ANTIBODIES THAT BIND Nectin-4 AND USES THEREOF

Abstract

The invention relates to an antibody and an antigen binding fragment which specifically bind to Nectin-4, and a kit containing the antibody or the antigen binding fragment. In addition, the invention also relates to a nucleic acid encoding the antibody, a host cell comprising the nucleic acid, and a method for preparing the antibody. The invention also relates to the diagnostic and prognostic use of said antibody that specifically binds to Nectin-4.

Description

Technical Field

[0001] The present invention relates to the field of antibody medicine. Specifically, 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 antigen-binding fragment. In addition, the present invention relates to a nucleic acid encoding the antibody and a host cell containing the nucleic acid, as well as a method for preparing the antibody. The present invention also relates to the diagnostic and prognostic uses of the antibody that binds to nectin-4. 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 about 66KD. It is a member of the Nectin family in the immunoglobulin superfamily. Its extracellular domain is composed of three Ig-like domains (VCC type) and participates in the formation and maintenance of adhesion junctions together with cadherins.

[0003] Nectin-4 is specifically expressed in embryos, placentas, and tumor cells. It is not expressed or expressed at very low levels in normal tissues and organs. It is a classic tumor marker in clinical practice and an ideal target for antibody-drug conjugates (ADCs). Antibody-drug conjugates targeting nectin-4 bind to nectin-4 overexpressed on the surface of tumor cells to form ADC-receptor conjugates; then, ADC enters the cell through target-mediated endocytosis, and then the linker is cut off by tissue proteases in the lysosomes, releasing toxic small molecules to achieve specific killing of tumor cells. PADCEV targeting nectin-4 TM (Also known as enfortumab vedotin-ejfv; Chinese name: Vitin-Enfortumab) is an ADC drug, which is a human IgG1 monoclonal antibody enfortumab targeting the nectin-4 protein coupled with the cytotoxic agent MMAE (monomethyl auristatin E, a microtubule disruptor) (Pia M. Challita-Eid et al. (2016) Cancer Res. 76 (10): 3003-13). It has been approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma. However, PADCEV targeting nectin-4 TM It is for therapeutic use only and cannot be used for immunohistochemistry (IHC) staining, so it has no diagnostic value.

[0004] There is a need in the art for anti-nectin-4 antibodies that specifically recognize nectin-4, which can detect the presence and / or expression level of nectin-4 in a sample with high sensitivity (e.g., by IHC detection), thereby diagnosing a disease or evaluating the prognosis, which is of great significance for improving the survival rate of cancer patients, prolonging the survival period, avoiding excessive chemotherapy, and improving the quality of life. Therefore, the use of anti-nectin-4 antibodies with good specificity and high sensitivity in diagnosis and prognosis has extremely high application value. Summary of the invention

[0005] The present invention provides an anti-nectin-4 antibody with good specificity and high sensitivity, which can reliably and sensitively detect nectin-4. The antibody or antigen-binding fragment specifically binding to nectin-4 of the present invention has one or more of the following characteristics:

[0006] (a) In the ELISA assay, it has the ability to specifically bind only to Nectin-4, and has no specific binding to 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;

[0007] (b) having the ability to bind to Nectin-4 molecules expressed on cells as measured in a flow cytometry assay;

[0008] (c) can specifically detect Nectin-4 protein when measured in Western blotting; and when an excess of Nectin-4 protein is added, the binding of Nectin-4 protein to the membrane is inhibited; and / or

[0009] (d) in immunohistochemical staining, specifically staining tissues expressing nectin-4 molecules, for example, the tissues are selected from tonsils, salivary glands, brain, cerebellum, placenta, bladder, lung, breast, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureter, ovary, fallopian tube, cervix, endometrium, skin, kidney, prostate, pancreas, thyroid, spleen and liver.

[0010] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment that specifically binds to nectin-4, comprising a heavy chain variable region and a light chain variable region, wherein:

[0011] (a) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO:5 or a variant of HCDR1 as set forth in SEQ ID NO:5 with no more than 2 amino acid changes, HCDR2 as set forth in SEQ ID NO:6 or a variant of HCDR2 as set forth in SEQ ID NO:6 with no more than 2 amino acid changes, and HCDR3 as set forth in SEQ ID NO:7 or a variant of HCDR3 as set forth in SEQ ID NO:7 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 as set forth in SEQ ID NO:8 or a variant of LCDR1 as set forth in SEQ ID NO:8 with no more than 2 amino acid changes, LCDR2 as set forth in SEQ ID NO:9 or a variant of LCDR2 as set forth in SEQ ID NO:9 with no more than 2 amino acid changes, and LCDR3 as set forth in SEQ ID NO:10 or a variant of LCDR3 as set forth in SEQ ID NO:10 with no more than 2 amino acid changes;

[0012] (b) the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO:15 or a variant of HCDR1 set forth in SEQ ID NO:15 with no more than 2 amino acid changes, HCDR2 set forth in SEQ ID NO:16 or a variant of HCDR2 set forth in SEQ ID NO:16 with no more than 2 amino acid changes, and HCDR3 set forth in SEQ ID NO:17 or a variant of HCDR3 set forth in SEQ ID NO:17 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 set forth in SEQ ID NO:18 or a variant of LCDR1 set forth in SEQ ID NO:18 with no more than 2 amino acid changes, LCDR2 set forth in SEQ ID NO:19 or a variant of LCDR2 set forth in SEQ ID NO:19 with no more than 2 amino acid changes, and LCDR3 set forth in SEQ ID NO:20 or a variant of LCDR3 set forth in SEQ ID NO:20 with no more than 2 amino acid changes; or

[0013] (c) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:25 or a variant of HCDR1 shown in SEQ ID NO:25 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:26 or a variant of HCDR2 shown in SEQ ID NO:26 with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:27 or a variant of HCDR3 shown in SEQ ID NO:27 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 shown in SEQ ID NO:28 or a variant of LCDR1 shown in SEQ ID NO:28 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:29 or a variant of LCDR2 shown in SEQ ID NO:29 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:30 or a variant of LCDR3 shown in SEQ ID NO:30 with no more than 2 amino acid changes.

[0014] In some embodiments, the amino acid changes are conservative amino acid modifications.

[0015] In some embodiments, the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 comprises

[0016] (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0017] (b) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 13, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 14, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0018] (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:23, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0019] 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-chain Fv, single-chain Fab or diabody.

