A monoclonal antibody targeting nectin4, related nucleic acids, vectors, host cells and use in the preparation of antitumor drugs

Abstract

This invention discloses a monoclonal antibody targeting NECTIN4, its associated nucleic acid, vector, host cell, and its application in the preparation of antitumor drugs, belonging to the field of biomedical technology. The monoclonal antibody comprises a specific heavy chain variable region VH and a light chain variable region VL. The HCDR1-3 amino acid sequences of VH are shown in SEQ ID NO:1-3, and the LCDR1-3 amino acid sequences of VL are shown in SEQ ID NO:4-6. This invention also provides the nucleic acid encoding the antibody (SEQ ID NO:10), a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector. This Nectin4 monoclonal antibody can specifically target and bind to the NECTIN4 protein on the surface of tumor cells, inhibiting tumor cell cycle progression, proliferation, migration, and invasion by regulating the NF-κB pathway. As a naked antibody, it exhibits significant tumor-targeting inhibitory effects, especially for esophageal cancer, and can also be used for targeted therapy of other malignant tumors such as breast cancer and lung cancer. This monoclonal antibody has a better safety profile than ADC drugs, providing a new option for tumor targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a monoclonal antibody targeting NECTIN4, its related nucleic acid, vector, host cell, and its application in the preparation of anti-tumor drugs. Background Technology

[0002] NECTIN4 (also known as Nectin-4 or PVRL4) is a member of the immunoglobulin superfamily (IgSF) and belongs to the transmembrane cell adhesion molecule family, playing an important role in the Nectin protein family. From a tissue expression perspective, NECTIN4 is highly expressed in embryonic and placental tissues, while its expression level is extremely low in normal adult tissues. This significant difference in expression provides an important prerequisite for its potential as a target for tumor therapy. In the field of malignant tumors, the expression characteristics of NECTIN4 undergo significant alterations—its expression is reactivated and significantly increased, a phenomenon widely observed in various malignant tumors, including esophageal cancer, bladder cancer, and breast cancer. This makes NECTIN4 an important potential target in cancer research. In particular, the high expression of NECTIN4 is especially prominent in esophageal cancer (especially esophageal squamous cell carcinoma, ESCC). Previous studies have confirmed that the expression level of NECTIN4 in esophageal cancer is closely related to tumor invasiveness, lymphatic metastasis, and poor patient prognosis, further clarifying its close association with the development and progression of esophageal cancer. From a mechanistic perspective, NECTIN4 can promote tumor cell proliferation, tumor-associated angiogenesis, lymphangiogenesis, and tumor metastasis by activating key signaling pathways such as PI3K / AKT. Furthermore, its extracellular domain may enter the circulatory system after being cleaved by the ADAM protease, participating in the regulation of the tumor microenvironment and influencing tumor progression from multiple dimensions. Given the high specificity of NECTIN4 expression in tumor tissues and its low expression in normal tissues, antibody-drug conjugates (ADCs) targeting this target have become a current research hotspot. Several NECTIN4 ADC drugs have already entered clinical trials, such as Padcev and 9MW2821. These drugs can be used not only as monotherapy for cancer treatment but also to explore their feasibility in combination with immunotherapies such as PD-1 inhibitors. In terms of clinical progress, some NECTIN4 ADC drugs have entered Phase III clinical trials for urothelial carcinoma, and related drugs for esophageal cancer have received FDA Fast Track designation and orphan drug designation, demonstrating promising application prospects. However, current antibody research targeting NECTIN4 still has significant limitations. In terms of indication exploration, existing NECTIN4-targeted therapies (such as Padcev and 9MW2821) mainly focus on urothelial carcinoma, cervical cancer, and triple-negative breast cancer. Targeted therapy research for esophageal cancer is still in its early stages, lacking specific targeting regimens for this cancer type. At the mechanism of action level, most existing NECTIN4 antibodies rely on ADCs (antibody-drug conjugates) to exert their effects, primarily achieving tumor killing through the delivery of cytotoxic drugs. However, research on the direct regulatory mechanisms of the NECTIN4 signaling pathway itself is insufficient, failing to fully elucidate the direct impact of antibodies on target signaling function. Regarding treatment strategies, current research mainly focuses on ADC drugs and their combination therapies. The development of naked antibody monotherapy targeting NECTIN4 is significantly insufficient, and related research remains relatively scarce. Summary of the Invention

