Nectin-4 protein binding molecule as well as preparation method and application thereof

Abstract

The invention provides a Nectin-4 protein binding molecule as well as a preparation method and application thereof. The binding molecule has excellent binding performance, can realize specific cell membrane Nectin-4 binding, and has excellent binding specificity and detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay, and more specifically, this invention relates to a Nectin-4 protein-binding molecule, its preparation method and application. Background Technology

[0002] The Nectin family is a class of calcium-independent cell adhesion molecules, including Nectin-1, Nectin-2, Nectin-3, and Nectin-4. Nectin-4, also known as poliovirus receptor-like protein 4 (PVRL4), is a 66 kDa type I transmembrane protein whose main function is to promote cell-cell contact. Nectin-4, along with cadherin, participates in the formation and maintenance of adhesion junctions. Nectin-4 is also a receptor for several measles viruses. Nectin-4 mutations are a cause of type I ectodermal dysplasia syndactyly, a rare autosomal recessive genetic disorder.

[0003] Human Nectin-4 protein is primarily expressed in the placenta, and weakly to moderately expressed in normal human tissues such as skin, bladder, salivary glands, esophagus, and breast. Studies have shown that Nectin-4 is overexpressed in various malignant tumors, including urothelial carcinoma, breast cancer, and cervical cancer, and is associated with cancer progression and poor prognosis. Therefore, Nectin-4 is a very attractive drug target.

[0004] Currently, there are almost no antibodies on the market that can specifically and sensitively detect Nectin-4, especially anti-Nectin-4 monoclonal antibodies that can be used in immunohistochemistry (IHC). Existing anti-Nectin-4 monoclonal antibodies suffer from non-specific reactions and cross-reactivity, and it is difficult to overcome the problems of non-specificity and low sensitivity, which may lead to false positive or false negative results.

[0005] Therefore, the preparation of anti-Nectin-4 monoclonal antibodies with high specificity, high sensitivity and wide application range has important practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to provide a Nectin-4 protein-binding molecule, its preparation method, and its application.

[0007] In a first aspect of the invention, an antibody (including its antigen-binding fragment) that binds to Nectin-4 is provided, the antibody having a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of heavy chain CDR1 is as shown in SEQ ID NO:4, the amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO:6; the amino acid sequence of light chain CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO:8, and the amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO:9.

[0008] In one or more preferred embodiments, the antibody comprises: a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:2, or having more than 80% (e.g., 85%, 90%, 93%, 95%, 97%, or 99%) similarity to the amino acid sequence shown in SEQ ID NO:2; a light chain variable region having the amino acid sequence shown in SEQ ID NO:3, or having more than 80% (e.g., 85%, 90%, 93%, 95%, 97%, or 99%) similarity to the amino acid sequence shown in SEQ ID NO:3; and retaining binding activity.

[0009] In one or more preferred embodiments, the antibody has the property of specifically binding to Nectin-4 on the cell membrane.

[0010] In one or more preferred embodiments, the antibody comprises: a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody; or, the antibody comprises: Fab, Fab'-SH, Fv, Fd, scFv, or (Fab')2 fragment.

[0011] In another aspect of the invention, an isolated polynucleotide or a construct containing the polynucleotide is provided, the polynucleotide encoding an antibody binding to Nectin-4 as described above; preferably, the construct is an expression vector.

[0012] In another aspect of the invention, an antibody expression system is provided, the expression system comprising the aforementioned construct or genome in which an exogenous polynucleotide is integrated; preferably, the expression system is a cell (expression system).

[0013] In one or more embodiments, the cell (host cell) is a eukaryotic cell or a prokaryotic cell.

[0014] In one or more embodiments, the eukaryotic cells include mammalian cells, yeast cells, etc.

[0015] In one or more embodiments, the mammalian cells include (but are not limited to): HEK-293 cells, Chinese hamster ovary (CHO) cells, Vero cells, NSO cells, BHK cells, PER-C6 cells, etc.

[0016] In one or more embodiments, the prokaryotic cells include (but are not limited to) Escherichia coli cells.

[0017] In another aspect of the present invention, a method for preparing any of the antibodies described above is provided, comprising: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby obtaining the antibody; preferably, further comprising purifying and isolating the antibody.

[0018] In one or more preferred embodiments, a reagent is used to prepare cells that specifically target and express Nectin-4; preferably, the cells are cells whose cell membranes express Nectin-4; preferably, the antibody in the reagent is further modified with a conjugate.

[0019] In one or more preferred embodiments, the conjugate includes a detectable marker; more preferably, the detectable marker includes (but is not limited to) one or more of the following: fluorescent dyes (such as fluorescein isothiocyanate, Cy3 or Cy5), radioactive isotopes, horseradish peroxidase (HPR), alkaline phosphatase, ferritin, colloidal gold, and biotin.

[0020] In one or more preferred embodiments, the conjugate comprises: a cytotoxin or a bioactive protein (including a second antibody or multiple antibodies).

[0021] In another aspect of the invention, a reagent is provided that specifically targets cells expressing Nectin-4, comprising any of the antibodies described above, and a conjugate operatively linked thereto.

[0022] In one or more preferred embodiments, the conjugate includes a detectable marker; more preferably, the detectable marker includes (but is not limited to) one or more of the following: fluorescent dyes (such as fluorescein isothiocyanate, Cy3 or Cy5), radioactive isotopes, horseradish peroxidase (HPR), alkaline phosphatase, ferritin, colloidal gold, and biotin.

[0023] In one or more preferred embodiments, the conjugate comprises: a cytotoxin or a bioactive protein (including a second antibody or multiple antibodies).

[0024] In one or more embodiments, the antibody and the conjugate can be operatively linked by means of covalent connection, coupling, attachment, cross-linking, etc.