[0020] In some embodiments, the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 comprises

[0021] (a) SEQ ID NO:1, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:2, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; e.g., the heavy chain sequence set forth in SEQ ID NO:1, and the light chain sequence set forth in SEQ ID NO:2;

[0022] (b) SEQ ID NO:11, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO:12, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; e.g., the heavy chain sequence set forth in SEQ ID NO:11, and the light chain sequence set forth in SEQ ID NO:12; or

[0023] (c) SEQ ID NO:21, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO:22, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; for example, the heavy chain sequence set forth in SEQ ID NO:21, and the light chain sequence set forth in SEQ ID NO:22.

[0024] 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.

[0025] In some embodiments, the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 is an IgG class antibody.

[0026] In a second aspect, the present invention provides a nucleic acid encoding the anti-nectin-4 antibody or antigen-binding fragment described in the first aspect above, a vector (preferably, an expression vector) comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector. In some embodiments, the host cell is prokaryotic or eukaryotic, for example, selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In some embodiments, the host cell is a HEK 293 cell or a CHO cell.

[0027] In a third aspect, the present invention provides a method for preparing the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect, the method comprising culturing the host cell described in the second aspect under conditions suitable for expressing a nucleic acid encoding the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect, and recovering the expressed anti-Nectin-4 antibody or its antigen-binding fragment from the culture.

[0028] In a fourth aspect, the present invention provides a kit comprising the anti-nectin-4 antibody or antigen-binding fragment thereof described in the first aspect.

[0029] In a fifth aspect, the present invention provides a use of the anti-nectin-4 antibody or antigen-binding fragment thereof described in the first aspect for detecting the level of nectin-4 in a sample.

[0030] In a sixth aspect, the present invention provides a method for diagnosing cancer in a subject using the anti-nectin-4 antibody or antigen-binding fragment thereof according to the first aspect.

[0031] In some embodiments, the present invention provides a method for determining a subject's eligibility for treatment with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., a therapeutic anti-Nectin-4 antibody or an antigen-binding fragment thereof) using the anti-Nectin-4 antibody or antigen-binding fragment thereof described in the first aspect above.

[0032] In some embodiments, the present invention provides a method for prognosing a subject suffering from cancer using the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect above, wherein the subject suffering from cancer is a subject suffering from cancer who has been treated with an anti-tumor agent (e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment)). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In conjunction with the instruction manual Figure 1 When reading together, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0034] Figure 1 : Shows the binding of antibody MM11, antibody R012, antibody 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) detected by ELISA.

[0035] Figure 2 :Shows the binding of antibody MM11, antibody R012, antibody R036 to nectin-4 expressing cells detected by flow cytometry (FACS method). In the figure, Blank means only the secondary antibody is added to human prostate cancer cells (PC-3), and antibody MM11, antibody R012 or antibody R036 as the primary antibody is not added; NC means only PC-3 cells and blank buffer are added; "PC-3-Nectin4" is a PC-3 cell line expressing nectin-4.

[0036] Figure 3 : Western blotting was used to detect the reactivity of antibody MM11, antibody R012, and antibody R036 with cell lysates expressing nectin-4. In the figure, lane M: protein molecular weight marker; lane 1: PC-3 cell lysate; lane 2: PC-3-nectin-4 cell lysate. Figure 3 The upper panels show that the antibody MM11, the antibody R012, and the antibody R036 react with the cell lysate expressing Nectin-4, but do not react with the cell lysate not expressing Nectin-4; Figure 3In the small figure below, "MM11 (Inhibition)" and "R012 (Inhibition)" indicate the results after the cell lysate was incubated with "MM11 antibody" or "R012 antibody" respectively after transfer to the membrane, and an excess of recombinant Nectin-4 protein was added; "Gapdh" indicates the reaction of incubating the cell lysate with anti-glyceraldehyde-3-phosphate dehydrogenase antibody after transfer to the membrane, which serves as an internal reference for Western blotting.

[0037] Figure 4 : Shows the immunohistochemical staining results of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control and representative normal human tissues (scale bar: 200 μm).

[0038] Figure 5 : Shows the results of immunohistochemical staining of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human bladder cancer tissues (scale bar: 200 μm).

[0039] Figure 6 : Shows the results of immunohistochemical staining of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human lung cancer tissues (scale bar: 200 μm).

[0040] Figure 7 : Shows the results of immunohistochemical staining of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human breast cancer tissues (scale bar: 200 μm).

[0041] Figure 8 : Shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on representative normal human tissues (scale bar, 100 μm).

[0042] Fig. 9 : Shows the results of immunohistochemical staining of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on human prostate cancer tissues (scale bar, 50 μm).

[0043] Fig.10: Shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on human lung cancer tissues (scale bar, 50 μm).

[0044] Fig.11 : Shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on human triple-negative breast cancer tissues (scale bar, 50 μm). DETAILED DESCRIPTION

[0045] Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular methods and experimental conditions described herein as such methods and conditions may vary.

[0046] I. Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0049] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0050] In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0051] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, or chimeric antibodies. Antibodies can belong to any class (e.g., IgG, IgE, IgM, IgD, IgA).

[0052] Exemplary antibodies of the present disclosure are immunoglobulin G (IgG) type antibodies comprising the following four polypeptide chains cross-linked by interchain disulfide bonds: two heavy chains (HC) and two light chains (LC). The amino terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids that is primarily responsible for 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.

[0053] VH region and VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), with more conservative regions interspersed therebetween, called framework regions (FRs). CDRs are exposed on the surface of the protein and are important regions of the antigen-binding specificity of antibodies. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the heavy chain are called "HCDR1, HCDR2 and HCDR3" and the three CDRs of the light chain are called "LCDR1, LCDR2 and LCDR3". CDRs contain most of the residues that form specific interactions with the antigen. Amino acid residues can be assigned to CDRs according to well-known numbering schemes, including those in 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, 274, 937-958 (1998)), and North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 275, 947-959 (1999). Biology, 406, 228-256 (2011)) or those described in IMGT (ImMunoGeneTic international database available from www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212).

[0054] However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different numbering schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different numbering schemes are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different numbering schemes (e.g., different numbering scheme rules or combinations).

[0055] The CDRs of the antibodies of the present invention can be manually evaluated to determine the boundaries according to any numbering scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0056] An "antigen-binding fragment" is a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), 2 , Fv, single-chain Fv (scFv), single-chain Fab, diabody, single-domain antibody (sdAb, nanobody), camel Ig, Ig NAR, F(ab)′ 3 Fragment, Bi-scFv, (scFv) 2 , miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv proteins ("dsFv"). The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. For a more detailed description, see also: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Journal of Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0057] The terms "Nectin family proteins" and "Nectin family" are used interchangeably and are cell adhesion molecules that form physical connections between adjacent cells to achieve 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. Among them, the expression of Nectin-4 protein in healthy humans is mainly limited to placenta and embryonic tissues. Compared with healthy adult tissues, many types of tumor cells have high expression of Nectin-4 protein. Nectin-4 protein is a tumor-associated antigen.