[0003] The purpose of this invention is to provide a monoclonal antibody targeting NECTIN4, its related nucleic acid, vector, host cell, and its application in the preparation of antitumor drugs, so as to solve the problem of the lack of NECTIN4 targeting strategies in the field of esophageal cancer, provide a naked antibody that can directly regulate the NF-κB pathway, fill the gap in naked antibody research and development, and thus help cancer treatment.

[0004] The objective of this invention is achieved through the following technical solution: Technical Solution 1: A monoclonal antibody targeting NECTIN4, comprising a heavy chain variable region VH and a light chain variable region VL; wherein VH comprises HCDR1, HCDR2 and HCDR3; and wherein VL comprises LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is shown in SEQ ID NO:2; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; The amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

[0005] As some possible embodiments of this application, the mature peptide amino acid sequence of the heavy chain is shown in SEQ ID NO: 7, and the mature peptide amino acid sequence of the light chain is shown in SEQ ID NO: 8.

[0006] As some possible embodiments of this application, the monoclonal antibody includes chimeric antibodies, humanized antibodies, fully human antibodies, and their antigen-binding fragments; the antigen-binding fragment is selected from at least one of the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and bispecific di-scFv.

[0007] As some possible embodiments of this application, the amino acid sequence of the monoclonal antibody includes the mature peptide amino acid sequence of the heavy chain as shown in SEQ ID NO: 7, the mature peptide amino acid sequence of the light chain as shown in SEQ ID NO: 8, and the constant region as shown in SEQ ID NO: 9.

[0008] Technical Solution 2: Nucleic acid expressing a monoclonal antibody targeting NECTIN4, the nucleotide sequence of which is shown in SEQ ID NO:10.

[0009] Technical Solution 3: A vector containing a monoclonal antibody targeting NECTIN4.

[0010] Technical Solution 4: Host cells containing nucleic acids or vectors.

[0011] Technical Solution 5: The use of monoclonal antibodies, nucleic acids, vectors or host cells targeting NECTIN4 in the preparation of targeted drugs for the prevention or treatment of tumors.

[0012] As some possible implementations of this application, the tumor includes at least one of esophageal cancer, breast cancer, papillary thyroid carcinoma, diffuse large B-cell lymphoma, lung cancer, cervical cancer, or nasopharyngeal carcinoma.

[0013] As one possible implementation of this application, the tumor is esophageal cancer.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention successfully screened and obtained multiple monoclonal antibodies targeting NECTIN4 by immunizing mice with the NECTIN4 protein. These antibodies can specifically recognize and bind to the NECTIN4 protein, demonstrating significant potential in targeted tumor therapy. Unlike existing NECTIN4-targeted therapies, which mainly focus on urothelial carcinoma and other similar diseases, this invention is the first to achieve NECTIN4-targeted therapy for esophageal cancer, effectively filling the gap in specific treatment options for this target in esophageal cancer and providing esophageal cancer patients with a novel targeted therapy option.

[0015] Regarding its mechanism of action, this invention's antibody overcomes the limitations of existing NECTIN4 antibodies, which primarily rely on ADCs (anti-cytotoxic drugs) to kill tumors through cytotoxic drug delivery. This antibody does not require conjugation to a cytotoxic payload and can exert targeted therapeutic effects on its own. It specifically acts on the NF-κB signaling pathway, inhibiting esophageal cancer cell cycle progression to block proliferation and significantly reducing cancer cell migration and invasion capabilities, effectively curbing tumor progression. Simultaneously, this mechanism avoids the "bystander effect" that ADC drugs may cause, achieving more precise regulation of esophageal cancer. This study is the first to reveal the molecular mechanism by which NECTIN4 monoclonal antibodies regulate esophageal cancer through the NF-κB pathway, compensating for the shortcomings of existing technologies in understanding the function of the NECTIN4 signaling pathway itself.