[0025] In one or more embodiments, when the conjugate is a cytotoxin, a radioisotope, or a bioactive protein, the antibody has the function of specifically targeting cells expressing Nectin-4 and carrying the cytotoxin or bioactive protein to the cells.

[0026] In one or more embodiments, the cytotoxicant is an antitumor toxin, including (but not limited to): maytansine, orlistatine, monomethylaurestatin, maytansine alkaloids, dolalastatin, cazithromycin, methotrexate, vinca disine, taxanes such as docetaxel, paclitaxel, lalotaxel, tescetaxel or octataxel, trichosporon toxins, CC1065, DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines or SN-38, etc.

[0027] In another aspect of the invention, the use of any of the antibodies or reagents described above in the preparation of formulations, kits, or pods for detecting Nectin-4 expression-related diseases is provided; preferably, the Nectin-4 expression-related diseases include (but are not limited to): urothelial carcinoma, breast cancer, esophageal cancer, head and neck squamous cell carcinoma, cervical cancer, and lung cancer; preferably, the tumors include tumors expressing Nectin-4; preferably, the tumors include (but are not limited to): urothelial carcinoma, breast cancer, esophageal cancer, head and neck squamous cell carcinoma, cervical cancer, and lung cancer.

[0028] In another aspect of the invention, a kit or pharmaceutical kit is provided, comprising any of the antibodies, reagents, or compositions thereof described above.

[0029] In one or more embodiments, the kit or cassette further includes a container for containing the antibody, reagent, or composition, wherein the container may be one or more.

[0030] In one or more embodiments, the kit or cassette further contains a solid-phase carrier, or the antibody is immobilized (coated) on the solid-phase carrier.

[0031] In one or more embodiments, the solid-phase carrier includes (but is not limited to): a chip (including a liquid-phase chip, a microfluidic chip), a test pad (such as a colloidal gold test pad), a test strip (including a test strip, such as a colloidal gold test strip), a glass slide, a well plate (such as a coated plate), microspheres, and magnetic beads.

[0032] In one or more embodiments, the kit or medicine box also contains instructions for use (packaging insert) describing how to use the antibody, reagent, or composition.

[0033] In one or more embodiments, the kit may also include (but is not limited to) any or more of the following reagents: diluent (such as sample and antibody diluent), secondary antibody (such as Polymer), chromogenic agent (such as DAB chromogenic reagent), washing solution, calibrator, quality control, stop solution, luminescent solution, antigen retrieval agent, dewaxing agent, and blocking reagent.

[0034] In one or more embodiments, the kit may further include: sample collection tubes, instructions for use, etc.

[0035] In another aspect of the present invention, a method for in vitro detection of Nectin-4 antigen is provided, the method comprising: analyzing the presence of Nectin-4 antigen in a sample to be tested using any of the antibodies or reagents described above; preferably, the method comprises: immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), or flow cytometry.

[0036] In one or more preferred embodiments, the method is an immunohistochemistry method, comprising: providing a sample (e.g., a slide) and labeling and staining it with the antibodies described above in this invention.

[0037] In one or more preferred embodiments, the method is an enzyme-linked immunosorbent assay (ELISA) based on the antibody or bispecific antibody of the present invention; for example, it includes: (a) contacting the sample to be tested with a first antibody, thereby causing the Nectin-4 antigen in the sample to bind to the first antibody on a solid-phase carrier, forming a solid-phase carrier with a binary complex of "Nectin-4 antigen-first antibody"; (b) adding a second antibody to the system of (a), thereby forming a solid-phase carrier with a ternary complex of "second antibody-Nectin-4 antigen-first antibody"; and the second antibody carries a label; (c) detecting the label in the ternary complex, thereby determining the presence and amount of Nectin-4 antigen in the sample to be tested; with the additional condition that steps (a) and (b) can be performed sequentially or simultaneously.

[0038] In one or more preferred embodiments, the method for detecting Nectin-4 antigen is an in vitro method.

[0039] In one or more preferred embodiments, the method for detecting Nectin-4 antigen is a non-diagnostic method (e.g., performing protein expression analysis on cells, not with the direct purpose of obtaining disease diagnosis results).

[0040] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0041] Figure 1The purified recombinant protein was analyzed using the SDS-PAGE method.

[0042] Figure 2 1. Immunoblotting analysis using ADx024S monoclonal antibody. Lane 1: A549 (-); Lane 2: T47D (+); Lane 3: MCF-7 (+); Lane 4: SKBR3 (+); Lane 5: Nectin-4 recombinant protein; Lane 6: Nectin-1 recombinant protein; Lane 7: Nectin-2 recombinant protein; Lane 8: Nectin-3 recombinant protein.

[0043] Figure 3 Immunohistochemical staining was performed using the ADx024S monoclonal antibody on cell line samples HEK293, HEK293 cells transiently overexpressing Nectin4 plasmid, human breast adenocarcinoma MDA-MB-231 cells, human breast adenocarcinoma SKBR3 cells, and human breast duct carcinoma T47D cells. The commercially available monoclonal antibodies Nectin-4 (A-9) and Nectin-4 (EPR15613-68) were used as comparative antibodies.