[0058] The term "binding" or "specific binding" used when referring to antigens and antibodies means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.

[0059] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

[0060] The comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0061] The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).

[0062] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0063] For polypeptide sequences, "conservative modifications" or "conservative amino acid modifications" include substitutions, deletions or additions to a polypeptide sequence that result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, species homologs and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for 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, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative amino acid modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0064] As used herein, "vector" means a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0065] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably in the present invention and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and the progeny derived therefrom, without considering the number of passages. The progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cells screened or selected in the initial transformed cells are included herein.

[0066] As used herein, "subject", "individual" or "subject" refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention, treatment and / or diagnosis of a disease or condition. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice and rats. The term includes human subjects having a disease or at risk of having a disease.

[0067] A "biological sample" from a subject refers to a collection of cells, tissues or body fluids obtained from an individual or subject. The source of a tissue or cell sample can be a solid tissue, such as a fresh, frozen and / or preserved organ or tissue sample or a biopsy sample or a puncture sample; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial 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 tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor tissue section samples or frozen tumor samples.

[0068] The terms "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" refer to the level of a sample, cell, tissue or standard used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part of the body of the same subject or individual, and is a healthy and / or non-diseased tissue or cell. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy tissue or cell of an individual who is not the subject. The presence or expression level of Nectin-4 from a "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" is referred to as the "reference presence or expression level".

[0069] 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 bridge between the primary antibody and a subsequent reagent (if a subsequent reagent is required).

[0070] Immunofluorescence (IF) technology is the earliest developed technology in labeling immunology. It is based on the principle of antigen-antibody reaction. First, the known antigen or antibody is labeled with a fluorescent group, and then the fluorescent antibody (or antigen) is used as a probe to examine the corresponding antigen (or antibody) in the cell or tissue. The antigen-antibody complex formed in the tissue or cell contains labeled fluorescein. When the specimen is observed under a fluorescence microscope, the fluorescein emits bright fluorescence (yellow-green or orange-red) under the irradiation of external excitation light, and the tissue cells where the fluorescence is located can be seen, thereby determining the nature and location of the antigen or antibody, and using quantitative technology to measure the content.

[0071] Immunoprecipitation (IP) is a method that uses antigen-antibody specific reactions to purify and enrich a specific protein. The main processes include capture and fixation.

[0072] Co-Immunoprecipitation (Co-IP) is an extension of immunoprecipitation. Co-IP is a method developed by utilizing the specific binding of antigenic proteins and antibodies and the phenomenon that bacterial proteins "Protein A / G" specifically bind to the Fc fragment of antibodies (immunoglobulins). Usually, Protein A / G is pre-bound to Argarose beads, and reacted with a solution containing antigenic proteins (such as cell lysate) and antibodies against bait protein X. The Prorein A / G on the Argarose beads adsorbs the bait protein X through the antibody, and the bait protein X separates the target protein Y from the cell lysate by interacting with the target protein Y, thereby achieving the purpose of co-immunoprecipitation.

[0073] II. Antibodies of the present invention that specifically bind to nectin-4

[0074] The present invention provides antibodies that specifically bind to nectin-4. In some embodiments, the antibodies of the present invention are monoclonal antibodies, for example, they can be derived from any eukaryotic clone, prokaryotic clone or phage clone. Monoclonal antibodies can be produced by hybridoma technology, recombinant technology, phage display technology, B cell clone screening technology, synthetic technology (e.g., CDR transplantation) or other technology combinations known in the art.

[0075] Methods for producing and purifying antibodies 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 or rabbits or other animals can be immunized with human nectin-4 and the antibodies produced can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art. Similarly, phage libraries can be screened to screen thousands of Fab fragments for interaction with human nectin-4 and the interactors produced can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art.

[0076] The terms "antibody binding to Nectin-4 protein", "antibody binding to Nectin-4", "antibody against Nectin-4 protein", "anti-Nectin-4 antibody", "isolated antibody binding to Nectin-4 protein", "Nectin-4 antibody" and "Nectin-4 protein antibody" are used interchangeably herein and refer to antibodies of the present invention that are capable of specifically binding to Nectin-4 protein, whereby the antibodies can be used as diagnostic agents and / or detection agents targeting Nectin-4 protein.

[0077] The isolated anti-nectin-4 antibodies and antigen-binding fragments of the present invention specifically bind to the nectin-4 protein, and comprise a heavy chain variable region and a light chain variable region, wherein:

[0078] (a) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO:5 or a variant of HCDR1 as set forth in SEQ ID NO:5 with no more than 2 amino acid changes, HCDR2 as set forth in SEQ ID NO:6 or a variant of HCDR2 as set forth in SEQ ID NO:6 with no more than 2 amino acid changes, and HCDR3 as set forth in SEQ ID NO:7 or a variant of HCDR3 as set forth in SEQ ID NO:7 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 as set forth in SEQ ID NO:8 or a variant of LCDR1 as set forth in SEQ ID NO:8 with no more than 2 amino acid changes, LCDR2 as set forth in SEQ ID NO:9 or a variant of LCDR2 as set forth in SEQ ID NO:9 with no more than 2 amino acid changes, and LCDR3 as set forth in SEQ ID NO:10 or a variant of LCDR3 as set forth in SEQ ID NO:10 with no more than 2 amino acid changes;

[0079] (b) the heavy chain variable region comprises HCDR1 set forth in SEQ ID NO:15 or a variant of HCDR1 set forth in SEQ ID NO:15 with no more than 2 amino acid changes, HCDR2 set forth in SEQ ID NO:16 or a variant of HCDR2 set forth in SEQ ID NO:16 with no more than 2 amino acid changes, and HCDR3 set forth in SEQ ID NO:17 or a variant of HCDR3 set forth in SEQ ID NO:17 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 set forth in SEQ ID NO:18 or a variant of LCDR1 set forth in SEQ ID NO:18 with no more than 2 amino acid changes, LCDR2 set forth in SEQ ID NO:19 or a variant of LCDR2 set forth in SEQ ID NO:19 with no more than 2 amino acid changes, and LCDR3 set forth in SEQ ID NO:20 or a variant of LCDR3 set forth in SEQ ID NO:20 with no more than 2 amino acid changes; or

[0080] (c) the heavy chain variable region comprises HCDR1 as set forth in SEQ ID NO:25 or a variant of HCDR1 as set forth in SEQ ID NO:25 with no more than 2 amino acid changes, HCDR2 as set forth in SEQ ID NO:26 or a variant of HCDR2 as set forth in SEQ ID NO:26 with no more than 2 amino acid changes, and HCDR3 as set forth in SEQ ID NO:27 or a variant of HCDR3 as set forth in SEQ ID NO:27 with no more than 2 amino acid changes; the light chain variable region comprises LCDR1 as set forth in SEQ ID NO:28 or a variant of LCDR1 as set forth in SEQ ID NO:28 with no more than 2 amino acid changes, LCDR2 as set forth in SEQ ID NO:29 or a variant of LCDR2 as set forth in SEQ ID NO:29 with no more than 2 amino acid changes, and LCDR3 as set forth in SEQ ID NO:30 or a variant of LCDR3 as set forth in SEQ ID NO:30 with no more than 2 amino acid changes;

[0081] Wherein the amino acid change is an addition, deletion or substitution of an amino acid, for example, the amino acid change is a conservative amino acid substitution. The CDR is a CDR according to the Kabat numbering scheme.