[0016] From a treatment strategy perspective, current research and development of NECTIN4-targeted drugs mainly focuses on ADC drugs and combination therapy regimens, with a lack of treatment options for naked antibody monotherapy. The NECTIN4 monoclonal antibody provided in this invention, as a naked antibody, possesses significant targeted anti-tumor activity and can be used independently as monotherapy for esophageal cancer, effectively simplifying the clinical medication process. Furthermore, this antibody can synergize with PD-1 / PD-L1 inhibitors, and its synergistic mechanism differs from the combination mode of existing ADC drugs, providing a new pathway for targeted-immunotherapy combination therapy. Because it does not carry exogenous toxic loads, this monoclonal antibody is significantly safer than ADC drugs, and in the future, it can be further developed into a bifunctional antibody or used as an ADC platform for technological expansion, showing broad prospects in both clinical application and drug development. Attached Figure Description

[0017] Figure 1 Immunofluorescence detection of endogenous 6His-nectin4 in antiserum of immunized mice; Figure 2 : Endogenous cell detection was performed using the supernatant of monoclonal cells; Figure 3 Immunofluorescence detection of endogenous 6His-nectin4 in monoclonal hybridoma cell lines; Figure 4 Comparison of monoclonal antibody sequencing results with VH; Figure 5 Comparison of monoclonal antibody sequencing results with VL; Figure 6 Results of monoclonal antibody purity testing; Figure 7 : Results of immunofluorescence assay for monoclonal antibodies; Figure 8 Results of Western blot analysis of monoclonal antibody protein. Detailed Implementation

[0018] Example 1: Preparation, protein expression and purification of NECTIN4 recombinant antigen.

[0019] The gene sequence of NECTIN4 was found on the NCBI website. The obtained DNA sequence was cloned into the pET28b expression vector, and the cDNA sequence was cloned into the pET28b expression vector. Different E. coli strains (BL21, T7E), induction temperatures (16°C, 37°C), and induction times (4 hours, 16 hours) were tested. The optimal expression conditions (37°C, 4 hours, BL21 strain) were determined by SDS-PAGE analysis. After lysing the bacterial cells, soluble components (NPE) and inclusion bodies (DPE) were separated by centrifugation. The inclusion bodies were dissolved using denaturing buffer containing 8M urea. The His-tagged protein was purified using a Ni column. The target protein was separated by different buffers (binding, washing, elution), and finally, urea was removed by dialysis. The purity was verified by SDS-PAGE, and the concentration was determined by the Bradford method to obtain the purified protein (final concentration 1.50 mg / ml, stored in PBS buffer).

[0020] Given the need to prepare mouse monoclonal antibodies for Western blotting and immunofluorescence (IF) experiments, after comprehensive consideration, the prokaryotic system domain Gly 32-Asp331 was used to express the protein. The vector used was pET28b, with a GS linker and a 6*His tag added to the N-terminus. The restriction enzyme sites were NdeI (gene sequence SEQ ID NO: 11) / EoRI (gene sequence SEQ ID NO: 12). After expression and purification testing, the optimal expression and purification conditions were selected for scale-up production for immunization, screening, and titer detection. The expressed protein 6His-nectin4 (gene sequence SEQ ID NO: 13) contains 321 amino acids (AAs) and has a size of 34.5 kDa; pI 5.8.

[0021] Expressed sequence: MGSSHHHHHHSSGLVPRGSHMGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLA ASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVD.

[0022] The specific experimental steps are as follows: 1. Antibody production.

[0023] Cell line: 22-D9-B3-H2; Antibody production method: supernatant culture; Antibody purification method: ProG.