[0044] Figure 4 The results of immunohistochemical detection using ADx024S monoclonal antibody on paraffin-embedded tissues of normal human breast and tonsils were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0045] Figure 5 The results of immunohistochemical detection using ADx024S monoclonal antibody on paraffin-embedded tissue of normal human esophagus and parotid gland were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0046] Figure 6 The results of immunohistochemical detection using ADx024S monoclonal antibody on normal human prostate and cervical paraffin tissue were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0047] Figure 7 The results of immunohistochemical detection using ADx024S monoclonal antibody on normal human bladder and paraffin-embedded skin tissue were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0048] Figure 8 Immunohistochemical staining results of paraffin-embedded tissues from human cervical cancer and head and neck squamous cell carcinoma using ADx024S monoclonal antibody; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0049] Figure 9Immunohistochemical staining results of paraffin-embedded tissues from human esophageal cancer, breast cancer, urothelial carcinoma, and lung cancer using the ADx024S monoclonal antibody; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0050] Figure 10 The results of immunohistochemical detection using ADx024S monoclonal antibody on paraffin tissue microarrays for human cervical cancer and esophageal squamous cell carcinoma were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0051] Figure 11 The results of immunohistochemical detection using ADx024S monoclonal antibody on paraffin tissue microarrays of human head and neck squamous cell carcinoma and breast cancer were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody.

[0052] Figure 12 The results of immunohistochemical detection using ADx024S monoclonal antibody on paraffin tissue microarrays for human urothelial carcinoma and non-small cell lung cancer were analyzed; the commercially available monoclonal antibody Nectin-4 (A-9) was used as a comparative antibody. Detailed Implementation

[0053] Through in-depth research, the inventors have developed a monoclonal antibody against Nectin-4 protein (named Monoclonal Antibody ADx024S). This monoclonal antibody exhibits excellent immunogen-binding properties and, targeting a specific epitope of the Nectin-4 protein, can achieve specific cell membrane binding to Nectin-4. The antibody of this invention possesses excellent binding specificity and detection sensitivity.

[0054] Those skilled in the art will understand that an antigen may contain a variety of epitopes (antigenic determinants). Therefore, more than one antibody can be obtained against the same antigen, and these antibodies may have different binding properties (such as specificity). Therefore, those skilled in the art need to compare and screen for the same antigen to find a monoclonal antibody suitable for specific binding. Due to the spatial structure of antigens, many epitopes are contained within the spatial structure, making it difficult to find antibodies that can specifically bind to epitopes that are stably located outside the spatial structure. The inventors of this invention have conducted in-depth research and isolated monoclonal antibodies with high specificity and sensitivity to Nectin-4, particularly suitable for immunohistochemistry.

[0055] In this invention, the target of detection is the Nectin-4 protein, whose expression indicates specific symptoms. Existing anti-Nectin-4 monoclonal antibodies suffer from non-specific reactions and cross-reactivity, making it difficult to overcome the challenges of non-specificity and low sensitivity. This invention provides a novel detection reagent to address these technical difficulties.

[0056] As used in this article, “sample to be tested” can encompass a variety of sample types, including cells (including ex vivo), tissues, and body fluids.

[0057] As used herein, "specificity" means that the antibody can bind to Nectin-4 or a fragment thereof. More specifically, it refers to an antibody that can bind to Nectin-4 or its fragment but does not recognize or bind to other unrelated molecules. Even more specifically, it refers to an antibody that can bind to Nectin-4 or its fragment on the cell membrane but does not recognize or bind to other molecules.

[0058] As used herein, the terms "first monoclonal antibody," "first antibody," and "primary antibody" are used interchangeably and refer to the anti-Nectin-4 monoclonal antibody of the present invention.

[0059] As used herein, the terms "detection antibody," "second monoclonal antibody," "second antibody," "enzyme-labeled antibody," and "secondary antibody" are used interchangeably and refer to antibodies that specifically target Nectin-4 and correspond to the first antibody. For the antigen Nectin-4, the corresponding first and second antibodies are different and can simultaneously bind to different epitopes (antigenic determinants) of Nectin-4.

[0060] As used in this article, “sample to be tested” encompasses a variety of sample types, including various objects that require Nectin-4 content detection.

[0061] As used herein, the term "marker" or "detectable marker" refers to a marker located on or linked to (indirectly linked to) the monoclonal antibody or second monoclonal antibody of the present invention, used to determine the presence and amount of Nectin-4 in a sample to be tested. For example, the marker may be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0062] As used in this article, "recombinant" refers to proteins, genetic engineering vectors, or cells obtained (or mass-produced) through genetic engineering.

[0063] In this invention, a human Nectin-4 protein fragment (amino acids 32-349 in Q96NY8-1) is selected as the immunogen. The amino acid sequence of the immunogen is shown in SEQ ID NO:1. The Nectin-4 protein fragment provides suitable epitopes, which is beneficial for the screening of antibodies in this invention. The immunogen is fused with a linker and a tag to facilitate purification and other recombinant operations. The Nectin-4 protein fragment can be a chemically synthesized product or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. The polynucleotide encoding the polypeptide can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods.

[0064] The anti-Nectin-4 protein monoclonal antibody of the present invention comprises a binding domain containing a complementary determining region (CDR) selected from the following: heavy chain CDR1 as shown in SEQ ID NO:4, heavy chain CDR2 as shown in SEQ ID NO:5, and heavy chain CDR3 as shown in SEQ ID NO:6; light chain CDR1 as shown in SEQ ID NO:7, light chain CDR2 as shown in SEQ ID NO:8, and light chain CDR3 as shown in SEQ ID NO:9.

[0065] In a preferred embodiment, the anti-Nectin-4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:3.

[0066] In some implementations, the antibodies provided herein are antibody fragments. Antibody structures can be modified to construct molecules with similar antigen affinity but different structures, such as Fab, F(ab')2, Fab', Fab'-SH, Fv fragments, single-chain antibodies (e.g., scFv), single-domain antibodies, antibody-drug conjugates, bifunctional antibodies, etc. Fab' fragments refer to Fab with a small number of amino acid residues (including one or more cysteine ​​residues from the antibody hinge region) added to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH refers to Fab' with free thiol groups on the cysteine ​​residues in the constant region.