[0082] In some embodiments, the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention comprises:

[0083] (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0084] (b) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 13, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 14, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0085] (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence of SEQ ID NO:23, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0086] In some embodiments, the antibodies of the invention bind to mammalian nectin-4, such as human nectin-4. In some embodiments, the nectin-4 antibodies of the invention bind to one or more extracellular domains of nectin-4.

[0087] In some embodiments, the antibodies of the invention have one or more of the following properties:

[0088] (a) In the ELISA assay, it has the ability to specifically bind only to Nectin-4, and has no specific binding to 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;

[0089] (b) having the ability to bind to Nectin-4 molecules expressed on cells as measured in a flow cytometry assay;

[0090] (c) can specifically detect Nectin-4 protein when measured in Western blotting; and when an excess of Nectin-4 protein is added, the binding of Nectin-4 protein to the membrane is inhibited; and / or

[0091] (d) in immunohistochemical staining, specifically staining tissues expressing nectin-4 molecules, for example, the tissues are selected from tonsils, salivary glands, brain, cerebellum, placenta, bladder, lung, breast, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureter, ovary, fallopian tube, cervix, endometrium, skin, kidney, prostate, pancreas, thyroid, spleen and liver.

[0092] III. Nucleic acids of the present invention and host cells containing the same

[0093] In one aspect, the present invention provides a nucleic acid encoding any of the above Nectin-4 antibodies or antigen-binding fragments thereof or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. 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 preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0094] The present invention also encompasses nucleic acids that hybridize to the above-described nucleic acids under stringent conditions or nucleic acids encoding polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions or insertions compared to the above-described nucleic acids.

[0095] In one embodiment, one or more vectors comprising the nucleic acid described above are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes but is not limited to a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC).

[0096] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in a culture medium and screened for suitable activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the specific expression vector and mammalian host cell used. Variation or optimization.

[0097] IV. Production and purification of antibodies of the invention

[0098] In one embodiment, the present invention provides a method for preparing a Nectin-4 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the Nectin-4 antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the Nectin-4 antibody, and optionally isolating the Nectin-4 antibody. In a certain embodiment, the method further comprises recovering the Nectin-4 antibody from the host cell or host cell culture.

[0099] In order to recombinantly produce the antibodies of the present invention, the nucleic acid encoding the Nectin-4 antibody of the present invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to the nucleic acid encoding the Nectin-4 antibody of the present invention.

[0100] The antibodies of the invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0101] V. Activity Assays for Antibodies of the Invention

[0102] The nectin-4 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0103] In some embodiments, antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, FACS, and the like.

[0104] Cells used in any of the above in vitro assays include cells that naturally express Nectin-4 or cell lines engineered to express Nectin-4. The cell lines engineered to express Nectin-4 are cell lines that do not normally express Nectin-4 but express Nectin-4 after transfection of DNA encoding Nectin-4 into the cells.

[0105] VI. Methods and kits for diagnosis and detection

[0106] In some embodiments, any of the Nectin-4 antibodies provided herein can be used to detect the presence and / or level of Nectin-4 in a biological sample.

[0107] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

[0108] In one embodiment, a nectin-4 antibody is provided for use in a diagnostic or detection method to detect and / or assess the presence and / or level of nectin-4 for diagnostic or prognostic purposes, for example, to determine the efficacy of a given treatment regimen.

[0109] In one aspect, methods of detecting the presence and / or level of Nectin-4 in a biological sample are provided. In some embodiments, the method comprises detecting the presence and / or level of Nectin-4 protein in a biological sample.

[0110] In some embodiments, Nectin-4 is human Nectin-4. In certain embodiments, the method comprises contacting a biological sample with a Nectin-4 antibody as described herein under conditions that allow the Nectin-4 antibody to bind to Nectin-4, and detecting whether a complex is formed between the Nectin-4 antibody and Nectin-4. The formation of a complex indicates the presence of Nectin-4. The method can be an in vitro or in vivo method. In one embodiment, the Nectin-4 antibody is used to select a subject suitable for treatment with a therapeutic Nectin-4 antibody, for example, wherein Nectin-4 is a biomarker for selecting the subject.

[0111] In one embodiment, the antibodies of the invention can be used to diagnose cancers or tumors that express Nectin-4 protein, such as evaluating the treatment or progression, diagnosis and / or staging of solid tumors (e.g., sarcomas and carcinomas of multiple organ systems, such as those affecting the esophagus, lung, breast, ovary, lymphoid, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelial, bladder cells, prostate), pharynx, central nervous system (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma) and pancreas), hematological cancers (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma) that express Nectin-4 protein in a subject. In some embodiments, the cancer diagnosed with the anti-Nectin-4 antibody can be in the early, intermediate or advanced stage or is a metastatic cancer, for example, including but not limited to carcinoma in situ, metastatic cancer that expresses Nectin-4 protein. In some embodiments, the cancer is selected from colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial carcinoma, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, prostate cancer.

[0112] In some embodiments, a labeled nectin-4 antibody is provided. Labels include, but are not limited to, labels that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as labels that are indirectly detected, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 1. 3 H and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0113] In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with a nectin-4 antibody. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

[0114] In some embodiments, nectin-4 is detected prior to treatment, eg, prior to initiation of treatment; or prior to a treatment after a treatment interval.

