Antibodies targeting nectin-4 and uses thereof

Abstract

The application discloses an antibody targeting Nectin-4 and application thereof. The antibody targeting Nectin-4 comprises at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is INVMG, the amino acid sequence of the CDR2 is TITSGGSTNYADSVKG, and the amino acid sequence of the CDR3 is DRMDGSIWYDY. The antibody of the application has strong binding force with the Nectin-4 antigen, and has the advantages of strong tumor targeting, fast tumor enrichment and long tumor retention.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to antibodies targeting Nectin-4 and their applications. Background Technology

[0002] Nectin-4 (also known as poliovirus receptor-associated 4, PVRL-4) is an immunoglobulin-like molecule. Nectin-4 works in conjunction with other linker proteins such as Nectin-1, Nectin-2, and Nectin-3 to participate in cell-cell adhesion. Compared to other Nectins, Nectin-4 is specifically enriched in human embryonic and placental tissues, and its expression significantly decreases in adulthood. Recent studies have found that Nectin-4 is particularly overexpressed in various malignant tumors, including breast cancer, lung cancer, colorectal cancer, pancreatic cancer, and ovarian cancer, and acts as a tumor-associated inducer. In some types of cancer, overexpression of Nectin-4 is associated with various aspects of tumor progression, such as proliferation, angiogenesis, epithelial-to-mesenchymal transition, metastasis, DNA repair, tumor recurrence, and poor prognosis (Subhajit et al., 2021).

[0003] Although Nectin-4 has shown potential as a therapeutic target in cancer treatment, existing Nectin-4 antibodies still have some issues to overcome in terms of efficacy, safety, drug resistance, and mechanism of action. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an antibody or its antigen-binding fragment targeting Nectin-4, which has a strong binding affinity to the Nectin-4 antigen and offers advantages such as strong tumor targeting, rapid tumor enrichment, and long-lasting retention.

[0005] Antibodies or antigen-binding fragments targeting Nectin-4

[0006] The present invention provides an antibody or antigen-binding fragment thereof targeting Nectin-4, comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domains are CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is INVMG (SEQ ID NO: 1), the amino acid sequence of CDR2 is TITSGGSTNYADSVKG (SEQ ID NO: 2), and the amino acid sequence of CDR3 is DRMDGSIWYDY (SEQ ID NO: 3).

[0007] In some embodiments, the antibody or its antigen-binding fragment comprises at least one immunoglobulin single variable domain, said immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 1;

[0008] The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. The single variable structure of the immunoglobulin specifically binds to the Nectin-4 antigen or fragments thereof.

[0009] This article provides antibodies or antigen-binding fragments thereof targeting Nectin-4, including any one or any combination of CDR1, CDR2 and CDR3 mentioned above.

[0010] In some implementations, the aforementioned antibody or its antigen-binding fragment, and the CDR1, CDR2, and CDR3 of the immunoglobulin's single variable domain are defined according to the Kabat numbering system.

[0011] In some implementations, the immunoglobulin single variable domain further includes a frame region, preferably an alpaca-derived frame region or a human-derived frame region.

[0012] In some embodiments, the amino acid sequence of the single variable domain of the immunoglobulin in the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, or has at least 80%, at least 85%, or at least 90% sequence identity.

[0013] In some embodiments, the amino acid sequence of the single variable domain of the immunoglobulin is as shown in SEQ ID NO:1, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1 and does not involve any alteration to the CDR sequence.

[0014] In some embodiments, the antibody or its antigen-binding fragment is a nanobody, an Fc fusion antibody, a heavy chain antibody, a monoclonal VHH antibody, a bispecific antibody, a multispecific antibody, or a VHH polymer.

[0015] In some implementations, the antibody is a nanobody.

[0016] In some embodiments, at least one immunoglobulin single variable domain in the aforementioned antibody or its antigen-binding fragment is a VHH. In some embodiments, the antibody or its antigen-binding fragment comprises one or more (e.g., 2, 3, 4, 5, 6) of the aforementioned immunoglobulin single variable domains, which may be the same or different and may form VHH polymers, such as dimers or multimers.

[0017] In some embodiments, the antibody or its antigen-binding fragment further comprises a His tag and / or Cys.

[0018] In some implementations, the aforementioned antibody or its antigen-binding fragment also includes an immunoglobulin Fc region.

[0019] In some implementations, the immunoglobulin Fc region is derived from human or mouse sources.

[0020] In some implementations, the immunoglobulin Fc region is IgG Fc.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises a human immunoglobulin Fc region. For example, the immunoglobulin Fc region is the Fc region of human IgG1, IgG2, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of wild-type IgG or a variant thereof.

[0022] In some implementations, the Fc region is an Fc region that enhances effector function, for example, enhancing antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC) with enhanced effector function.

[0023] An exemplary IgG1 Fc region includes substitutions having the following: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 2 80H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; or any combination of the above positions. The mutations are defined according to the EU numbering system.

[0024] An exemplary IgG1 Fc region includes substitutions having the following: S239D; S239E; S239K; F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D 280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of the above positions.

[0025] In some embodiments, the Fc region contained in the aforementioned antibody or its antigen-binding fragment can cause the binding protein to form a dimer molecule, while prolonging the in vivo half-life of the binding protein.

[0026] In some implementations, VHH antibodies are directly linked to the Fc region of immunoglobulins to form VHH-Fc antibodies.

[0027] In some embodiments, the immunoglobulin single variable domain in the aforementioned antibody or its antigen-binding fragment is linked to the Fc region via a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length, without secondary or higher-level structures. For example, the linker is a flexible linker, such as G4S, GS, GAP, etc.

[0028] In some embodiments, the antibody or its antigen-binding fragment is a heavy chain antibody, which further comprises an immunoglobulin Fc region, the amino acid sequence of which is shown in SEQ ID NO: 7, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7 and maintains the function of the heavy chain constant region.

[0029] In some embodiments, the aforementioned antibody or its antigen-binding fragment further comprises the aforementioned His tag sequence. The His tag sequence is a short sequence containing consecutive histidine residues, such as 6 His, 8 His, and 10 His.

[0030] In some embodiments, the aforementioned antibody or its antigen-binding fragment is contained in a Cys terminus at the C-terminus or N-terminus.