[0067] In some embodiments, the invented antibody may be modified into a chimeric antibody or further shaped into a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, humanized antibodies may also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues derived from the non-human antibody, for example, to repair or improve antibody specificity or affinity.

[0068] This invention also includes variants of the anti-Nectin-4 antibody. For example, antibody variants prepared to further improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, residue deletions and / or insertions and / or substitutions within the antibody's amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain a final construct that can possess desired characteristics, such as antigen binding.

[0069] Amino acids can be grouped according to their common side chain properties for preferred amino acid substitution, for example, substitution with amino acids of the same / property: (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0070] After the antibodies are disclosed in this invention, those skilled in the art can artificially prepare them using bioengineering techniques known in the art. The monoclonal antibodies of this invention can be obtained using Nectin-4 or its fragments or functional regions through conventional immunoassay techniques. Alternatively, they can be prepared using recombinant methods or synthesized using a peptide synthesizer.

[0071] To recombinantly produce anti-Nectin-4 antibodies, the nucleic acids encoding the antibody (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. These nucleic acids can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding both the heavy and light chains of the antibody.

[0072] Suitable host cells for cloning or expressing antibody-encoding vectors include the eukaryotic or prokaryotic cells listed herein. After expression, the antibody in the soluble fraction can be isolated from the cells and further purified.

[0073] In a preferred embodiment, the anti-Nectin-4 monoclonal antibody further comprises a modifying conjugate, which includes several detectable markers. A variety of detectable markers conventionally used in the art for binding with detection antibodies can be employed. Any marker capable of binding to the monoclonal antibody of the present invention and, after appropriate processing, accurately indicating the presence and amount of the target protein in the sample to be tested is acceptable.

[0074] When antibodies are labeled for detection, the labeling methods can include fluorescent labeling, chemiluminescent labeling, radioactive labeling, enzyme-linked labeling, biotin / avidin labeling, magnetic bead labeling, or nanoparticle labeling.

[0075] The detectable markers described in this invention may include, but are not limited to: fluorescent markers, chromogenic markers, colloidal gold; such as: enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More specifically, examples include horseradish peroxidase (HPR), alkaline phosphatase, glucose oxidase, biotin, β-D-galactosidase, urease, catalase, glucosyl amylase, fluorescein isothiocyanate, radioactive isotopes, Cy3, or Cy5. More than one marker may also be included.

[0076] When using some of the enzyme markers shown above, it is also necessary to use some substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be reported by means of color development, etc.

[0077] The substrate corresponding to the label, such as the enzyme, can be catalyzed by the label to produce a colorimetric reaction, displaying a recognition signal indicating the binding of the secondary antibody to the target protein. Examples of such substrates include: o-phenylenediamine (OPD), tetramethylbenzidine (MB), and ABTS for horseradish peroxidase; p-nitrophenyl phosphate (p-NPP) for alkaline phosphatase; phycoerythrin (streptavidin-phycoerythrin, also known as PE-labeled streptavidin, SA-R-PE) for biotin; and so on. Those skilled in the art can select a suitable substrate based on the type and characteristics of the label used.

[0078] The marker can be directly applied to the detection antibody; or, the marker can be applied to the anti-antibody of the specific anti-second antibody. Those skilled in the art can select a suitable marker according to the type and characteristics of the antibody used.

[0079] For detection and / or analytical purposes, the labeling used to label antibodies depends on the specific detection / analytical technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labeling for detection / analytical techniques and / or methods known in the art is well known to those skilled in the art.

[0080] As a specific example, the detectable marker is HRP, which acts as a colorimetric marker and generates a signal when combined with OPD, MB, or ABTS.

[0081] The anti-Nectin-4 antibody described in this invention can also be linked to one or more cytotoxins, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof), or radioisotopes. For example, antibody-drug conjugates (ADCs) can be formed, wherein the antibody is bound to one or more drugs, including but not limited to maytansine; orlistatine, such as the monomethyl orlistatine drug portions DE and DF (MMAE and MMAF); dolalastatin; cazithromycin or its derivatives; methotrexate; vinca disine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; or trichothecene, etc.

[0082] The anti-Nectin-4 antibody of the present invention can also be linked to one or more enzymatically active toxins or fragments thereof, including but not limited to diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain, and trichothecotoxin.

[0083] The anti-Nectin-4 antibody described in this invention can also bind to one or more radioactive atoms to form radioactive conjugates. Examples of various radioactive isotopes include At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu.

[0084] After obtaining the anti-Nectin-4 antibody of the present invention, those skilled in the art can sensitively detect the presence or concentration of Nectin-4 in a sample through various methods, employing techniques commonly used in the field of immunology. These include qualitative and quantitative detection methods. Preferably, the qualitative detection includes: identifying the presence of Nectin-4 using immunoblotting or immunofluorescence methods; for example, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, and immunofluorescence; more specifically, methods such as ELISA, immunogold immunofluorescence strips, immunofluorescence strips, and homogeneous enzyme immunoassay to measure Nectin-4. The application results are ideal.

[0085] Compared to some existing anti-Nectin-4 antibodies, the anti-Nectin-4 antibody of this invention exhibits highly ideal specificity, specifically binding to Nectin-4 on the cell membrane without nuclear staining. Nuclear staining is considered non-specific. Many cell membrane targets are primarily detected through cell membrane staining using IHC. For example, Nectin-4 ADCs target Nectin-4 on the cell membrane. If a Nectin-4 IHC antibody cannot accurately assess the staining of Nectin-4 on the cell membrane, it cannot be used for retrospective testing in clinical trials, CTA enrollment, or companion assays.

