically bind to human Nectin-4 protein.

[0130] Example 5. Immunohistochemical staining using Nectin-4 rabbit monoclonal antibody Multiple tumor tissue samples and normal human tissue samples were selected. After fixation in neutral formalin, the samples were prepared into paraffin-embedded tissue sections. Normal human tissues included tissues and organs from multiple sources, while tumor tissues included three bladder cancer tissues, two lung cancer tissues, and three breast cancer tissues.

[0131] Nectin-4 expression in tissue samples was assayed using an anti-Nectin-4 monoclonal antibody. A commercially available rabbit anti-Nectin-4 monoclonal antibody (purchased from abcam, clone number: EPR15613-68) was used as a positive control. All antibodies were diluted to the required concentration using antibody diluent (catalog number P010A01) purchased from MEDx Translational Medicine (Suzhou) Co., Ltd. Immunohistochemical staining was assayed using a fully automated immunohistochemical staining system (Leica BOND III) according to the protocol detailed in the table below.

[0132] [Table 3]

[0133] Note: The peroxidase inactivation reagent, goat anti-rabbit IgG polymer HRP, DAB chromogenic reagent, and hematoxylin stain were all included in the Leica BOND Polymer Refine Detection Kit (catalog number DS9800). All reagents were diluted to their respective concentrations and prepared for direct use.

[0134] The results of immunohistochemical staining are shown in Table 3, Figures 4, 5, 6, and 7. In normal and tumor tissues with fluctuating Nectin-4 expression levels, the three antibodies EPR15613-68, R012, and R036 showed essentially identical results. The target protein, Nectin-4, was localized to the cell membrane and / or cytoplasm, consistent with protein localization reported in the literature, and the antibodies did not induce nonspecific staining. In normal tissues, placental trophoblast cells, cutaneous epithelium, and tonsillar crypt epithelium showed significant Nectin-4 positive staining, while other normal tissues and organs were substantially Nectin-4 negative. In most tumor tissues, tumor cells were Nectin-4 positive, but in some small populations of tumor tissue, tumor cells were Nectin-4 negative, and normal cells within the tumor tissue were also Nectin-4 negative.

[0135] [Table 4]

[0136] Example 6. Immunohistochemical staining using anti-Nectin-4 mouse monoclonal antibody Multiple tumor tissue samples and normal human tissue samples were selected. After fixation in neutral formalin, the samples were prepared into paraffin-embedded tissue sections. Normal human tissues included tissues and organs from multiple sources, while tumor tissues included three prostate cancer tissues, three lung cancer tissues, and three triple-negative breast cancer tissues. Nectin-4 expression in the tissue samples was assayed using an anti-Nectin-4 monoclonal antibody. Mouse monoclonal antibody M22-321b41.1 was used as a positive control. M22-321b41.1 was the anti-Nectin-4 mouse monoclonal antibody in the patent application published in Chinese Patent Application No. 111051345. All antibodies were diluted to the usable concentration using antibody diluent (catalog number P010A01) purchased from MEDx Translational Medicine (Suzhou) Co., Ltd. and prepared for use. Immunohistochemical staining was performed using a fully automated immunohistochemical staining system (Leica BOND III) according to the protocol detailed in the table below.

[0137] [Table 5]

[0138] Note: The peroxidase inactivation reagent, rabbit anti-mouse post primary antibody, goat anti-rabbit IgG-polymer HRP, DAB chromogenic reagent, and hematoxylin stain were all included in Leica's BOND Polymer Refine Detection Kit (catalog number DS9800). All reagents were diluted to their respective concentrations and prepared for direct use.

[0139] The results of immunohistochemical staining are shown in Table 5, Figures 8, 9, 10, and 11. In normal tissues, antibodies M22-321b41.1 and MM11 showed essentially identical results. In normal tissues, salivary glands and cutaneous epithelium in some individuals showed weak Nectin-4 positivity, and crypt epithelium of the tonsils and bronchi of the lungs showed significant Nectin-4 positivity, but Nectin-4 was substantially negative in other normal tissues and organs. In most tumor tissues, tumor cells showed Nectin-4 positivity, while normal cells within the tumor remained Nectin-4 negative. When tumor tissues with different Nectin-4 expression levels were stained using antibodies MM11 and M22-321b41.1, the target protein Nectin-4 was localized to the cell membrane and / or cytoplasm, consistent with the protein's localization in the literature, and the antibodies did not induce nonspecific staining. In some tumor tissues, particularly in triple-negative breast cancer tumors, antibody MM11 showed a significantly stronger staining signal than antibody M22-321b41.1.

[0140] [Table 6]

[0141] The above describes exemplary embodiments of the present invention. Those skilled in the art will understand that these disclosures are illustrative and that various other substitutions, adaptations, and modifications can be made within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described herein.

[0142] [Table 7] TIFF2026520735000014.tif242170TIFF2026520735000015.tif165170

Claims

1. An anti-Nectin-4 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, (a) The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 5, HCDR2 shown in SEQ ID NO: 6, and HCDR3 shown in SEQ ID NO: 7, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9, and LCDR3 shown in SEQ ID NO:

10. (b) The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 15, HCDR2 shown in SEQ ID NO: 16, and HCDR3 shown in SEQ ID NO: 17, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 20, or (c) The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 25, HCDR2 shown in SEQ ID NO: 26, and HCDR3 shown in SEQ ID NO: 27, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO:

