CLDN3 monoclonal antibody and application thereof

Abstract

The invention discloses a CLDN3 monoclonal antibody and application thereof.The antibody comprises a light chain and a heavy chain and is characterized in that the amino acid sequence of the light chain is shown as SEQ ID No: 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No: 2; the nucleotide sequence of the light chain is as shown in SEQ ID No: 3, and the nucleotide sequence of the heavy chain is as shown in SEQ ID No: 4. The CLDN3 monoclonal antibody provided by the invention has high specificity, high affinity and stability, can effectively recognize a target antigen CLDN3 on a gastric cancer cell line mkn-45, can effectively inhibit proliferation of mkn-45 gastric cancer cells, and provides an important basis for development of CLDN3-targeted antibody drugs.

Description

Technical Field

[0001] This invention relates to the field of antibody bioengineering technology, specifically to a CLDN3 monoclonal antibody or a fragment thereof, DNA molecule, vector, recombinant, application, drug and pharmaceutical composition. Background Technology

[0002] The application of monoclonal antibodies in the field of tumor treatment has attracted much attention, largely due to their high affinity, high specificity, long serum half-life and metabolic stability.

[0003] Antitumor antibody drugs exert their therapeutic effects through several different but non-contradictory mechanisms, ranging from simple blockade to activation and enhancement of the body's innate immune response. Due to their high affinity and specificity, antibodies interact with targeted cell populations, competitively binding to receptors or ligands on target cells, thereby blocking ligand-receptor interactions and downregulating signal transduction. They can also inhibit receptor polymerization, which is the fundamental principle behind the development of monoclonal antibodies in oncology. However, research has found that monoclonal antibody-mediated immune effector functions play a crucial role in the treatment of various antibodies. Immune effector functions are mediated by the interaction between the Fc region of antibody molecules (formed by CH2 and CH3 of the heavy chain) and Fc γ receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb) on immune cells and complement C1q proteins, resulting in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which in turn mediate tumor killing. Besides directly killing tumor cells through antibody immune effector functions, the activation of T cells to attack tumors by inhibiting immunosuppressive pathways such as programmed death receptor 1 / programmed death ligand 1 (PD-1 / PD-L1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) has been widely used in the treatment of various tumors, ushering in a new era of tumor immunotherapy. In addition, type II monoclonal antibodies targeting CD20 can also exert therapeutic effects through a direct cytotoxic mechanism, without mediating the accumulation of CD20 molecules on the target cell surface, but rather inducing programmed death of malignant B cells through a mechanism independent of caspase activity.

[0004] Twenty-five years have passed since rituximab, the first monoclonal antibody approved for cancer treatment, was developed. Anti-tumor antibody drugs have become an important component of cancer therapy, but they still face many challenges, such as antibody resistance, potential immune-related adverse reactions induced by antibody drugs, high production costs, and complex administration methods. Therefore, more research and clinical data are needed to further advance personalized precision antibody therapy, ultimately aiming to improve efficacy, reduce toxicity, and improve patient prognosis.

[0005] Claudin3 (CLDN3) is a member of the Claudins family of transmembrane tight junction proteins. With a relative molecular mass of 20 kDa-34 kDa, CLDN3 is one of the most important skeletal proteins in tight junctions. CLDN3 plays a crucial role in maintaining the function of intercellular physical barriers and participating in molecular-cell communication; it is an important component in the formation of the blood-brain barrier, intestinal barrier, and blood-testis barrier.

[0006] Aberrant expression of the CLDN3 gene is associated with various diseases, such as inflammatory bowel disease, abnormal liver metabolism and biliary barrier function, gestational hypertension, and leukoencephalopathy. In the field of oncology, research has found that CLDN3 plays a crucial role in the development and progression of many cancers, including liver cancer, breast cancer, colon cancer, and ovarian cancer. Studies have also shown that CLDN3 is highly expressed in gastric cancer, and higher CLDN3 expression contributes to the development of intestinal-type gastric adenocarcinoma, potentially serving as a prognostic indicator and a target biomarker for chemotherapy. Furthermore, research has found that CLDN3 can suppress immune responses by inhibiting CD8+ T cell activation-related chemokines, suggesting that CLDN3 may be a potential target for immunotherapy.

[0007] Our previous research revealed that CLDN3 is a transmembrane protein, and we confirmed its high expression in gastric cancer tissue, making it an ideal target for monoclonal antibody therapy. Monoclonal antibodies targeting CLDN3 can effectively inhibit the proliferation of gastric cancer cells, providing a new strategy for the treatment of gastric cancer. Summary of the Invention

[0008] In previous research, the inventors discovered that tight junction protein 3 (CLDN3) is a transmembrane protein highly expressed in gastric cancer tissue, making it an ideal target for monoclonal antibody therapy for gastric cancer. To achieve the above objectives, this invention provides CLDN3 monoclonal antibodies or fragments thereof, as well as applications of DNA molecules, vectors, recombinants, antibodies or fragments thereof, and pharmaceuticals and pharmaceutical compositions. Optimizing the antibody sequence can improve its specificity, affinity, and stability, thereby enhancing therapeutic efficacy.