[0086] This invention also provides a kit for detecting Nectin-4, the kit containing the monoclonal antibody described herein. In some embodiments, one of the monoclonal antibodies is immobilized on a solid-phase support, and the other serves as the detection antibody. As an example, the solid-phase support used may be a microtiter plate (ELISA plate).

[0087] To eliminate false positives and false negatives, quality controls (restrictions) can be set up during the testing process. Furthermore, to obtain quantitative results, multiple Nectin-4 standards with known concentrations can be set up during the testing process. Conventional methods can be used to set up the standards. For quantitative detection, a standard curve can be constructed using the aforementioned standards, including plotting the OD values ​​of the standards on the ordinate (Y-axis) and the standard concentrations on the abscissa (X-axis) to create a quantitative standard curve for the Nectin-4 kit. Therefore, based on the OD values ​​obtained from the test sample, the concentration of Nectin-4 in the test sample can be calculated using the standard curve.

[0088] Furthermore, to facilitate testing, the kit preferably includes other auxiliary reagents, such as those commonly used in immunohistochemical methods. The properties and preparation methods of these reagents are well known to those skilled in the art. These reagents include, but are not limited to, chromogenic agents, washing buffers, stop solutions, and sensitizing diluents. These reagents can be adjusted according to different detection methods. The kit may also include instructions for use.

[0089] The main advantages of this invention are that the anti-Nectin-4 antibody described herein has high affinity and high specificity in recognizing Nectin-4, especially cell membrane Nectin-4, exhibiting very high specificity, sensitivity, and accuracy. Currently, there are almost no antibodies on the market capable of specifically and sensitively detecting Nectin-4, especially anti-Nectin-4 monoclonal antibodies applicable to immunohistochemistry (IHC), and this invention changes this situation.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0091] Example 1: Preparation of Immunogen

[0092] The amino acid sequence of the human Nectin-4 protein is shown in Uniprot ID: Q96NY8-1. After in-depth analysis, the inventors selected the protein fragment of human Nectin-4 (aa 32-349 in Q96NY8-1; i.e., SEQ ID NO:1) as the immunogen. The immunogen was prepared by using an artificially synthesized protein gene and adding a linker (GGGSGGGS) and an 8-histidine tag (SEQ ID NO:2) to the C-terminus of the protein.

[0093] The sequence of the immunogen is as follows (SEQ ID NO:1):

[0094] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGT TSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSAS

[0095] The sequence of the immunogen, linker, and tag fusion is as follows (SEQ ID NO:10):

[0096] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSR SFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASGGGSGGGS HHHHHHH

[0097] Overexpression was performed via transient transfection using the HEK293 system, and the overexpression supernatant was purified by Ni column affinity.

[0098] The purified recombinant protein was purified by SDS-PAGE (>=80% purity) and tested for endotoxin (<30 EU / mg). Once the protein met the requirements, it was used as an immunogen for immunization of experimental animals. The SDS-PAGE results are shown below. Figure 1 As shown.

[0099] Example 2: Immunoassay and ELISA Detection

[0100] Rabbits were immunized five times and once as a booster. The first five immunizations were performed subcutaneously at multiple sites on the back of the rabbits, with each rabbit receiving 0.4 mg of recombinant Nectin-4 protein (the immunogen prepared in Example 1 above) and adjuvant. The booster was administered intravenously (IV). The doses of the initial and booster immunizations were twice those of the other immunizations. Serum from the rabbits on day 10 after the fourth immunization was collected and the titer was determined using an indirect ELISA method. The serum titer was required to be greater than 0.2 at a ratio of 1:64000. Rabbits meeting the titer requirements were selected for the B cell screening stage.

[0101] In addition, the serum from rabbits after the fourth immunization was purified by antigen affinity and tested using Western blotting (positive material was T-47D, negative material was A549). The tests showed that the rabbits met the titer requirements and the Western blotting showed a clear target band. After IV enhancement, the spleen was harvested for B cell screening.

[0102] Example 3: B cell screening

[0103] Four days after booster immunization, spleens were harvested. B cells capable of secreting specific antibodies were isolated by antigen coating and adsorption. In the initial screening stage, six 96-well plates were plated, and the supernatant from 576 wells of B cells was sent for ELISA. The supernatant from the 120 wells of positive clones with the strongest indirect ELISA was selected for sample processing for IHC and WB detection.

[0104] The supernatant from the positive clones was used to perform IHC on breast cancer samples and normal tissue samples, and 29 clones with positive signals on breast cancer sections were initially selected. These 29 clones were transferred into 24-well plates for culture, and the expanded supernatant was used to further verify WB (T-47D / A549) and IHC (breast cancer tissue).

[0105] Using IHC breast cancer slide positivity and WB material positivity as evaluation criteria, five clones were finally selected to enter the small-scale transformation stage for validation.

[0106] Example 4, Recombinant Small Transformation and Identification

[0107] Antibody mRNA was obtained by lysing B cells of the target clone. The nucleic acids encoding the heavy and light chains of the B cells secreting specific antibodies were amplified by RT-PCR. The obtained nucleic acids encoding each heavy and light chain were constructed into the pcDNA3.4 vector using homologous recombination. The vector was transformed into TOP10 competent cells and cultured overnight at 37°C. Single colonies were picked and sequenced to obtain the nucleic acid sequences encoding the heavy and light chains. Plasmids containing different heavy and light chain nucleic acid sequences were paired and transfected into HEK293 cells to express a small amount of recombinant antibody. The supernatant was collected three days later for further verification by Western blotting and IHC.

[0108] Based on the validation results, one of the clones showed particularly excellent specificity in IHC and WB, and the inventors named it the monoclonal antibody ADx024S.

[0109] Example 5: Structural analysis of monoclonal antibody ADx024S

[0110] The monoclonal antibody ADx024S was sequenced and analyzed, including the sequence structure of its light and heavy chains and complementarity-determining regions.