[0024] 2. Antibody quality testing.

[0025] 2.1 Antibody titer test (results are shown in Table 1).

[0026] Detection method: Indirect ELISA; Coating antigen: 6His-nectin4; Coating concentration: 5 μg / ml, 100 μl / well; Coating diluent: CBS, pH 9.6; Secondary antibody: Peroxidase AffiniPure Goat Anti-Mouse IgG (FC); Antibody titer: The highest dilution ratio at which the detected value / negative control ratio is ≥2.1; Blank control: PBS.

[0027] Table 1: 2.2 SDS-PAGE detection (e.g.) Figure 6 ).

[0028] Example 2: Immunization of mice.

[0029] The antigen was emulsified thoroughly with either Freund's complete adjuvant or Freund's incomplete adjuvant by sonication. Freund's complete adjuvant was used for the first emulsification, and Freund's incomplete adjuvant was used from the second immunization onwards. No adjuvant was added for the initial pulse immunization. The immunization interval was 14 days, and blood samples were collected 7 days after the booster immunization. Five mice were immunized with the antigen four times in total. Each mouse received 50 μg of antigen per immunization. The antigen was a recombinant protein of 6His-nectin4. The antigen was mixed with an equal volume of Freund's adjuvant and administered subcutaneously. The immunization protocol was two points on the back and one point intraperitoneally, each injected with 1 / 3 of the volume, avoiding injection into the left spleen. Immunization was performed every two weeks. After four immunizations, blood was collected from the tail, and the serum titer was detected by ELISA. The binding effect of the antiserum on NECTIN4, endogenously expressed in KYSE410 cells, was detected by immunofluorescence.

[0030] The mouse antiserum with the highest titer was from mouse number 1. The titer of the recombinant 6His-nectin4-immunized mouse antiserum is shown in Table 2. Table 2: The antiserum from all five mice was able to bind to the recombinant antigen 6His-nectin4 from KYSE410 cells. The results of ELISA detection of the mouse serum after 4 immunizations are shown in Table 3.

[0031] Table 3: Five mice were immunized with each antigen for a total of four times, followed by a pre-fusion shock immunization. The antiserum titers of all five mice were >128000.

[0032] Antibody titer: A positive value is defined as a detectable value / negative control ratio ≥ 2.1.

[0033] Example 3: Cell fusion, subcloning screening and line establishment.

[0034] Mice with good serum immunization and validation results were selected (number 1). Spleens were harvested for cell fusion. Positive hybridoma cells were obtained using HAT medium. The positive hybridoma cells were subcloned using the limiting dilution method to obtain monoclonal cell lines. Two to three rounds of indirect ELISA screening were performed during each subcloning period. After obtaining the positive monoclonal cell lines that met the requirements, cell lines were established.

[0035] Indirect ELISA detection results of the titer of the supernatant from the established plants: Finally, the supernatants of nine cell lines with high titers were selected for endogenous cell testing. Endogenous cell testing was performed on the supernatants of monoclonal cell lines, and the results were analyzed based on (e.g., Figure 2 , Figure 3(and Table 4), select the 22-D9-B3-H2 monoclonal strain for monoclonal antibody production.

[0036] Table 4: Example 4: Monoclonal cell screening (limiting dilution method).

[0037] The first round of subclones were screened using limiting dilution monoclonal assay. Antibody typing (antibody subtype IgG1) was performed based on a titer >128000 (see Table 5) (see Table 6) (see Table 6) (see Table 6). Antibody purity was also assessed (the antibody band was single, indicating high purity; after opening the disulfide bonds with reducing loading buffer, bands of approximately 55kD and 25kD were observed, indicating the antibody was IgG). Figure 6 ).

[0038] Table 5: Table 6: Example 5: Large-scale production and purification of monoclonal antibodies (supernatant culture, ProG purification).

[0039] Antibodies were produced using supernatant culture and purified using the ProG method. The specific steps are as follows: (1) Centrifuge 5000g of the sample to be purified for 5 min and collect the supernatant.