[0115] In some embodiments, the present invention provides a method for diagnosing a nectin 4-related disease, such as cancer, in a subject, the method comprising:

[0116] 1) contacting a biological sample from the subject with an anti-nectin-4 antibody or an antigen-binding fragment thereof of the present invention as a capture agent, wherein the biological sample is a tissue sample, a cell sample or a body fluid sample of the subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue;

[0117] 2) detecting the binding of the anti-Nectin-4 antibody or antigen-binding fragment thereof of the present invention to a biological sample from the subject; and optionally

[0118] 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 presence or expression level of Nectin-4;

[0119] Detection of Nectin-4 and / or detection of an expression level of Nectin-4 in the sample that is significantly higher than a reference level indicates a Nectin-4-related disease, such as cancer, in the subject;

[0120] Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

[0121] 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., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or an antigen-binding fragment thereof), the method comprising:

[0122] 1) contacting a biological sample from the subject with an anti-nectin-4 antibody or an antigen-binding fragment thereof of the present invention as a capture agent, wherein the biological sample is a tissue sample, a cell sample or a body fluid sample of the subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue;

[0123] 2) detecting binding of the antibody or antigen-binding fragment thereof of the invention to a biological sample from the subject; and optionally

[0124] 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 presence or expression level of Nectin-4;

[0125] Detection of Nectin-4 and / or detection of an expression level of Nectin-4 in the sample higher than a reference level indicates that the subject can be treated 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 an antigen-binding fragment thereof);

[0126] Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

[0127] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising a prior step of diagnosing cancer or determining the eligibility of a patient for treatment with an anti-Nectin-4 antibody, the prior step comprising performing an immunohistochemistry (IHC) assay, an ELISA assay, a flow cytometry (FACS) assay, a Western blot, an immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP) on a biological sample from the subject using an anti-Nectin-4 antibody as disclosed herein, for example, the prior step comprises:

[0128] 1) contacting the biological sample with the anti-nectin-4 antibody or antigen-binding fragment thereof of the present invention as a capture agent, wherein the biological sample is a tissue sample, cell sample or body fluid sample of a subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., paraffin section or frozen section) sample of a tumor or healthy tissue;

[0129] 2) detecting binding of the antibody or antigen-binding fragment thereof to the biological sample; and optionally

[0130] 3) determining the expression of Nectin-4 in the biological sample, wherein the expression level of Nectin-4 in the biological sample is compared with a reference expression level of Nectin-4;

[0131] Optionally, the expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

[0132] In some embodiments, the present invention provides a method for prognosing a subject with cancer. Wherein the subject with cancer has been treated with an anti-tumor agent, the anti-tumor agent includes but is not limited to metabolic inhibitors, antibiotic anti-cancer agents, plant alkaloid anti-cancer agents, topoisomerase inhibitors, anti-tumor alkylating agents, monoclonal antibodies, ADCs, etc., the method comprising:

[0133] 1) contacting a biological sample from 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., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or an antigen-binding fragment thereof)) with an anti-Nectin-4 antibody or an antigen-binding fragment thereof of the present invention as a capture agent, wherein the biological sample is a tissue sample, a cell sample or a body fluid sample of the subject, for example, a tissue sample of a tumor or healthy tissue, blood, serum, urine, saliva, or tissue fluid sample, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue;

[0134] 2) detecting the binding of the antibody or antigen-binding fragment thereof of the present invention to a biological sample from the subject;

[0135] 3) determining the presence or expression level of Nectin-4 in a biological sample from the subject,

[0136] Detection of Nectin-4 and / or detection of an expression level of Nectin-4 in the sample higher than a reference level indicates a poor prognosis; or, absence of Nectin-4 in the sample or detection of a level of Nectin-4 lower than a reference presence or expression level indicates a good prognosis,

[0137] Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

[0138] In some embodiments, the invention also provides a kit comprising an anti-nectin-4 antibody of the invention, and optionally a package insert instructing the use of the kit.

[0139] In some embodiments, a method for treating a tumor is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample containing cancer cells) for the presence of Nectin-4, thereby determining a Nectin-4 value, comparing the Nectin-4 value with a control value (e.g., the value of Nectin-4 in a sample from a healthy individual), and if the Nectin-4 value is greater than the control value, administering to the subject a therapeutically effective amount of a Nectin-4 antibody (e.g., a therapeutic anti-Nectin-4 antibody), optionally in combination with one or more other therapies, thereby treating the tumor.

[0140] The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention.

[0141] Example

[0142] Example 1. Preparation and sequencing of nectin-4 antibody

[0143] The nectin-4 antibody of the present invention can 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 this example, and can also be prepared by other methods known in the art.

[0144] A human nectin-4 fragment, for example, a fragment containing the extracellular domain of human nectin-4 (SEQ ID NO: 31) is used as an antigen for immunization. The antigen is applied together with Freund's complete adjuvant (CFA) for the first immunization. Freund's incomplete adjuvant (IFA) is used for the booster immunization. The emulsion containing the antigen is administered subcutaneously.

[0145] 1.1. Preparation by hybridoma method:

[0146] Balb / c mice were immunized with recombinant nectin-4 extracellular protein (see UniProt database accession number Q96NY8, 32-349 (SEQID NO: 32), prepared using a mammalian cell expression system). When the immunized mouse serum had a high anti-nectin-4 antibody titer, the mouse spleen was aseptically removed to prepare a spleen cell suspension, which was then fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and dispensed into 96-well cell culture plates. The plates were placed at 37°C and 5% CO 2 The cells were cultured in an incubator to form hybridoma cell lines. The best mouse monoclonal antibody for nectin-4 in immunohistochemical testing was obtained by screening and named MM11.

[0147] The sequence information of antibody MM11 is as follows: the heavy chain constant region is a mouse IgG1 type antibody sequence, the heavy chain variable region and the light chain variable region are shown in italics and underlined, and the complementarity determining region (CDR) sequence defined according to the KABAT naming system is further shown in bold.

[0148] Heavy chain amino acid sequence

[0149]

[0150] Light chain amino acid sequence

[0151]

[0152] 1.2. Preparation by phage display technology:

[0153] Recombinant Nectin-4 extracellular protein (see UniProt database accession number Q96NY8, position 32-349) was used to immunize Japanese white rabbits. Mononuclear cells were obtained from the blood of the immunized rabbits, and then total RNA was extracted from the mononuclear cells to generate a cDNA gene library of Nectin-4 rabbit antibodies. A phage library was formed from the cDNA gene library of Nectin-4 rabbit antibodies, and biopanning was performed to screen out Nectin-4 rabbit monoclonal antibodies that performed well in immunohistochemical tests, which were named R012 and R036, respectively.