[0111] The amino acid sequence of the heavy chain variable region of the rabbit anti-human Nectin-4 monoclonal antibody (underlined portion indicates CDR region) (SEQ ID NO:2):

[0112] QEQLKETGGGLVQPGGSLTLSKASGIDLR SHGIN WVRQAPGKGLEWIG SIASDDGSTYYANSVKG RFTVSSDNAQNTVFLQMTSLTASDTATYFCAR GRRNYLDL WGPGTLLTVSS

[0113] In this heavy chain, the CDRs are as follows:

[0114] CDR1: SHGIN (SEQ ID NO:4);

[0115] CDR2:SIASDDGSTYYANSVKG(SEQ ID NO:5);

[0116] CDR3: GRRNYLDL (SEQ ID NO: 6).

[0117] The amino acid sequence of the light chain variable region of the rabbit anti-human Nectin-4 monoclonal antibody (underlined portion indicates CDR region) (SEQ ID NO:3):

[0118] AQVLTQTPSSVSAAVGGTVTINC QSSQSIYNNNYLS WYQQKPGQPPKLLIY DASTLAS GVP

[0119] SRFKGSGSGTQFTLTISGVQCDDAATYYC LGIYCDSDCYTS FGGGTEVVVKGDP

[0120] In this light chain, the CDRs are as follows:

[0121] The light chain of the anti-Nectin-4 monoclonal antibody includes (the CDR region is determined according to the Kabat CDR definition):

[0122] CDR1: QSSQSIYNNNYLS(SEQ ID NO:7);

[0123] CDR2:DASTLAS(SEQ ID NO:8);

[0124] CDR3: LGIYCDSDCYTS (SEQ ID NO:9).

[0125] Example 6: Production, purification, and identification of monoclonal antibody ADx024S

[0126] Preparation of plasmid encoding the ADx024S clone antibody:

[0127] The pcDNA3.4 plasmid containing the heavy and light chains of the ADx024S nucleic acid sequence was co-transfected into HEK293 cells (25 ml) using a transfection reagent. The culture supernatant was collected, purified using Protein A, and replaced with buffer (PBS pH 7.4, 50% glycerol, 0.05% BSA, 0.03% Proclin 300). The purified antibody was diluted at a certain ratio and identified by Western blotting and IHC.

[0128] The results showed that the purified antibody behaved identically to that observed in the small-scale transformation. The purified antibody was stored at -20°C.

[0129] Example 7: Immunoblot detection based on monoclonal antibody ADx024S

[0130] 1. Experimental Procedure

[0131] The ADx024S monoclonal antibody was subjected to Western blotting reactions with recombinant Nectin-1, recombinant Nectin-2, recombinant Nectin-3, recombinant Nectin-4, T47D, and A549 cell lysates to verify the specificity of the monoclonal antibody. Specifically, each protein was added to 4× Loading Buffer and denatured at 95°C for 10 min. 10 ng of recombinant protein and 100 ng of cell lysate were loaded.

[0132] After SDS-PAGE, the protein was transferred to a PVDF membrane; after blocking with 5% skim milk powder (prepared with 1×TBST) at room temperature for 2 hours, it was incubated overnight at 4°C with the ADx024S monoclonal antibody (0.4ug / ml).

[0133] The following day, after washing, HRP-labeled goat anti-rabbit IgG (1:2000) was added and incubated at room temperature for 1 hour, followed by ECL chemiluminescence color development.

[0134] 2. Test Results

[0135] like Figure 2 As shown, the ADx024S monoclonal antibody can be used in immunoblotting experiments and specifically recognizes endogenous Nectin-4 and recombinant Nectin-4 protein, but does not recognize Nectin-1, Nectin-2, or Nectin-3, and does not exhibit any cross-reactivity.

[0136] Example 8: Immunological analysis of cell lines

[0137] 1. Experimental Procedure

[0138] In this embodiment, the cell lines used were HEK293, HEK293 cells transiently overexpressing Nectin4 plasmid, human breast adenocarcinoma MDA-MB-231, human breast adenocarcinoma SKBR3, and human breast ductal carcinoma T47D. The paraffin-embedded cell blocks were sourced from Haixing Biotechnology. After paraffin sectioning and baking, Nectin-4 mRNA in situ hybridization was performed using ACD (trade name: RNAscope). TM (Probe-Hs-NECTIN4, catalog number 562061) to determine whether a sample expresses Nectin-4.

[0139] Immunohistochemical staining was performed using the ADx024S monoclonal antibody (2ug / ml) and commercially available Nectin-4 antibody (EPR15613-68, catalog number ab192033, purchased from Abcam, 0.65ug / ml) and Nectin-4 antibody (A-9, catalog number sc-515093, purchased from Santa Cruz, 2ug / ml) on a Leica BOND-MAX fully automated immunostaining system. The specific procedures are shown in Table 1.

[0140] Table 1

[0141]

[0142]

[0143] 2. Test Results

[0144] According to the scoring guidelines in the RNAscope manual, the scoring method for Nectin-4 mRNA in situ hybridization is as follows:

[0145] 0 means that there are 0 or fewer than 1 staining points per 10 cells;

[0146] 1+ means that there are 1-3 signal points in each cell;

[0147] 2+ indicates that each cell contains 4-10 signal points or very few clusters of signal points;

[0148] 3+ means that each cell has more than 10 signal points or less than 10% of the cells have clusters of signal points;

[0149] 4+ means that each cell has more than 10 signal points or more than 10% of the cells have clusters of signal points.

[0150] In this embodiment, staining scores of 1 / 2 / 3 / 4+ are considered as positive expression of Nectin-4 mRNA.