[0031] In some embodiments, the antibody or its antigen-binding fragment is constructed by linking the variable region of the antibody to a His-Cys tag, thus creating a VHH-His-Cys antibody.

[0032] In some specific implementations, the aforementioned antibody or its antigen-binding fragment contains the HHHHHHC (SEQ ID NO: 9) sequence.

[0033] In some embodiments, the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO: 8.

[0034] In some implementations, the KD value of the aforementioned antibody or its antigen-binding fragment binding to Nectin-4 can be ≤1×10⁻⁶. -7 M.

[0035] In some implementations, the antibodies or antigen-binding fragments described above bind to tumor cells. For example, the detection method in Example 3.

[0036] In some implementations, the antibodies or antigen-binding fragments described above have endocytic activity. For example, the detection method in Example 4.

[0037] In some implementations, the aforementioned antibodies or their antigen-binding fragments described herein are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0038] In some embodiments, the antibody or its antigen-binding fragment described herein, compared to SEQ ID NO: 1, contains one or more amino acid substitutions, such as conserved amino acid substitutions, for example, containing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conserved amino acid substitutions, which may occur in the CDR region and / or FR region.

[0039] In some embodiments, the antibody or its antigen-binding fragment is provided that binds to or competitively binds to the same epitope as the immunoglobulin single variable domain in the aforementioned antibody or its antigen-binding fragment targeting Nectin-4 described herein.

[0040] In some embodiments, the antibody or its antigen-binding fragment is provided that blocks the binding of the immunoglobulin single variable domain in the aforementioned antibody or its antigen-binding fragment targeting Nectin-4 to Nectin-4 (e.g., human Nectin-4).

[0041] In some implementations, an antibody or antigen-binding fragment thereof targeting Nectin-4 is provided, the binding of which to Nectin-4 (e.g., human Nectin-4) is blocked by a single variable immunoglobulin domain in the aforementioned Nectin-4 binding protein.

[0042] As previously stated, and in this document, "at least 90% identity" includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity.

[0043] In another aspect, the present invention provides an antibody conjugate comprising an effector molecule and the antibody or antigen-binding fragment thereof targeting Nectin-4 disclosed herein.

[0044] In some embodiments, the effector molecule is selected from one or more of cytokines, lectins, enzymes, radioisotopes, antitumor agents, immunomodulatory agents, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, and metal ions.

[0045] In some embodiments, the antibody conjugate comprises the antibody targeting Nectin-4 disclosed in this invention or its antigen-binding fragment, and further comprises one or more of the following: His tag, Cys, cytokines, lectins, enzymes, radioisotopes, antitumor agents, immunomodulatory agents, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, and metal ions.

[0046] In some embodiments, antibody conjugates are provided herein, comprising an antibody targeting Nectin-4 or an antigen-binding fragment thereof and an effector molecule. The effector molecule is a molecule capable of exhibiting the desired target activity; for example, the effector molecule is selected from radioisotopes, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0047] In some embodiments, the effector molecule is a marker. As an example, the marker can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such markers are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., IR808, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. The markers covered herein can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing the enzyme with a substrate and detecting the reaction products produced by the enzyme's action on the substrate, while calorimetric markers are detected by simple, visually appealing colored markers.

[0048] In some implementations, the markers described above can be linked to the antibodies described herein using connectors of varying lengths to reduce potential steric hindrance.

[0049] In some implementations, the markers described above can be linked to the antibodies described herein using chelating agents such as NOA, DOTA, etc.

[0050] In some implementations, the effector molecule is a cytotoxic drug.

[0051] In some implementations, the C-terminus of the antibody in the antibody conjugate is linked to a His-Cys tag.

[0052] In some embodiments, the antibody conjugate has an amino acid sequence as shown in SEQ ID NO: 8.

[0053] In some embodiments, the antibody conjugate is an antibody probe, and the label of the antibody probe is preferably IR808.

[0054] In some specific embodiments, the antibody conjugate is a conjugate of the antibody or its antigen-binding fragment coupled with IR808.

[0055] Nucleic acid and vector

[0056] In another aspect, this invention provides an isolated nucleic acid that encodes an antibody or antigen-binding fragment thereof targeting Nectin-4, as described in this invention. The nucleic acid herein may be RNA, DNA, or cDNA. According to some embodiments herein, the nucleic acid is an isolated nucleic acid.

[0057] The nucleic acids described herein may also be in vector form, may exist within a vector and / or may be part of a vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be, in particular, an expression vector, that is, a vector that provides an antibody targeting Nectin-4 or its antigen-binding fragment for expression in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system).

[0058] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid as provided in the present invention. In some embodiments, the recombinant expression vector is a plasmid, a bacteriophage, or a viral vector.

[0059] In some embodiments, the viral vector is a retroviral vector, an adenovirus vector, or an adeno-associated virus vector, and the retroviral vector is, for example, a lentiviral vector.

[0060] The vectors described herein are generally not naturally occurring. However, portions of the vectors may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. Suitable vectors include those designed for amplification and expansion or for expression, or both of the above. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0061] This expression vector typically contains at least one nucleic acid described herein, operatively linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a specific host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the antibody targeting Nectin-4 described herein, or its antigen-binding fragment, include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0062] The nucleic acids described herein may be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides described herein, and / or may be isolated from suitable natural sources.

[0063] Transformation

[0064] Another aspect of the present invention provides a transformant comprising the isolated nucleic acid or recombinant expression vector as provided in the present invention.

[0065] This article provides recombinant host cells that express or are able to express one or more of the antibodies targeting Nectin-4 described herein or their antigen-binding fragments and / or contain the nucleic acids or vectors described herein.

[0066] In some embodiments, the host cell of the transformant is a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0067] Bacterial cells include, for example, strains of Gram-negative bacteria (such as Escherichia coli). Escherichia coli ) strains, Proteus spp. ( Proteus ) strains and Pseudomonas spp. Pseudomonas ) strains) and Gram-positive bacterial strains (e.g., Bacillus spp.) Bacillus ) strains, Streptomyces ( Streptomyces ) strains, Staphylococcus spp. Staphylococcus) strains and Lactococcus spp. Lactococcus (strain) cells.