[0154] The sequence information of antibody R012 and antibody R036 is as follows, the heavy chain constant region is a rabbit IgG antibody sequence, the heavy chain variable region and the light chain variable region are shown in italics and underlined, and the complementary determining region (CDR) sequence defined according to the KABAT naming system is further shown in bold:

[0155] Antibody R012

[0156] Heavy chain amino acid sequence

[0157]

[0158]

[0159] Light chain amino acid sequence

[0160]

[0161] Antibody R036

[0162] Heavy chain amino acid sequence

[0163]

[0164] Light chain amino acid sequence

[0165]

[0166] Example 2. ELISA method to detect the binding ability of antibodies to Nectin.4 and its family proteins

[0167] In order to verify the antigen binding specificity of the Nectin-4 antibody obtained in Example 1, an enzyme-linked immunosorbent assay (ELISA) was used to detect whether there was a cross reaction between the antibody and other Nectin family proteins.

[0168] The nectin family of proteins includes the following 9 proteins: Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, Nectin-like-5. The specific information is as follows:

[0169]

[0170]

[0171] The ELISA test method was performed as follows:

[0172] 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 diluted to 5 μg / ml with coating buffer (carbonate buffer, pH 9.6), and 100 μl / well was coated on a 96-well plate at 2-8°C overnight;

[0173] Blocking: Take out the plate and pat dry, wash the plate twice with PBST at 200 μl / well, pat dry. Add 3% BSA / PBST at 200 μl / well and incubate at room temperature for 1.5 hours to implement blocking;

[0174] Sample addition: Take out the plate and pat dry, wash the plate twice with PBST at 200 μl / well, and pat dry. Dilute the Nectin-4 antibodies (antibody MM11, antibody R012, antibody R036) prepared in Example 1 to 10 μg / ml, 1 μg / ml, 100 ng / ml and 10 ng / ml respectively with 1% BSA / PBST, use 1% BSA / PBST dilution as blank control, add 100 μl / well to 96-well plate, and incubate at room temperature for 2 h;

[0175] Add the secondary antibody: remove the plate and pat dry, wash the plate 6 times with PBST at 200 μl / well, pat dry. Dilute goat anti-mouse IgG-HRP (Jackson, catalog number: 115-035-003) at a ratio of 1:20000 or dilute goat anti-rabbit IgG (H+L) antibody-HRP (ThermoFisher, catalog number: 31460) at a ratio of 1:10000, 100 μl / well, incubate at room temperature for 1 hour;

[0176] Color development: Take out the plate and pat dry, wash the plate 6 times with PBST at 200 μl / well, pat dry. Add TMB substrate at 100 μl / well and incubate at room temperature for about 5 minutes;

[0177] Stop the reaction and read the plate: add 1 mol / L H 3 PO 4 Stop the reaction and read the plate. Using 650 nm as the reference wavelength, read and record the absorbance of the well plate at a wavelength of 450 nm (OD450 nm-OD650 nm).

[0178] ELISA test results:

[0179] The ELISA test results are shown in Table 1 and Figure 1 As shown, mouse antibody MM11, rabbit antibody R012 and rabbit antibody R036 can specifically bind to recombinant human nectin-4 protein and have no cross-reaction with other proteins of the same family.

[0180] Table 1. Cross-reaction results of antibodies with Nectin-4 protein and Nectin family proteins

[0181]

[0182]

[0183]

[0184] Example 3. Analysis of antibody binding to nectin-4 expressing cells by flow cytometry

[0185] In order to verify the binding specificity of the nectin-4 antibody obtained in Example 1 to the nectin-4 expressing cells, flow cytometry (FACS) was used to detect the binding of the antibody to the nectin-4 expressing cells.

[0186] The flow cytometry assay was performed as follows:

[0187] Cell treatment:

[0188] Human prostate cancer cells (PC-3) were purchased from ATCC and do not express nectin-4. Human prostate cancer cells (PC-3) were used as a negative control.

[0189] PC-3-Nectin-4 cells are genetically engineered cell lines constructed by Myvicon, and are PC-3 cell lines expressing Nectin-4. Specifically, the full-length gene of human Nectin-4 (NCBI gene ID: 81607, 1533bp) was knocked into PC-3 cells and a stably transfected cell line with high expression of Nectin-4 was obtained through monoclonal cell screening. Flow cytometry detection confirmed that Nectin-4 was highly expressed on the cell surface, and the cell line was named PC-3-Nectin-4 cells.

[0190] PC-3-Nectin 4 cells grown to the logarithmic phase and PC-3 cells as negative control were digested with trypsin, centrifuged at 200 g for 5 min, and the cells were collected and prepared into a 5×10 5 1 ml of cell suspension was pipetted into the flow tubes, i.e. 5×10 5 cells / flow cytometry tube, then centrifuge at 350 g for 5 min at 4°C, discard the supernatant, and wash twice with pre-cooled PBS for later use.

[0191] Antibody sample dilution and incubation:

[0192] Antibodies MM11, R012 and R036 were diluted to 5 μg / ml with PBS, and 200 μl of antibody dilution was taken to resuspend the above cells respectively, and the cells were gently mixed and incubated on ice in the dark for 1 hour.

[0193] Add fluorescent secondary antibody:

[0194] The flow tube was centrifuged at 4°C, 350g for 5 min, the supernatant was discarded, 1 ml of pre-cooled PBS was added to each tube to wash the cells twice, and then 500 μl of the secondary antibody was added. For antibody MM11, the secondary antibody was goat anti-mouse IgG Fc-FITC (1:200) (Abcam, catalog number ab150113); for antibody R012 and antibody R036, the secondary antibody was goat anti-rabbit IgG H&L-Alexa 488 (1:2000) (Abcam, catalog number ab150077), resuspend the cells, incubate on ice for 60 min, centrifuge at 350 g for 5 min at 4°C, discard the supernatant, then add 1 ml of pre-cooled PBS to each tube to wash the cells twice, and resuspend in 500 μl PBS.

[0195] Detection:

[0196] Use a flow cytometer to detect each flow tube, follow the relevant instrument operations, and import the results into the corresponding storage path.

[0197] FACS test results:

[0198] FACS test results Figure 2 As shown in the figure, the detection of PC-3-Nectin4 cells using antibody MM11, antibody R012 and antibody R036 all produced high fluorescence signals, while the detection of PC-3 cells had no obvious fluorescence signals. The results showed that Nectin-4 antibodies MM11, R012 and R036 can specifically recognize and bind to Nectin-4 expressed on PC-3-Nectin 4 cells, and do not recognize other proteins on the cells.

[0199] Example 4. Western blotting to detect the reactivity of antibodies with nectin-4 expressing cell lysates

[0200] Western blotting was used to detect whether the Nectin-4 antibody obtained in Example 1 specifically recognized the Nectin-4 protein. In addition, during Western blotting, an inhibition experiment was performed by adding an excess of Nectin-4 protein to further confirm the specificity of the Nectin-4 antibody obtained in Example 1.

[0201] The Western blot assay was performed as follows:

[0202] Human prostate cancer cells (PC-3) were purchased from ATCC, and flow cytometry was used to confirm that PC-3 cells did not express nectin-4.