30. Anti-Nectin-4 antibody or its antigen-binding fragment.

2. Including heavy chain variable region and light chain variable region, (a) The heavy chain variable region includes the sequence of Sequence ID No. 3, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and the light chain variable region includes the sequence of Sequence ID No. 4, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, (b) The heavy chain variable region includes the sequence of SEQ ID NO: 13, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region includes the sequence of SEQ ID NO: 14, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or (c) The heavy chain variable region includes the sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and the light chain variable region includes the sequence of SEQ ID NO: 24, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, For example, the anti-Nectin-4 antibody or its antigen-binding fragment, (a) A heavy chain variable region containing the sequence of sequence number 3, and a light chain variable region containing the sequence of sequence number 4, (b) A heavy chain variable region containing the sequence of sequence number 13, and a light chain variable region containing the sequence of sequence number 14, or (c) A heavy chain variable region containing the sequence of sequence number 23, and a light chain variable region containing the sequence of sequence number 24, Includes, For example, the anti-Nectin-4 antibody or its antigen-binding fragment, (a) The heavy chain sequence of Sequence ID No. 1, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain sequence of Sequence ID No. 2, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, for example, the heavy chain sequence shown in Sequence ID No. 1 and the light chain sequence shown in Sequence ID No. 2, (b) The heavy chain sequence of Sequence ID No. 11, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain sequence of Sequence ID No. 12, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, for example, the heavy chain sequence shown in Sequence ID No. 11 and the light chain sequence shown in Sequence ID No. 12, or (c) The heavy chain sequence of Sequence ID No. 21, or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, and the light chain sequence of Sequence ID No. 22, or a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity therewith, for example, the heavy chain sequence shown in Sequence ID No. 21 and the light chain sequence shown in Sequence ID No. 22, The anti-Nectin-4 antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. The antigen-binding fragments are Fab, Fab', and F(ab'). ２ An anti-Nectin-4 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is Fv, single-stranded Fv, single-stranded Fab, or a diabody.

4. An anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, having one or more of the following characteristics: (a) When measured in an ELISA assay, it has the ability to specifically bind only to Nectin-4, and does not show specific binding to the Nectin family members Nectin-1, Nectin-2, Nectin-3, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5. (b) When measured in a flow cytometry assay, it has the ability to bind to the Nectin-4 molecule expressed on the cell. (c) When measured by Western blotting, Nectin-4 protein can be specifically detected, and when an excess of Nectin-4 protein is added, its binding to Nectin-4 protein on the membrane is inhibited, and / or (d) When measured by immunohistochemical staining, tissues expressing the Nectin-4 molecule are specifically stained, for example, the tissues are selected from the group consisting of tonsils, salivary glands, brain, cerebellum, placenta, bladder, lungs, mammary glands, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureters, ovaries, fallopian tubes, cervix, endometrium, skin, kidneys, prostate, pancreas, thyroid, spleen, and liver.

5. An isolated nucleic acid encoding an anti-Nectin-4 antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 4.

6. A vector comprising the nucleic acid described in claim 5, preferably the vector being an expression vector.

7. A host cell comprising the nucleic acid described in claim 6 or the vector described in claim 7, wherein the host cell is preferably a prokaryotic or eukaryotic cell, more preferably selected from the group consisting of Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof, and most preferably the host cell is a 293 cell or a CHO cell.

8. A method for preparing an anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4, comprising: culturing the host cells according to claim 7 under conditions suitable for expressing a nucleic acid encoding the anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4; and recovering the expressed anti-Nectin-4 antibody or its antigen-binding fragment from the culture medium.

9. A kit comprising an anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4.

10. Use of an anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4 for the preparation of a kit for detecting Nectin-4 in a sample.

11. A method for detecting Nectin-4 in a biological sample from a subject, 1) Contacting the biological sample with a capture agent, wherein the capture agent is an anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4; 2) Assay the binding of the scavenger to the biological sample, and optionally, 3) Determining the presence or expression level of Nectin-4 in the biological sample from the subject, and optionally comparing the presence or expression level of Nectin-4 in the biological sample from the subject with the presence or expression level of a reference Nectin-4. Includes, A method wherein the presence or expression level of Nectin-4 is optionally assayed using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

12. A method for determining the eligibility of a subject for treatment with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate, 1) Contacting a biological sample from the subject with a capture agent, wherein the capture agent is an anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1 to 4; 2) Assay on the binding of the scavenger from the subject to the biological sample, and optionally, 3) Determining the presence or expression level of Nectin-4 in the biological sample from the subject, and optionally comparing the presence or expression level of Nectin-4 in the biological sample from the subject with the presence or expression level of a reference Nectin-4. Includes, A method wherein the presence or expression level of Nectin-4 is optionally assayed using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

13. A method for predicting the prognosis of a subject with cancer, 1) Contacting a biological sample from a subject treated with an antitumor agent (e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate) with a capture agent, wherein the capture agent is an anti-Nectin-4 antibody or its antigen-binding fragment as described in any one of claims 1 to 4; 2) Assay on the binding of the scavenger from the subject to the biological sample, and optionally, 3) To determine the presence or expression level of Nectin-4 in the biological sample from the subject, Includes, The detection of Nectin-4 in the sample and / or a Nectin-4 expression level higher than the reference level indicates a poor prognosis, while the absence of Nectin-4 in the sample, or the detection of a Nectin-4 level lower than the reference level or expression level, indicates a good prognosis. A method wherein the presence or expression level of Nectin-4 is optionally assayed using an immunohistochemistry (IHC) assay, a Western blot assay, a fluorescence-activated cell sort (FACS) assay, an enzyme-linked immunosorbent assay (ELISA), an immunofluorescence (IF) assay, and / or a co-immunoprecipitation (Co-IP) assay.

14. The method according to any one of claims 11 to 13, wherein the biological sample is a tissue sample, cell sample, or bodily fluid sample from a subject, such as a tissue sample from tumor tissue or healthy tissue, a blood sample, a serum sample, a urine sample, a saliva sample, or a tissue fluid sample, such as a tissue section (e.g., paraffin section or frozen section) from tumor tissue or healthy tissue.

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