[0068] Fungal cells, for example, include Trichoderma ( Trichoderma Neurospora ( Neurospora ) and Aspergillus spp. Aspergillus Cells of species including yeast; or including yeast ( Saccharomyces (e.g., brewer's yeast) Saccharomyces cerevisiae )), genus *Fissionyomyces* ( Schizosaccharomyces (e.g., Saccharomyces cerevisiae) Schizosaccharomyces pombe ), Pichia pastoris ( Pichia (e.g., Pichia pastoris) Pichia pastoris ) and Pichia pastoris ( Pichia methanolica )) and Hansenula genus ( Hansenula ) of the species' cells.

[0069] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0070] However, this article may also use amphibian cells, insect cells, plant cells, and any other cells in the art used to express heterologous proteins.

[0071] Expression vectors can be transfected or introduced into suitable host cells. Various techniques can achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector and host cells used. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0072] Preparation method

[0073] This article provides a method for preparing an antibody or antigen-binding fragment targeting Nectin-4, the method comprising: using the transformant provided by this invention to obtain the antibody or antigen-binding fragment targeting Nectin-4 from a culture; specifically, expressing the target protein in a host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may be included, for example, purification using an A or G Sepharose FF column containing adjusted buffer to wash away non-specifically bound components, followed by elution of the bound antibody using a pH gradient, detection by SDS-PAGE, and collection. Optionally, filtration and concentration may be performed using conventional methods. Soluble mixtures and polymers may also be removed using conventional methods, such as molecular sieving or ion exchange. The obtained product should be immediately frozen, such as at -70°C, or lyophilized.

[0074] The engineered antibodies or antigen-binding fragments described in this paper can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Mammalian expression systems lead to antibody glycosylation, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0075] This article provides a method for preparing antibody conjugates, comprising linking the aforementioned Nectin-4-targeting antibody or its antigen-binding fragment with an effector molecule.

[0076] Another aspect of the present invention provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof targeting Nectin-4 as described in one aspect of the present invention.

[0077] Another aspect of the present invention provides a genetically modified cell comprising the chimeric antigen receptor as described in the present invention.

[0078] In some embodiments of the present invention, the genetically modified cells are eukaryotic cells, preferably isolated human cells.

[0079] In some embodiments of the present invention, the genetically modified cells are immune cells, such as T cells or NK cells.

[0080] Composition

[0081] Another aspect of the present invention provides a pharmaceutical composition comprising one or more of the antibody or antigen-binding fragment thereof provided by the present invention, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, and the chimeric antigen receptor; and pharmaceutically acceptable excipients.

[0082] In some embodiments, the pharmaceutical composition contains one or more of the antibody or antigen-binding fragment thereof as described above, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, and the chimeric antigen receptor, which are effective for the treatment, relief, or prevention of cancer.

[0083] In some implementations, the pharmaceutically acceptable excipient is a pharmaceutically acceptable excipient, diluent, or carrier.

[0084] Some embodiments provide a pharmaceutical composition comprising an antibody targeting Nectin-4 as described above for diagnosing cancer, or an antigen-binding fragment thereof, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0085] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of an antibody or antigen-binding fragment targeting Nectin-4 per unit dose, or the amount of antibody or antigen-binding fragment targeting Nectin-4 per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0086] In some embodiments, an article or product is provided comprising the aforementioned antibody targeting Nectin-4 or an antigen-binding fragment thereof. Optionally, the article comprises a container and a label. The container is, for example, a bottle, syringe, or test tube. The container contains a composition effective for treating a condition. A label on or attached to the container indicates that the composition is intended to treat the selected condition. The composition contains the aforementioned antibody targeting Nectin-4 or an antigen-binding fragment thereof.

[0087] In some embodiments, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof targeting Nectin-4 as described herein. The antibody or antigen-binding fragment targeting Nectin-4 may be an effective amount for treating or alleviating a disease (e.g., cancer) or for diagnosing a disease (e.g., cancer), and the pharmaceutical composition may also contain at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0088] use

[0089] In another aspect, the present invention provides the use of one or more of the antibodies or antigen-binding fragments thereof, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates, the chimeric antigen receptors, and the pharmaceutical compositions provided by the present invention in preparation reagents or kits.

[0090] In some implementations, the reagent or kit is used to detect Nectin-4 expression.

[0091] In some implementations, the reagent or kit is used for cancer diagnosis.

[0092] In some implementations, the reagent or kit is used for drug development.

[0093] In some embodiments of the present invention, the reagents or kits are used for in vivo imaging techniques, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry.

[0094] Another aspect of the present invention provides the use of one or more of the antibody or antigen-binding fragment thereof provided by the present invention, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, and the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer; wherein the cancer is a Nectin-4 expression-associated cancer.

[0095] This article provides methods for using the aforementioned antibodies targeting Nectin-4 or their antigen-binding fragments or antibody conjugates, polynucleotides, or compositions (including pharmaceutical compositions) for the treatment, relief, prevention, or diagnosis of diseases or conditions.

[0096] In some implementations, the aforementioned diseases associated with Nectin-4 expression are proliferative disorders or any other diseases or conditions characterized by uncontrolled cell growth (e.g., cancer; in this document, cancer and tumor may be used interchangeably), such as diseases associated with Nectin-4 expression or abnormal Nectin-4 expression (e.g., cancer).

[0097] In some implementations, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

[0098] In some implementation schemes, the aforementioned cancer is breast cancer or bladder cancer.

[0099] Detection

[0100] reagents or kits

[0101] Another aspect of the present invention provides a reagent or kit for detecting Nectin-4 and / or diagnosing cancers related to Nectin-4 expression. The reagent or kit comprises one or more of the antibody or antigen-binding fragment thereof provided by the present invention, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, and the pharmaceutical composition, which are contacted with a sample to be tested to detect the expression level of Nectin-4 in the sample. The method is not for diagnostic purposes.

[0102] In some embodiments of the present invention, the test sample is a cell or an animal.

[0103] In some embodiments of the present invention, the method employs in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry to detect Nectin-4.

[0104] In some embodiments, a kit is also provided containing the aforementioned antibody or antibody conjugate, polynucleotide, and may also include diagnostic instructions for use. The kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the antibody or antibody conjugate may be formulated as a pharmaceutical composition.