[0203] PC-3-Nectin-4 cells are genetically engineered cell lines constructed by Myvicon, and are PC-3 cell lines expressing Nectin-4. Specifically, the full-length gene of human Nectin-4 (NCBI gene ID: 81607, 1533bp) was knocked into PC-3 cells and a stably transfected cell line with high expression of Nectin-4 was obtained through monoclonal cell screening. Flow cytometry detection confirmed that Nectin-4 was highly expressed on the cell surface, and the cell line was named PC-3-Nectin-4 cells.

[0204] Protein extraction: Resuscitate PC-3-Nectin 4 cells and PC-3 cells, grow them in cell culture flasks in a cell culture incubator to the appropriate cell density, discard the culture medium, and add pre-cooled PBS to wash twice. Then add RIPA lysis buffer (Radioimmunoprecipitation assay buffer) (Thermo, catalog number 89901) containing protease inhibitors to lyse the cells, scrape the cells with a cell scraper, lyse on ice for 30 minutes, and centrifuge at 12000rpm, 4°C for 10 minutes. Take the supernatant after centrifugation into a new centrifuge tube, determine the protein content by BCA method and adjust to the appropriate concentration. After adding 5× loading buffer and mixing evenly, heat in a 97°C metal bath for 10 minutes to completely denature the protein; use glyceraldehyde-3-phosphate dehydrogenase (GAPDH (glyceraldehyde-3-phosphate dehydrogenase)) as an internal reference for Western blotting;

[0205] Electrophoresis: The loading amount of the two cell lysates was 4.6 μg, and the electrophoresis conditions were: 120 V, 80 min;

[0206] Transfer: Wet transfer was used, and the wet transfer conditions were: 0.35A, 90min;

[0207] Blocking: Incubate the membrane with 5% BSA / TBST at room temperature for 1 h with shaking;

[0208] Add primary antibody:

[0209] (1) diluting the nectin-4 antibodies prepared in Example 1 (antibody MM11, antibody R012, and antibody R036, respectively) to 20 ng / ml with 5% BSA / TBST, adding them as the primary antibody, and incubating the membrane with shaking at 2-8° C. overnight; or

[0210] (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 and added as the primary antibody; 200 μg / ml recombinant human nectin-4 (McVancon, Lot: 20210101) was added for pre-incubation for 1 h, and then the membrane was incubated with shaking at 2-8°C overnight;

[0211] Add the secondary antibody: After rewarming for 30 minutes, wash the membrane three times with TBST, each time for 5 minutes, dilute the goat anti-mouse IgG-HRP (Jackson, catalog number: 115-035-003) as the secondary antibody at a ratio of 1:50000 with 5% BSA / TBST, and incubate at room temperature for 1 hour with shaking; or dilute the goat anti-rabbit IgG (H+L) antibody-HRP (ThermoFisher, catalog number: 31460) as the secondary antibody at a ratio of 1:25000 with 5% BSA / TBST, and incubate at room temperature for 1 hour with shaking;

[0212] Color development: After the second antibody incubation is completed, wash the membrane three times with TBST, 5 minutes each time, add 1:1 prepared ECL luminescent solution for color development, and collect the luminescent signal.

[0213] Western blot test results:

[0214] Western blotting results Figure 3 As shown, the detection band of GAPDH as an internal reference is approximately at 36kDa, which ensures the accuracy and reliability of the experimental results. Specific bands that bind to antibodies MM11, R012, and R036 can be found in cells with positive expression of Nectin-4, but no specific bands are found in cells with negative expression of Nectin-4.

[0215] The specific bands binding to antibodies MM11 and R012 in nectin-4 positive cells disappeared after adding excess recombinant nectin-4 protein, indicating that anti-nectin-4 antibodies can specifically bind to human nectin-4 protein.

[0216] Example 5. Immunohistochemical staining of nectin-4 rabbit monoclonal antibody

[0217] Multiple tumor tissues and normal human tissue samples were selected, fixed with neutral formalin, and prepared into paraffin tissue sections. The normal human tissues included tissues and organs from multiple sources; the tumor tissues included: 3 bladder cancers, 2 lung cancers, and 3 breast cancers.

[0218] Anti-Nectin-4 monoclonal antibody was used to detect the expression of Nectin-4 in tissue samples, and commercial Nectin-4 rabbit monoclonal antibody (purchased from abcam, clone number: EPR15613-68) was used as a positive control. All antibodies were diluted to working concentrations using antibody diluent, which was purchased from Maijie Translational Medicine Research (Suzhou) Co., Ltd., catalog number P010A01. Immunohistochemical staining was detected using a fully automated immunohistochemical staining system (Leica BOND III), and the specific procedures are shown in the table below.

[0219] Table 2. Immunohistochemical staining procedures for nectin-4 rabbit monoclonal antibody

[0220]

[0221] Note: Peroxidase inactivation reagent, goat anti-rabbit IgG-polymer HRP, DAB color development reagent, and hematoxylin staining solution are all included in Leica's BOND Polymer Refine Detection Kit, catalog number DS9800. All reagents have been diluted to working concentrations and can be used directly.

[0222] The results of immunohistochemical staining are shown in Table 3. Figure 4 , Figure 5 , Figure 6 and Figure 7As shown in the figure, the three antibodies EPR15613-68, R012 and R036 performed basically the same in normal tissues and tumor tissues with different nectin-4 expression levels. The target protein nectin-4 was localized on the cell membrane and / or cytoplasm, which was consistent with the protein localization reported in the literature, and no nonspecific staining was produced. In normal tissues, the trophoblast cells of the placenta, the epithelium of the skin and the crypt epithelium of the tonsils were obviously positive for nectin-4 staining, and the rest of the normal tissues and organs were basically negative. In most tumor tissues, tumor cells were positive for nectin-4, a small part was negative for nectin-4, and normal cells in tumor tissues were negative for nectin-4.

[0223] Table 3. Immunohistochemical staining results of nectin-4 rabbit monoclonal antibody on normal human tissues

[0224]

[0225]

[0226] Example 6. Immunohistochemical staining of nectin-4 mouse monoclonal antibody

[0227] Multiple tumor tissues and normal human tissue samples were selected, fixed with neutral formalin, and prepared into paraffin tissue sections. The normal human tissues included tissues and organs from multiple sources; the tumor tissues included: 3 cases of prostate cancer, 3 cases of lung cancer, and 3 cases of triple-negative breast cancer tissues. Anti-Nectin-4 monoclonal antibody was used to detect the expression of Nectin-4 in tissue samples, and mouse monoclonal antibody M22-321b41.1 was used as a positive control. M22-321b41.1 is a patented Nectin-4 mouse monoclonal antibody, and the patent publication number is CN111051345A. All antibodies were diluted to working concentrations using antibody diluent. The antibody diluent was purchased from Maijie Translational Medicine Research (Suzhou) Co., Ltd., item number P010A01. Immunohistochemical staining was detected in a fully automatic immunohistochemical staining system (Leica BOND III). The specific procedures are shown in the following table.