[0040] (2) Add the supernatant to the protein G column and collect it in a 15 ml centrifuge tube.

[0041] (3) After loading the sample, wash the column with 10 column volumes of PBS.

[0042] (4) While washing the column, prepare 1.5 ml sterile EP tubes and add 50 μl of 1 M pH 9.0 Tris-HCl to each collection tube beforehand.

[0043] (5) Elute with 1 ml of 0.1 M glycine solution (pH 2.5) and collect the eluent in a 1.5 ml EP tube. Immediately after collection, invert the EP tube to mix and test the pH of the eluent with pH paper to determine if it is neutral.

[0044] (6) After elution, add 3 column volumes of 0.1M glycine solution (pH 2.5) to the protein G column to completely elute the column. Then wash the column with 5 column volumes of dihydrochloric acid and 2 column volumes of 20% ethanol. After the column has been completely washed, cap the bottom of the column tightly and add 2 column volumes of 20% ethanol to store the column at 4°C.

[0045] (7) Centrifuge the eluted antibody at 12000 rpm for 2-3 min at room temperature, discard the precipitate, take the supernatant and measure the OD value to calculate the antibody concentration. Formula: Antibody concentration (mg / ml) = OD280 / 1.4 × dilution factor. If the concentration cannot be displayed, it means that the concentration is too high. Use a multi-channel pipette to aspirate 200ul back into the original tube, then aspirate 20ul of the original antibody solution, add 180ul of 1*PBS to dilute 10 times, mix well, and perform detection.

[0046] (8) Calculate the antibody concentration based on the measured OD280, and run 2ug of antibody on the gel.

[0047] (9) Take antibody samples and send them for ELISA testing.

[0048] Example 6: Antibody titer detection.

[0049] Antibody titers were detected using a simplified ELISA method. Coating antigen: 6His-nectin 4; Coating concentration: 5 μg / ml, 100 μl / well; Coating diluent: CBS, pH 9.6; Secondary antibody: Peroxidase Affini Pure Goat Anti-Mouse IgG (FC); Antibody titer: the highest dilution ratio when the detected value / negative control is ≥2.1; Pre-immunization serum was used as a negative control, and PBS was used as a blank control. Details are shown in Table 7.

[0050] Table 7: Example 7: Monoclonal antibody immunofluorescence detection.

[0051] Cells were seeded into 6-well culture plates with coverslips, ensuring the coverslips were completely immersed in the culture medium, and cultured at 37°C in a 5% CO2 incubator for 2-3 days. When cells covered the bottom 2 / 3 of the plate, the plate was removed and the slides were soaked in PBS three times for 3 minutes each time. After fixing with 4% paraformaldehyde for 15 minutes, normal goat serum was added to the slides, and the plates were blocked at room temperature for 30 minutes. First antibody was added to each slide, and the plates were placed in a humidified chamber and incubated overnight at 4°C. The slides were then washed three times with PBST for 3 minutes each time, and fluorescent secondary antibody was added. After incubation in a humidified chamber at 20-37°C for 1 hour, the slides were soaked and washed three times with PBST for 3 minutes each time. DAPI was added and the plates were incubated in the dark for 5 minutes. The plates were then blocked four times with a blocking solution containing an anti-fluorescence quencher for 5 minutes each time. Finally, strong fluorescence signals (e.g., DAPI) were observed under a fluorescence microscope. Figure 7 As shown in the figure, the results indicate that the 22-D9-B3-H2 monoclonal antibody can effectively bind to the NECTIN4 protein on the surface of heavy chain cells.

[0052] Example 8: Detection of monoclonal antibody protein immunoblotting.