[0105] This document provides the use of antibodies targeting Nectin-4 or their antigen-binding fragments or antibody conjugates, polynucleotides, and compositions thereof for detection. This document also provides methods, systems, or apparatus for in vivo or in vitro detection of Nectin-4, comprising treating a sample with the aforementioned antibodies targeting Nectin-4 or their antigen-binding fragments or antibody conjugates, polynucleotides, and compositions thereof.

[0106] Another aspect of the present invention provides a method for detecting Nectin-4 expression, wherein the method uses one or more of the antibody or its antigen-binding fragment provided by the present invention, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, the pharmaceutical composition, and the reagent or kit to detect Nectin-4 expression, and the method is for non-diagnostic purposes.

[0107] In some implementations, Nectin-4 protein expression can be detected in tissue samples, playing a crucial role in non-diagnostic and therapeutic research and applications. In basic research, scientists investigate the expression patterns and functions of Nectin-4 in different cell types, tissues, and organs to explore its roles in cell adhesion, signal transduction, and intercellular communication. In drug development, Nectin-4 expression detection not only helps identify drug targets but also assesses the impact of drug candidates on Nectin-4 expression to measure their potential efficacy. In disease model studies, by altering Nectin-4 expression levels in animal models, researchers can observe changes in disease progression and understand its role in disease development. Furthermore, while the exploration of Nectin-4 as a potential biomarker is not for diagnostic purposes, it helps researchers gain a more comprehensive understanding of the biological characteristics of diseases. In terms of therapeutic target validation and cell therapy development, Nectin-4 expression detection provides important evidence for preclinical research and clinical trials. For example, in CAR-T cell therapy, it can be used to detect whether Nectin-4 is suitable as a target for tumor cells.

[0108] In some implementations, the method includes the following steps:

[0109] (1) Contact the sample with an antibody or antigen-binding fragment targeting Nectin-4;

[0110] (2) Detect the complex formed between the antibody targeting Nectin-4 or its antigen-binding fragment and the sample;

[0111] (3) Detect the complex.

[0112] In some embodiments, the method further includes the step of contacting a reference sample (e.g., a control sample) with a reagent. The extent of complex formation is determined by comparison with the reference sample. A change in complex formation in the sample or subject (e.g., a statistically significant change) compared to the control sample or subject indicates the presence of Nectin-4 in the sample.

[0113] Another aspect of the present invention provides a system for detecting Nectin-4 expression, the system comprising:

[0114] The present invention provides one or more of the following: the antibody or its antigen-binding fragment, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, the pharmaceutical composition, and the reagent or kit; for specifically binding to Nectin-4 protein;

[0115] A sample processing device for receiving and processing a sample to be tested, the sample containing or potentially containing Nectin-4 protein;

[0116] The detection platform is selected from one of the following platforms: immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, Western blot, and flow cytometry.

[0117] A signal detection and quantification device is used to detect the signal generated after an antibody or its antigen-binding fragment targeting Nectin-4 binds to the Nectin-4 protein in a sample, and to perform quantitative analysis on this signal;

[0118] Data analysis and processing software is used to analyze data output from signal detection and quantification devices to determine the expression level of Nectin-4 protein in samples; and,

[0119] Control and control components, including positive and negative controls, are used to validate the accuracy and specificity of the detection system.

[0120] In some embodiments, the sample processing apparatus includes one or more of the steps of fixation, permeation, slicing, or cell lysis.

[0121] In some embodiments, the signal detection and quantification device includes an enzyme substrate reaction detector, a fluorescence detector, a radioactive counter, or an optical density scanner.

[0122] In some implementations, the data analysis and processing software is capable of providing a visual representation of Nectin-4 protein expression levels.

[0123] Another aspect of the present invention provides a detection device for detecting Nectin-4 protein expression, the detection device comprising:

[0124] An antibody or antigen-binding fragment immobilization module targeting Nectin-4 is used to immobilize an antibody or antigen-binding fragment targeting Nectin-4 so as to specifically bind to the Nectin-4 protein;

[0125] The sample processing module is used to receive biological samples and perform preprocessing steps such as fixation, permeation, sectioning, or cell lysis.

[0126] The detection module is used to bind the processed sample to an antibody or its antigen-binding fragment targeting Nectin-4 and to detect the binding event. The detection module is selected from one of the following: immunohistochemical detection module, immunofluorescence detection module, enzyme-linked immunosorbent assay (ELISA) detection module, Western blot detection module, and flow cytometry detection module.

[0127] The signal detection and quantification unit, coupled to the detection module, is used to detect signals generated by combined events and provide quantitative analysis of the signals.

[0128] The data analysis and processing unit, coupled to the signal detection and quantification unit, is used to receive and analyze signal data to determine the expression level of Nectin-4 protein;

[0129] The control and control unit provides positive and negative controls to verify the accuracy and specificity of the detection device.

[0130] In some implementations, the antibody targeting Nectin-4 or its antigen-binding fragment immobilization module comprises one or more microarrays of immobilized antibodies.

[0131] In some embodiments, the signal detection and quantification unit includes at least one detector selected from enzyme substrate reaction detectors, fluorescence detectors, radioactive counters, and optical density scanners.

[0132] In some implementations, the data analysis and processing unit includes a user interface for displaying visualizations of Nectin-4 protein expression levels.

[0133] Another aspect of the present invention provides a method for diagnosing, preventing, improving, or treating diseases, conditions, or symptoms associated with Nectin-4 expression, the method comprising administering to a subject in need an effective amount of one or more of the following: the Nectin-4 conjugate, the nucleic acid, the recombinant expression vector, the transformant, the pharmaceutical composition, and the reagent or kit provided by the present invention.

[0134] In some implementations, the disease, condition, or symptom associated with Nectin-4 expression is selected from one or both of breast cancer and bladder cancer.

[0135] As used herein, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeuticly effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0136] Combinations of drugs containing antibodies targeting Nectin-4 or their antigen-binding fragments may be used for the prophylactic treatment of breast and / or bladder cancer. For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0137] In another aspect, the present invention provides one or more of the following: the antibody or antigen-binding fragment thereof, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, the pharmaceutical composition, and the reagent or kit, for the diagnosis, prevention, improvement, or treatment of diseases, conditions, or symptoms associated with Nectin-4 expression.