[0151] In situ hybridization of Nectin-4 mRNA showed that HEK293 and MDA-MB-231 were Nectin-4 negative cell lines, while Nectin-4 overexpression in HEK293, T47D, and SKBR3 resulted in Nectin-4 positive cell lines. Commercially available monoclonal antibodies Nectin-4 (A-9) and Nectin-4 (EPR15613-68) were used as comparative antibodies.

[0152] Immunohistochemical results are shown in Table 2 and Figure 3 As shown.

[0153] Table 2

[0154]

[0155] *Positive mRNA in situ hybridization results are signal sites.

[0156] According to Table 2 and Figure 3 The ADx024S monoclonal antibody showed negative staining in negative cell lines HEK293 and MDA-MB-231, but showed clear positive staining of the cell membrane in positive cell lines Nectin-4 transtransgenic overexpression HEK293, T47D, and SKBR3, indicating that it has very ideal specificity and high sensitivity.

[0157] The monoclonal antibody Nectin-4 (A-9) showed no cell membrane staining in positive cell lines T47D and SKBR3, and exhibited non-specific cytoplasmic staining, indicating that the antibody had insufficient sensitivity and poor specificity.

[0158] The monoclonal antibody Nectin-4 (EPR15613-68) showed strong cell membrane staining in the negative cell lines HEK293 and MDA-MB-231, and non-specific staining of the cell nucleus was observed in all cell lines, indicating poor antibody specificity.

[0159] Therefore, the ADx024S monoclonal antibody obtained in this invention has excellent specificity and high sensitivity.

[0160] Example 9: Immunohistochemical staining of normal tissue microarrays

[0161] 1. Experimental Procedure

[0162] In this embodiment, the FDA999w chip from Xi'an Taibos was used. The normal tissue microarray included 33 types of normal tissue samples; each type of tissue included 3 different case samples. The 33 types of normal tissues included: brain, cerebellum, adrenal gland, ovary, pancreas, parathyroid gland, pituitary gland, testis, thyroid gland, mammary gland, spleen, tonsils, thymus, bone marrow, bronchus, bronchioles, alveoli, heart, esophagus, stomach, small intestine, colon, liver, salivary glands, kidney, prostate, cervix, endometrium, bladder, skeletal muscle, skin, peripheral nerves, and mesothelial cells.

[0163] After baking normal tissue microarray slides, the Nectin-4 mRNA in situ hybridization technology was first used (company: ACD, trade name: RNAscope). TM Probe-Hs-NECTIN4 (catalog number 562061) was used to determine whether the sample expressed Nectin-4. Next, immunohistochemical detection was performed using the ADx024S monoclonal antibody (2ug / ml) described in this invention and commercially available A-9 (2ug / ml) on a Leica BOND-MAX fully automated immunostaining system. The specific procedure is shown in Table 1.

[0164] 2. Test Results

[0165] The cell membrane staining is shown in Table 3.

[0166] Table 3

[0167]

[0168]

[0169] According to Table 3 and Figure 4-7 The results of in situ hybridization of the ADx024S monoclonal antibody and Nectin-4 mRNA described in this invention are completely consistent. The ADx024S monoclonal antibody showed clear cell membrane staining in normal breast, tonsils, esophagus, parotid gland, prostate, cervix, bladder, and skin. Furthermore, the detection results of the ADx024S monoclonal antibody are entirely consistent with the objective situation of Nectin-4 tissue expression in clinical practice.

[0170] Nectin-4 (A-9) stains the cell membrane in normal breast, tonsils, esophagus, prostate, cervix, bladder, and skin, but the clarity of the cell membrane staining is not as good as ADx024S. In the parotid gland, it shows no staining of the cell membrane and non-specific staining of the cytoplasm.

[0171] Example 10: Immunohistochemical staining of tumor tissue

[0172] 1. Experimental Procedure

[0173] This embodiment used 2 cases of cervical cancer, 2 cases of head and neck squamous cell carcinoma, 1 case of esophageal squamous cell carcinoma, 1 case of breast cancer, 1 case of urothelial carcinoma, and 1 case of lung cancer tissue as test subjects. After the samples were baked, Nectin-4 mRNA in situ hybridization technology (company: ACD, trade name: RNAscope) was first used. TM Probe-Hs-NECTIN4 (catalog number 562061) was used to determine whether the sample expressed Nectin-4. Then, the ADx024S monoclonal antibody (2ug / ml) and A-9 monoclonal antibody (2ug / ml) described in this invention were used to perform immunohistochemical detection on the LeicaBOND-MAX fully automated immunostaining system. The specific procedures are shown in Table 1.

[0174] 2. Test Results

[0175] The in situ hybridization results of Nectin-4 mRNA showed that all 8 tumor tissues expressed Nectin-4 mRNA, and immunohistochemical detection using ADx024S and A-9 showed that Nectin-4 was positive.

[0176] The results are as follows Figure 8 and 9 The staining intensity of the ADx024S monoclonal antibody described in this invention in the tumor region was significantly stronger than that of A-9, and its specificity was also significantly better than that of A-9. A-9 exhibited a large amount of non-specific staining in normal skeletal muscle cells of head and neck squamous cell carcinoma, such as... Figure 8 2. Head and neck squamous cell carcinoma.

[0177] Therefore, when using the same primary antibody concentration and staining procedure, the ADx024S monoclonal antibody staining described in this invention has significantly higher sensitivity and specificity compared to A-9.