[0053] The coding sequence of the target protein (Nectin4) was cloned into a mammalian expression vector carrying a His tag sequence to obtain a recombinant expression plasmid. This recombinant expression plasmid was transfected into the target cell line (Kyse410 cells) and cultured for a specific time to induce recombinant protein expression. Transfected cells were collected, lysed, and total protein was extracted, quantified, and prepared as a Western blotting sample. Detection was performed using a specific antibody against the target protein (Nectin4), which showed the sum of endogenous and exogenous target proteins. Simultaneous detection was performed using a specific antibody against the His tag, with the result serving as a "His control," specifically and uniquely indicating successful expression of the exogenous recombinant protein. The results showed that the "His control" exhibited a single, clear band of the expected molecular weight, and the target protein showed a significantly enhanced band at the corresponding position, indicating successful expression of the His-tagged recombinant protein in the cells (e.g., ...). Figure 8 (As shown).

[0054] sequence list SEQ ID NO: 1 GYTFTVYE SEQ ID NO: 2 IDPKTGGT SEQ ID NO: 3 TRSPY SEQ ID NO: 4 QSLLDSDGKTY SEQ ID NO: 5 LVS SEQ ID NO: 6 WQGTYFPQT SEQ ID NO: 7 QVQLQQSGAELVRPGASVTLSCKASGYTFTVYEMHWVKQTPIHGLEWIGAIDPKTGGTAYNRKFKGKATLTADRSSSTAYMELRSLTSEDSAVFYCTRSPYWGQGTLVTVSTAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO:8 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKINRVEAEDLGVYYCWQGTYFPQTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:9 QVQLQQSGAELVRPGASVTLSCKASGYTFTVYEMHWVKQTPIHGLEWIGAIDPKTGGTAYNRKFKGKATLTADRSSSTAYMELRSLTSEDSAVFYCTRSPYWGQGTLVTVSTAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH TFPAVLQSDLYTLSSSVTVPSSTWPSETTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIC TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLHSPGKDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYL VSKLDSGVPDRFTGSGSGTDFTLKINRVEAEDLGVYYCWQGTYFPQTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO:10 SEQ ID NO: 11 TTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCAT SEQ ID NO: 12 GAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGC SEQ ID NO: 13

Claims

1. A monoclonal antibody targeting NECTIN4, characterized in that: It includes an antibody heavy chain variable region VH and a light chain variable region VL; the VH includes HCDR1, HCDR2 and HCDR3; the VL includes LCDR1, LCDR2 and LCDR3; The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is shown in SEQ ID NO:2; The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 5; The amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody targeting NECTIN4 according to claim 1, characterized in that: The mature peptide amino acid sequence of the heavy chain is shown in SEQ ID NO: 7, and the mature peptide amino acid sequence of the light chain is shown in SEQ ID NO:

8.

3. The monoclonal antibody targeting NECTIN4 according to claim 1 or 2, characterized in that: The monoclonal antibody includes chimeric antibodies, humanized antibodies, fully human antibodies, and their antigen-binding fragments; the antigen-binding fragment is selected from at least one of the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and bispecific di-scFv.

4. The monoclonal antibody targeting NECTIN4 according to claim 3, characterized in that: The amino acid sequence of the monoclonal antibody includes the mature peptide amino acid sequence of the heavy chain as shown in SEQ ID NO: 7, the mature peptide amino acid sequence of the light chain as shown in SEQ ID NO: 8, and the constant region as shown in SEQ ID NO:

9.

5. A nucleic acid expressing the monoclonal antibody targeting NECTIN4 as described in any one of claims 1-4, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

10.

6. A vector comprising the nucleic acid of the monoclonal antibody targeting NECTIN4 as described in claim 5.

7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. Use of the monoclonal antibody targeting NECTIN4 according to any one of claims 1-4, the nucleic acid according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in the preparation of a targeted drug for the prevention or treatment of tumors.

9. The use according to claim 8, characterized in that: The tumor is at least one of the following: esophageal cancer, breast cancer, papillary thyroid carcinoma, diffuse large B-cell lymphoma, lung cancer, cervical cancer, or nasopharyngeal carcinoma.

10. The use according to claim 9, characterized in that: The tumor is esophageal cancer.

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