[0138] In some implementations, the disease, condition, or symptom associated with Nectin-4 expression is selected from one or both of breast cancer and bladder cancer.

[0139] In other embodiments, the in vivo detection method, system, or apparatus may include:

[0140] (1) Administering to a subject the aforementioned antibody targeting Nectin-4 or its antigen-binding fragment or antibody conjugate, polynucleotide, or combination thereof; and

[0141] (2) Detection of the formation of complexes between the aforementioned antibody or antibody conjugate, polynucleotide, and the composition and the subject.

[0142] Terminology Definition

[0143] To facilitate understanding of this article, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this article pertains.

[0144] “Nectin-4” or “PVRL-4” are used interchangeably and include variants, isotypes, species homologs of human Nectin-4, and analogs that share at least one common epitope with Nectin-4.

[0145] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0146] In this invention, the terms "single-domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "VHH domain" and "nanobody" are used interchangeably and all refer to nanobodies that specifically recognize and bind to Nectin-4.

[0147] The term "antibody" is used in the broadest sense to encompass a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0148] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all as defined by the Kabat numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTicsdatabase (IMGT, imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0149] The "immunoglobulin variable domain" is essentially composed of four "frame regions" (FR1, FR2, FR3, and FR4, respectively) and three "complementarity-determining regions" (CDR1, CDR2, and CDR3). Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

[0150] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNARs, domains, (single-domain) antibodies (e.g., dAbs™) that are VH domains or derived from VH domains, and (single-domain) antibodies (e.g., dAbs™) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. A specific example of a single variable domain of an immunoglobulin is the “VHH domain” (or simply “VHH”) as defined below.

[0151] The “VHH domain,” also known as a heavy chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) and the light chain variable domain (referred herein as the “VL domain”) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without requiring other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single-domain antibody," "VHH domain," "VHH," "VHH antibody fragment," "VHH antibody," and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. The "VHH domain" includes, but is not limited to, naturally occurring antibodies produced by camelids, humanized antibodies produced by camelids, or antibodies obtained through phage display technology.

[0152] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0153] The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0154] The VHH domain (alone or as part of a larger polypeptide) offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (such as Fab- or F(ab')2- fragments):

[0155] - Only a single domain is needed to bind to the antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of specially designed adapters).

[0156] The -VHH domain can be expressed by a single gene and does not require post-translational folding or modification;

[0157] -VHH domains can be easily modified into multivalent and multispecific formats;

[0158] - The VHH domain is highly soluble and has no tendency to aggregate;

[0159] The -VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without the use of refrigeration equipment, thus saving costs, time and the environment;

[0160] -VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production.

[0161] The -VHH domain is relatively small compared to conventional tetrapeptide chain antibodies (approximately 15 kDa, or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue penetration and allowing for higher dose administration compared to conventional tetrapeptide chain antibodies.

[0162] The -VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to the conventional VH domain), thereby reaching targets and epitopes that are inaccessible to conventional tetrapeptide chain structure antibodies.

[0163] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0164] Typically, the antibody or antigen-binding fragment targeting Nectin-4 described in this article will be preferably measured in a Biacore, KinExA, or Fortibio assay at a concentration of 10. -7 Up to 10 -10 moles per liter (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 Or a lower dissociation constant (KD), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 - 9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen it wants to bind to (i.e., Nectin-4). Any value greater than 10... -4 The KD value of M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described herein.

[0165] When “competition” is used in cases where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope, it means that there is competition between antigen-binding proteins, which is determined by the following assay: the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., Nectin-4 antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIAs with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves using a purified antigen (on a solid surface or cell surface) capable of binding to both an unlabeled detection antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the target antigen-binding protein. Typically, the target antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to an epitope adjacent to the epitope of the reference antigen-binding protein, the two epitopes spatially interfering with each other's binding. Further details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen.In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0166] Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Publication WO03 / 48731. Therefore, antibodies that compete with the antibody molecules described herein for binding to the same epitope on Nectin-4 can be obtained using conventional techniques known to those skilled in the art.

[0167] “Cross-reactivity” refers to, for example, the Nectin-4 binding protein described herein and Nectin-4 from different species. For instance, a single-domain antibody or derived protein of this paper that binds to human Nectin-4 may also bind to Nectin-4 from another species. Cross-reactivity is measured by detecting the specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or by binding or functional interactions with cells physiologically expressing Nectin-4. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0168] "Antigen" refers to a vertebrate molecule used for immunization to generate antibodies that recognize the antigen, or for screening expression libraries (e.g., phage, yeast, or ribosome display libraries, in particular). In this document, antigen is defined more broadly to include target molecules specifically recognized by antibodies, as well as portions or mimics of molecules used in immunization processes to generate antibodies or in library screening to select antibodies. For example, for the antibodies binding to human Nectin-4 discussed herein, monomers and multimers of human Nectin-4 (e.g., dimers, trimers, etc.), as well as truncated variants and other variants of human Nectin-4, are all referred to as antigens.

[0169] An epitope is a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed through ternary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a distinctive spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0170] "Conservative substitution" refers to the substitution with another amino acid residue that has properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

[0171] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same nucleotide or amino acid monomer—for example, if every position in two DNA molecules is occupied by the same nucleotide—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0172] "Nucleic acid molecules" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0173] "Vector" or "recombinant expression vector" means a construct capable of being delivered to a host cell and, in some embodiments, expressing one or more target genes or sequences. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0174] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides described herein. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that all progeny may not be exactly identical in DNA content due to intentional or unintentional mutations. Mutant progeny with the same function or biological activity as those screened in the originally transformed cells are included. Host cells may include microorganisms (e.g., bacteria), plant, or animal cells. Easily transformable bacteria include Enterobacteriaceae (…). enterobacteriaceae Members of, such as Escherichia coli ( Escherichia coli ) or Salmonella ( Salmonella strains of Bacillus; Bacillus family ( Bacillaceae For example, Bacillus subtilis ( Bacillus subtilis ); Pneumococcus ( Pneumococcus Streptococcus ( Streptococcus ) and Haemophilus influenzae ( Haemophilus influenzae Suitable microorganisms include Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) and Pichia pastoris ( Pichia pastoris Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), NS0 cells, and 293 cells.