[0178] Example 11: Immunohistochemical staining of tumor tissue microarray

[0179] 1. Experimental Procedure

[0180] This embodiment uses tissue microarrays of urothelial carcinoma, triple-negative breast cancer, cervical cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and non-small cell lung cancer. After the samples are baked, Nectin-4 mRNA in situ hybridization technology (company: ACD, trade name: RNAscope) is first used. TM Probe-Hs-NECTIN4 (catalog number 562061) was used to determine whether the sample expressed Nectin-4. Then, the ADx024S monoclonal antibody (2ug / ml) and A-9 monoclonal antibody (2ug / ml) described in this invention were used to perform immunohistochemical detection on the Leica BOND-MAX fully automated immunostaining system. The specific procedures are shown in Table 1.

[0181] 2. Test Results

[0182] The H-score (histochemical score) is a method for semi-quantitatively assessing immunohistochemical staining results. It converts the percentage of positive cells and their staining intensity within each slide into a corresponding numerical value. The scoring formula is: H-score = 0 × (percentage of negative tumor cells) + 1 × (percentage of weakly stained tumor cells) + 2 × (percentage of moderately stained tumor cells) + 3 × (percentage of strongly stained tumor cells), with a score range of 0-300.

[0183] RNAscope TM When using Probe-Hs-NECTIN4 for in situ hybridization detection of Nectin-4 mRNA, staining scores of 1 / 2 / 3 / 4+ were used as indicators of positive Nectin-4 mRNA expression. Results are shown in Table 4. Figure 10 , Figure 11 , Figure 12 As shown.

[0184] Table 4

[0185]

[0186] The results showed that, using immunohistochemical detection with ADx024S or A-9 and H-score for cell membrane staining, when an H-score greater than or equal to 1 indicated the presence of Nectin-4 protein expression, the positive rate of ADx024S was closer to that of Nectin-4 mRNA in situ hybridization, with a consistency greater than or equal to 90%. A-9 showed slightly lower positive rates in urothelial carcinoma, triple-negative breast cancer, cervical cancer, esophageal squamous cell carcinoma, and non-small cell lung cancer, and its consistency with Nectin-4 mRNA in situ hybridization was worse.

[0187] Therefore, when using the same primary antibody concentration and staining procedure, the ADx024S monoclonal antibody described in this invention has higher sensitivity than A-9.

[0188] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. An antibody that binds to Nectin-4, possessing both heavy chain and light chain variable regions, wherein... The amino acid sequence of heavy chain CDR1 is shown in SEQ ID NO:4, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:6; the amino acid sequence of light chain CDR1 is shown in SEQ ID NO:7, the amino acid sequence of heavy chain CDR2 is shown in SEQ ID NO:8, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID NO:

9.

2. The antibody binding to Nectin-4 as described in claim 1, characterized in that, The antibody comprises: an antibody whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO:2 or has more than 80% identity with the amino acid sequence shown in SEQ ID NO:2, whose light chain variable region has the amino acid sequence shown in SEQ ID NO:3 or has more than 80% identity with the amino acid sequence shown in SEQ ID NO:3, and which retains binding activity; Preferably, the antibody has the property of specifically binding to Nectin-4 on the cell membrane; Preferably, the antibody comprises: a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody; or, the antibody comprises: Fab, Fab'-SH, Fv, Fd, scFv, or (Fab')2 fragment.

3. An isolated polynucleotide or a construct containing the polynucleotide, wherein the polynucleotide encodes an antibody binding to Nectin-4 as described in any one of claims 1 to 2; preferably, the construct is an expression vector.

4. An antibody expression system, said expression system comprising the construct of claim 3 or the genome in which an exogenous polynucleotide of claim 3 is integrated; preferably said expression system is a cell.

5. A method for preparing the antibody according to any one of claims 1 to 2, comprising: The antibody is obtained by expressing it using the antibody expression system of claim 4 under suitable conditions for expression. Preferably, the method also includes purifying and isolating the antibody.

6. Use of the antibody according to any one of claims 1 to 2, for preparing a reagent that specifically targets cells expressing Nectin-4; preferably, the cells are cells whose cell membranes express Nectin-4; preferably, in the reagent, the antibody is further modified with a conjugate; Preferably, the conjugate includes a detectable marker; more preferably, the detectable marker includes one or more of the following: fluorescent dyes, radioactive isotopes, horseradish peroxidase (HPR), alkaline phosphatase, ferritin, colloidal gold, and biotin; Preferably, the conjugate comprises: Cytotoxins or bioactive proteins.

7. A reagent that specifically targets cells expressing Nectin-4, comprising the antibody as described in any one of claims 1 to 2, and a conjugate operatively linked thereto; Preferably, the conjugate includes a detectable marker; more preferably, the detectable marker includes one or more of the following: fluorescent dyes, radioactive isotopes, horseradish peroxidase, alkaline phosphatase, ferritin, colloidal gold, and biotin; Preferably, the conjugate comprises: Cytotoxins or bioactive proteins.

8. Use of the antibody according to any one of claims 1 to 2, or the reagent according to claim 7, in the preparation of formulations, kits, or cassettes for detecting Nectin-4 expression-related diseases; preferably, the Nectin-4 expression-related diseases include: Urothelial carcinoma, breast cancer, esophageal cancer, head and neck squamous cell carcinoma, cervical cancer, lung cancer; Preferably, the tumor includes tumors expressing Nectin-4; preferably, the tumor includes urothelial carcinoma, breast cancer, esophageal cancer, head and neck squamous cell carcinoma, cervical cancer, and lung cancer.

9. A kit or pharmaceutical kit comprising the antibody of any one of claims 1 to 3, the reagent of claim 7, or a composition containing the like.

10. A method for in vitro detection of Nectin-4 antigen, the method comprising: The presence of Nectin-4 antigen in the sample to be tested was analyzed using the antibody according to any one of claims 1 to 2 or the reagent according to claim 7. Preferably, the method includes: immunohistochemistry, Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry.

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