[0228] Table 4. Immunohistochemical staining procedures for Nectin-4 mouse monoclonal antibody

[0229]

[0230] Note: Peroxidase inactivation reagent, rabbit anti-mouse Post-primary, goat anti-rabbit IgG-polymer HRP, DAB color development reagent, and hematoxylin staining solution are all included in Leica's BOND Polymer Refine Detection Kit, catalog number DS9800. All reagents have been diluted to working concentrations and can be used directly.

[0231] The results of immunohistochemical staining are shown in Table 5. Figure 8 , Fig. 9 , Fig.10 and Fig.11 As shown in the figure, in normal tissues, the performance of antibody M22-321b41.1 and antibody MM11 is basically the same. In normal tissues, the salivary glands and skin epithelium of some individuals are weakly positive for Nectin-4, the tonsil crypt epithelium and the bronchus of the lungs are obviously positive for Nectin-4, and the rest of the normal tissues and organs are basically negative. In most tumor tissues, tumor cells are positive for Nectin-4, while normal cells in tumor tissues are negative for Nectin-4. In tumor tissues with different Nectin-4 expression levels, after staining with antibody MM11 and antibody M22-321b41.1, the target protein Nectin-4 is localized on the cell membrane and / or cytoplasm, which is consistent with the protein localization reported in the literature, and no nonspecific staining is produced. In some tumor tissues, especially triple-negative breast cancer, the staining signal of antibody MM11 is significantly stronger than that of antibody M22-321b41.1.

[0232] Table 5. Immunohistochemical staining results of nectin-4 mouse monoclonal antibody on normal human tissues

[0233]

[0234] The exemplary embodiments of the present invention are described above, and it should be understood by those skilled in the art that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0235] Exemplary sequences

[0236]

[0237]

[0238]

[0239]

[0240]

Claims

1. An anti-nectin-4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 5; HCDR2 as shown in SEQ ID NO: 6; and HCDR3 as shown in SEQ ID NO: 7; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 8; LCDR2 as shown in SEQ ID NO: 9; and LCDR3 as shown in SEQ ID NO: 10; (b) the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 15; HCDR2 as shown in SEQ ID NO: 16; and HCDR3 as shown in SEQ ID NO: 17; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 18; LCDR2 as shown in SEQ ID NO: 19; and LCDR3 as shown in SEQ ID NO: 20; or (c) the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 25; HCDR2 shown in SEQ ID NO: 26; and HCDR3 shown in SEQ ID NO: 27; the light chain variable region comprises LCDR1 shown in SEQ ID NO: 28; LCDR2 shown in SEQ ID NO: 29; and LCDR3 shown in SEQ ID NO:

30.

2. The anti-nectin-4 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein (a) the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; (b) the heavy chain variable region comprises a sequence of SEQ ID NO: 13, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 14, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or (c) the heavy chain variable region comprises a sequence of SEQ ID NO:23, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; For example, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises (a) the heavy chain variable region comprises the sequence of SEQ ID NO: 3, and the light chain variable region comprises the sequence of SEQ ID NO: 4; (b) the heavy chain variable region comprises the sequence of SEQ ID NO: 13, and the light chain variable region comprises the sequence of SEQ ID NO: 14; or (c) the heavy chain variable region comprises the sequence of SEQ ID NO: 23, and the light chain variable region comprises the sequence of SEQ ID NO: 24; For example, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises (a) SEQ ID NO:1, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and SEQ ID NO:2, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; e.g., the heavy chain sequence set forth in SEQ ID NO:1, and the light chain sequence set forth in SEQ ID NO:2; (b) SEQ ID NO:11, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO:12, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; e.g., the heavy chain sequence set forth in SEQ ID NO:11, and the light chain sequence set forth in SEQ ID NO:12; or (c) SEQ ID NO:21, or a heavy chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and SEQ ID NO:22, or a light chain sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; for example, the heavy chain sequence set forth in SEQ ID NO:21, and the light chain sequence set forth in SEQ ID NO:

22.

3. The anti-nectin-4 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single-chain Fv, single-chain Fab or diabody.

4. The 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) In the ELISA assay, it has the ability to specifically bind only to Nectin-4, and has no specific binding to 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) having the ability to bind to Nectin-4 molecules expressed on cells as measured in a flow cytometry assay; (c) can specifically detect Nectin-4 protein when measured in Western blotting; and when an excess of Nectin-4 protein is added, the binding of Nectin-4 protein to the membrane is inhibited; and / or (d) in immunohistochemical staining, specifically staining tissues expressing nectin-4 molecules, for example, the tissues are selected from tonsils, salivary glands, brain, cerebellum, placenta, bladder, lung, breast, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureter, ovary, fallopian tube, cervix, endometrium, skin, kidney, prostate, pancreas, thyroid, spleen and liver. 5 . A kit comprising the anti-nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 .

6. Use of the anti-nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for preparing a kit for detecting nectin-4 in a sample.

7. A method for detecting nectin-4 in a biological sample from a subject, the method comprising: 1) contacting the biological sample with a capture agent, wherein the capture agent is the anti-nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; 2) detecting binding of the capture agent to the biological sample; 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 presence or expression level of Nectin-4; Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

8. 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, the method comprising: 1) contacting the biological sample from the subject with a capture agent, wherein the capture agent is the anti-nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; 2) detecting binding of the capture agent to a biological sample from the 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 presence or expression level of Nectin-4; Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

9. A method for prognosing a subject suffering from cancer, the method comprising: 1) contacting a biological sample from a subject treated with an anti-tumor 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 the anti-nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; 2) detecting binding of the capture agent to a biological sample from the subject; and optionally 3) determining the presence or expression level of Nectin-4 in a biological sample from the subject, Detection of Nectin-4 and / or detection of an expression level of Nectin-4 in the sample higher than a reference level indicates a poor prognosis; or, absence of Nectin-4 in the sample or detection of a level of Nectin-4 lower than a reference presence or expression level indicates a good prognosis, Optionally, the presence or expression level of Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and / or co-immunoprecipitation (Co-IP).

10. according to the method described in any one of claims 7-9, wherein the biological sample is a tissue sample, a cell sample or a body fluid sample of a subject, for example, a tissue sample, blood, serum, urine, saliva, or a tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue.

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