[0175] "Pharmaceutical composition" means a mixture containing one or more antibodies described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0176] "Tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0177] The terms “cancer,” “cancerous,” “proliferative disorder,” and “tumor” are not mutually exclusive when used in this article.

[0178] "Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility or absence of such event or circumstance. "And / or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" herein includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to."

[0179] In this invention, the "non-diagnostic purpose" application scenarios include, but are not limited to: for example, detecting the presence of antigens (proteins containing the extracellular region of Nectin-4) in vitro in the laboratory; or using it as a positive antibody to screen other antibodies targeting Nectin-4; or competing with other antibodies targeting Nectin-4 to detect whether there is competition between the antibodies, i.e., whether the antigen epitopes are the same or similar, etc.

[0180] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0181] In this article, "subjects" and "patients" refer to mammals, especially primates, and particularly humans.

[0182] To facilitate understanding of this document, certain technical and scientific terms are specifically defined below. Unless based on common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the invention.

[0183] The reagents and raw materials used in this invention are all commercially available.

[0184] The positive and progressive effects of this invention are as follows: the antibody of this invention can specifically bind to HumanNectin, Cyno Nectin-4 and Mouse Nectin-4 with high affinity, and can also bind to Nectin-4 on the surface of human breast cancer cells MCF-7 cells with high affinity. The antibody also has the advantages of strong tumor targeting, rapid tumor enrichment and long retention. Attached Figure Description

[0185] Figure 1In vivo fluorescence imaging of the anti-Nectin-4 antibody conjugate 56-IR808. Detailed Implementation

[0186] The following embodiments are used to further describe the present invention, but these embodiments are not intended to limit the scope of the present invention.

[0187] Experimental methods not specifying specific conditions in the embodiments or test examples of this invention are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Methods in Contemporary Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents not specifying a particular source are commercially available, conventional reagents.

[0188] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0189] Example 1. Screening and preparation of anti-Nectin-4 antibodies

[0190] In this embodiment, human Nectin-4 (Acro, NE4-H52H3) with a His tag was used as an immunogen for alpaca immunization. Peripheral blood was collected, PBMCs were isolated from the peripheral blood, RNA was extracted from the PBMCs, and reverse transcription was performed to obtain total cDNA. A yeast library was constructed for antibody screening.

[0191] After two rounds of sorting, single-clone identification, sequencing, and sequence analysis yielded multiple unique VHH sequences binding to the human Nectin-4 antigen. The NB656-M1-56 sequence is shown below.

[0192] NB656-M1-56 Variable Area

[0193] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADRMDGSIWYDYWGQGTQVTVSS (SEQ ID NO: 1)

[0194] The CDR sequence is shown in Table 1 below.

[0195] Table 1. CDR Sequence List: (Kabat Numbering Rules)

[0196]

[0197] The above sequences were ligated to the human IgG1 Fc (including the hinge region) fragment to construct the VHH-Fc antibody. The plasmid was constructed, transiently transfected into 293 cells, and the cells were cultured to express the antibody. The antibody was purified using a Protein A column, washed with 1×PBS buffer, and eluted with 0.1 M glycine buffer (pH 2.5). The antibody was then dialyzed into 1×PBS buffer (pH 7.4). The human IgG1 Fc (including the hinge region) sequence is as follows:

[0198] > IgG1 Fc

[0199] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0200] >BS025-PC HC

[0201] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0202] > BS025-PC LC

[0203] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 6)

[0204] Example 2. Identification of the affinity between anti-Nectin-4 antibody and antigen Nectin-4

[0205] The binding ability of anti-Nectin-4 antibody to Nectin-4 antigen protein was detected by ELISA and BLI.

[0206] 1. ELISA

[0207] Experimental Methods: Human Nectin-4 his (Acro, NE4-H52H3), Cyno Nectin-4 his (Acro, NE4-C52H4), and Mouse Nectin-4 his (Acro, NE4-M52H3) were coated with 100 μL / well of (50 mM NaHCO3, pH 9.6) at a concentration of 2 μg / mL and incubated overnight at 4°C. The antigens were then washed three times with PBST solution. Blocking was performed with 5% milk at 37°C for 1 h. After washing once with PBST, the protein was serially diluted 1:5 from 100 nM (using 5% milk). 100 μL of each serially diluted antibody protein was added to the wells of an ELISA plate and incubated at 37°C for 1 h. The isotype control antibody (Isotype) used was purchased from Syd Labs, catalog number PA007165.m2a. Wash 5 times with PBST, then add the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted with blocking buffer accordingly. Wash the ELISA plate incubated with the secondary antibody 5 times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, and incubate at 37°C for 7 min. Stop the reaction by adding 50 μL / well of 1M HCl. OD 450 Readings, calculating EC 50 .

[0208] Experimental results: The results are shown in Table 2. The results show that the anti-NB656-M1-56 antibodies have a strong binding affinity to the Nectin-4 antigen, which is stronger than that of the control antibody BS025-PC.

[0209] Table 2. ELISA detection of anti-Nectin-4 antibody binding antigen

[0210]

[0211] 2. BLI

[0212] Experimental Method: First, the biosensor was immersed in PBST buffer for equilibration for 15 minutes. Then, it was immersed in a sample solution containing a known concentration of antibody (2 μg / ml). Next, the sensor with the immobilized antigen was immersed in buffer for baseline equilibration. Then, the biosensor with the immobilized antigen at a known concentration (200 nM, 100 nM, 50 nM, or 25 nM) was immersed in a sample solution containing the antibody to be tested. Finally, the sensor bound to the antibody was immersed in buffer for dissociation. The kinetic constants of the sample could be obtained by real-time monitoring of the biofilm thickness of the biosensor during the experiment using an Octet instrument. Specific steps are as follows:

[0213] (1) Turn on the power and operating software of the Octet K2 instrument (Sartorius) to initialize the instrument. This process takes about 1 minute.

[0214] (2) Add 200 μL of PBST to the well plate to pre-wet the sensor (the sensor is ProA sensor-Sartorius) for 15 min and set aside.

[0215] (3) Set the program to solidify the antibody for 60 seconds, bind the antigen for 180 seconds, and dissociate for 240 seconds;

[0216] (4) Prepare the sample according to the set procedure and add the sample into the detection plate;

[0217] (5) Place the sensor and sample plate into the instrument together and perform the tests sequentially;

[0218] (6) After the test is completed, retrieve the sensor, clean the sample plate, and turn off the instrument power.

[0219] (7) Use the analysis software DataAnalysis12 to perform fitting, and export the images and fitting affinity constant values.

[0220] Experimental results: The results are shown in Table 3, which show that the anti-Nectin-4 antibodies all have a strong binding affinity to the Nectin-4 antigen.

[0221] Table 3. Detection of anti-Nectin-4 antibody binding ability

[0222]

[0223] Example 3. In vitro cell binding assay of anti-Nectin-4 antibody

[0224] Experimental objective: To detect the binding ability of anti-Nectin-4 antibody using human breast cancer cells MCF-7 (Pronosa, CL-0149).

[0225] Experimental methods: MCF-7 cells were cultured in Pronox medium, washed twice with 1×PBS, and resuspended in 1×PBS to a cell concentration of 3×10⁻⁶ cells / mL. 5 Cells / μL, dispense 50μL of cells into each well of a PCR plate; dilute the antibody to be tested with 1×PBS to a starting concentration of 200 nM, perform 7 serial dilutions at a 1:5 ratio, and add 50μL / well to each well of the plate and incubate at 4°C for 1 hour; after incubation, wash the cells 3 times with 1×PBS, add 100μL of fluorescent secondary antibody (647 Anti-HumanIgG Fc) to each well, and incubate at 4°C for 1 hour; after incubation, wash the cells once with 1×PBS, transfer the cells to a 96-well cell plate, and analyze by flow cytometry.

[0226] Experimental results: The results are shown in Table 4. The results show that the anti-Nectin-4 antibodies have a strong binding force to the Nectin-4 antigen on human breast cancer cells MCF-7 cells, and the binding force is stronger than that of the control antibody BS025-PC.

[0227] Table 4. Binding of anti-Nectin-4 antibody to cell surface antigen Nectin-4 (EC) 50

[0228]

[0229] Example 4. Endocytotic activity of anti-Nectin-4 antibody

[0230] Experimental objective: To detect the endocytic activity of anti-Nectin-4 antibody using human breast cancer cells MCF-7.

[0231] Experimental method: After washing the cells twice with 1×PBS buffer, the cells were resuspended in 1×PBS buffer to a cell concentration of 3×10⁻⁶. 5 Cells / μL: Aliquot 50 μL of cells into PCR plates, one well per well. Dilute the antibody to be tested to 4 μg / mL with 1×PBS buffer, and add 50 μL / well to each plate to achieve a final antibody concentration of 2 μg / mL. Place 5 wells for each antibody. Incubate the antibody and cells at 4°C for 1 hour. After incubation, wash the cells once with 1×PBS buffer, resuspend the cells in 200 μL of 1×PBS buffer, and transfer the antibody and cells to 96-well cell culture plates at time gradients of 0 h, 0.5 h, 1 h, 2 h, and 4 h. Incubate at 37°C. After incubation, wash the cells once with 1×PBS buffer, add 100 μL of fluorescent secondary antibody (647 Anti-HumanIgG Fc) to each well, and incubate at 4°C for 1 hour. After incubation, wash the cells once with 1×PBS buffer, resuspend the cells in 200 μL of 1×PBS buffer, transfer the cells to 96-well cell culture plates, and analyze using flow cytometry.

[0232] Experimental results: The results are shown in Table 5. The anti-Nectin-4 antibody has endocytic activity.

[0233] Table 5. Endocytotic activity of anti-Nectin-4 antibody in MCF-7 cells

[0234]

[0235] Example 5. Anti-Nectin-4 antibody

[0236] The variable region of the antibody was linked to a His-Cys tag to construct the VHH-His-Cys antibody. The plasmid was constructed and transformed into *E. coli* BL21(DE3) strain. Single colonies were selected and activated overnight at 37°C in LB medium. The colonies were then transferred at a 1:100 ratio to 300 mL of LB medium and cultured. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mmol / L, and expression was induced at 30°C for 6 h. The culture system was centrifuged at 6,000 rpm for 5 min to collect the cells. The collected cells were resuspended in PBS, and polymyxin was added to a final concentration of 0.5 mM. The cells were lysed at 37°C for 2 h, followed by centrifugation at 8,000 rpm for 30 min. The filtered supernatant was conjugated with Ni Sepharose (GE) for 2 h. The supernatant was washed with Tris buffer containing 40 mM imidazole, and the target protein was eluted with buffer containing 250 mM imidazole. The protein was concentrated using ultrafiltration tubes, and the buffer was replaced with 0.1M NaHCO3 (pH 8.3). Protein purity was then confirmed to be >90% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The NB656-M1-56-His-Cys sequence is as follows:

[0237] >NB656-M1-56-His-Cys

[0238] EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADRMDGSIWYDYWGQGTQVTVSSHHHHHHC (SEQ ID NO: 8)

[0239] Example 6. Anti-Nectin-4 antibody conjugate

[0240] The anti-Nectin-4 antibody conjugate is obtained by conjugating anti-Nectin-4 antibody with anthocyanin dye derivative IR808 (also known as MHI-808). The specific preparation steps are as follows: Take 1 mg of anti-Nectin-4 antibody NB656-M1-56-His-Cys, dilute it to 1 mg / ml with 20 mM Tris-HCl buffer, pH 8.0, add 2.8 μL TCEP (100 mM) to prevent protein aggregation, and add about 32 μL of MHI-808 (5 mg / ml) for every 1 mg of protein according to the molar ratio of MHI-808:antiprotein = 2:1. React at 4℃ for 30 min, and dialyze in 20 mM Tris-HCl, pH 8.0 buffer (dialysis bag 5KD). Change the medium every 3-4 hours, for a total of 5 times.

[0241] The anti-Nectin-4 antibody conjugate 56-IR808 was obtained using the method described above.

[0242] Example 7. Targeting of the anti-Nectin-4 antibody conjugate

[0243] Experimental objective: To detect the tumor targeting and persistent tumor accumulation of anti-Nectin-4 antibody using bladder cancer cells T24 (Pronosai, CL-0277).

[0244] Experimental methods: First, T24 cells were cultured using Pronos-specific medium. Then, T24 cells (5 × 10⁻⁶) were resuspended in PBS. 6 (1 cell) was subcutaneously injected into mice (BALB / cNj-Foxn1) nu / Gpt, NO.D000521, female, Jicuiyaokang) right hind limb, constructing a T24 xenograft model. When the tumor volume reaches approximately 200-400 mm... 3 Ten T24 xenograft mice were intravenously injected with the corresponding anti-Nectin-4 antibody conjugate (concentration of 1 mg / ml, 100 μL per mouse). In vivo fluorescence imaging of the mice was performed using an in vivo imaging system (IVIS) at different time points (1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h).

[0245] Experimental results: The results are as follows Figure 1 As shown, the anti-Nectin-4 antibody conjugate 56-IR808 has the advantages of strong tumor targeting, rapid tumor enrichment, and long retention.

Claims

1. An antibody targeting Nectin-4, which is an immunoglobulin single variable domain or an Fc fusion antibody containing said immunoglobulin single variable domain, wherein the immunoglobulin single variable domain is VHH, and the immunoglobulin single variable domain contains CDR1, CDR2 and CDR3, characterized in that, The amino acid sequence of CDR1 is INVMG, the amino acid sequence of CDR2 is TITSGGSTNYADSVKG, and the amino acid sequence of CDR3 is DRMDGSIWYDY.

2. The antibody as described in claim 1, characterized in that, The immunoglobulin single variable domain also includes a framework region.

3. The antibody as described in claim 2, characterized in that, The frame region is either an alpaca-derived frame region or a human-derived frame region.

4. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 75% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

5. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 80% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

6. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 85% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

7. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 90% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

8. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 95% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

9. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 96% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

10. The antibody according to claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 97% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

11. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 98% identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

12. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin has at least 99% sequence identity with SEQ ID NO: 1 and does not involve any changes to the CDR sequence.

13. The antibody as described in claim 2, characterized in that, The amino acid sequence of the single variable domain of the immunoglobulin is shown in SEQ ID NO:

1.

14. The antibody according to any one of claims 1-13, characterized in that, When the antibody is an Fc fusion antibody, the antibody further comprises an immunoglobulin Fc region; the immunoglobulin Fc region is derived from human or mouse sources, and / or the immunoglobulin Fc region is IgG Fc.

15. The antibody as described in claim 14, characterized in that, The immunoglobulin Fc region is IgG1 Fc.

16. The antibody as claimed in claim 14, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 85% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

17. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 90% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

18. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 95% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

19. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 96% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

20. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 97% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

21. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 98% identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

22. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the immunoglobulin Fc region has at least 99% sequence identity with SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

23. The antibody as claimed in claim 16, characterized in that, The amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:

7.

24. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antibody as described in any one of claims 1-23; the antibody is a nanobody.

25. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody as described in any one of claims 1-23.

26. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 25.

27. The recombinant expression vector as described in claim 26, characterized in that, The recombinant expression vector is selected from plasmids, bacteriophages, and viral expression vectors.

28. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 25 or the recombinant expression vector as described in claim 26 or 27; the transformant is a non-animal variety or a non-plant variety.

29. The transformant as described in claim 28, characterized in that, The host cells of the transformant are selected from bacterial cells, insect cells, and mammalian cells.

30. The transformant as described in claim 29, characterized in that, The mammalian cells were 293 cells.

31. A method for preparing an antibody targeting Nectin-4, characterized in that, The method includes culturing the transformant as described in any one of claims 28-30 and obtaining the antibody from the culture.

32. An antibody conjugate, characterized in that, The antibody conjugate comprises an effector molecule and an antibody as described in any one of claims 1-23; the effector molecule is a radioactive isotope or a chromophore.

33. An antibody conjugate, characterized in that, The antibody conjugate comprises an effector molecule and an antibody as described in any one of claims 1-23; the effector molecule is a fluorophore.

34. An antibody conjugate, characterized in that, The antibody conjugate comprises an effector molecule and an antibody as described in any one of claims 1-23; the effector molecule is a chemiluminescent compound.

35. The antibody conjugate according to any one of claims 32-34, characterized in that, The antibody conjugate has a His-Cys tag attached to its C-terminus.

36. The antibody conjugate as described in claim 33, characterized in that, The amino acid sequence of the antibody with a C-terminus linked to a His-Cys tag is shown in SEQ ID NO:

8.

37. The antibody conjugate as described in claim 36, characterized in that, The antibody conjugate is an antibody probe.

38. The antibody conjugate as described in claim 37, characterized in that, The antibody probe is labeled with IR808.

39. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the antibody as described in any one of claims 1-23, the chimeric antigen receptor as described in claim 24, and the antibody conjugate as described in any one of claims 32-38; and pharmaceutically acceptable excipients.

40. A reagent or kit for detecting Nectin-4 and / or diagnosing Nectin-4 expression-related cancers, characterized in that, The reagent or kit comprises one or more of the antibodies as described in any one of claims 1-23, antibody conjugates as described in any one of claims 32-38, and pharmaceutical compositions as described in claim 39. The cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

41. The reagent or kit according to claim 40, characterized in that, The reagents or kits are used for in vivo imaging techniques, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry.

42. Use of one or more of the antibody as claimed in any one of claims 1-23, the nucleic acid as claimed in claim 25, the antibody conjugate as claimed in any one of claims 32-38, and the pharmaceutical composition as claimed in claim 39 in the preparation of a product for diagnosing cancer; wherein the cancer is a Nectin-4 expression-related cancer. in, The cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

43. A method for detecting Nectin-4 expression for non-diagnostic purposes, characterized in that, The method includes contacting one or more of the antibodies as described in any one of claims 1-23, antibody conjugates as described in any one of claims 32-38, pharmaceutical compositions as described in claim 39, and reagents or kits as described in claim 40 or 41 with a sample to be tested, and detecting the expression level of Nectin-4 in the sample to be tested.

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