[0009] The first objective of this invention is to provide a CLDN3 monoclonal antibody or a fragment thereof, the antibody comprising a light chain and a heavy chain, the amino acid sequence of the light chain being shown in SEQ ID No: 1, and the amino acid sequence of the heavy chain being shown in SEQ ID No: 2.

[0010] Preferably, the nucleotide sequence of the light chain is shown in SEQ ID No: 3, and the nucleotide sequence of the heavy chain is shown in SEQ ID No: 4.

[0011] A second object of the present invention is to provide a DNA molecule encoding a CLDN3 monoclonal antibody or a fragment thereof as described in any of the preceding claims.

[0012] A third object of the present invention is to provide a carrier comprising the DNA molecule as described above.

[0013] A fourth object of the present invention is to provide a recombinant comprising the DNA molecule as described above or the vector as described above.

[0014] The present invention also provides the use of the CLDN3 monoclonal antibody or fragment thereof as described above in the preparation of a cancer treatment drug, wherein the cancer is a cancer that highly expresses CLDN3.

[0015] Preferably, the cancer is at least one of gastric cancer, pancreatic cancer, and colorectal cancer.

[0016] Preferably, the cancer is stomach cancer.

[0017] The present invention further provides a medicament and a pharmaceutical composition thereof, wherein the medicament comprises a CLDN3 monoclonal antibody or a fragment thereof as described in any of the preceding claims. The pharmaceutical composition comprises the aforementioned medicament.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] 1. In this invention, a CLDN3 monoclonal antibody was obtained by hybridoma fusion combined with single B cell antibody screening technology. This CLDN3 monoclonal antibody has high specificity, high affinity and stability, and can effectively recognize the target antigen CLDN3 on the gastric cancer cell line mkn-45. It can also effectively inhibit the proliferation of mkn-45 gastric cancer cells, providing an important basis for the development of antibody drugs targeting CLDN3.

[0020] 2. The CLDN3 monoclonal antibody of the present invention has important significance and value in antibody structure and function, disease diagnosis and treatment, biotechnology and vaccine design, and immunological research. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0022] Figure 1 This is a schematic diagram showing the results of flow cytometry analysis of the obtained antibody molecules.

[0023] Figure 2 The image shows the antibody (monoclonal antibody-1H1F3E10) before and after purification in the example, detected by reducing SDS-PAGE electrophoresis.

[0024] Figure 3 A schematic diagram illustrating the results of screening and selecting individual hybridoma cells.

[0025] Figure 4 This is a schematic diagram of DNA gel electrophoresis for light and heavy chain amplification of a single hybridoma cell.

[0026] Figure 5 This is a high-resolution magnified schematic diagram of the purified CLDN3 antibody 1H1F3E10 after fluorescent labeling.

[0027] Figure 6 A schematic diagram of cell proliferation results obtained from a CCK-8 assay of antibody molecule 1H1F3E10.

[0028] Figure 7 This is a schematic diagram illustrating the principle of using a drug containing the CLDN3 antibody obtained in this invention to treat cancer. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0030] This invention provides a CLDN3 monoclonal antibody, which those skilled in the art can implement by appropriately modifying process parameters based on the content herein. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0031] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0032] An antibody is a protein composed of one or more polypeptides that can specifically bind to antigens. One form of antibody constitutes the basic structural unit of an antibody.

[0033] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0034] The present invention will be further illustrated below with reference to the embodiments:

[0035] Example 1:

[0036] This embodiment provides a method for preparing a CLDN3 monoclonal antibody, which is a monoclonal antibody that specifically binds to the human tight junction protein CLDN3, obtained by screening using hybridoma cell technology combined with single B cell technology. This antibody has high specificity, high affinity and stability.

[0037] The specific preparation method is as follows:

[0038] 1. Animal immunization

[0039] Two 6-8 week old female Balb / c mice were immunized with LNP-CLDN3 mRNA (purchased from Genscript Biotech). Each mouse received a single immunization dose of 10 μg, injected into the posterior thigh muscle. Two weeks later, the same dose was injected into the posterior thigh muscle. One week after the third immunization, blood was collected, and serum titer was determined using indirect ELISA. A titer was considered acceptable when the serum OD-450 of the immunized mice was twice or more than that of the control group. Mice with acceptable titers were selected for a pulse immunization one week later. One week after the pulse immunization, the spleens of these mice were harvested for hybridoma fusion. Mice with unacceptable titers were immunized again until a suitable titer was achieved, with a maximum of five immunizations.

[0040] 2. Blood collection and titer testing

[0041] Serum collection: One week after the third immunization, 50-60 μL of blood was collected from the orbital venous plexus of mice. After standing overnight at 4°C, the serum was centrifuged at 4000 rpm for 15 min at 4°C and the supernatant was separated for testing.

[0042] 3. ELISA combined with potency testing

[0043] Serum titer was determined using an indirect ELISA method. A titer was considered acceptable when the serum OD-450 of the immunized mice was twice or more than that of the control group mice. Mice with high titers were selected for hybridoma fusion.

[0044] The testing steps are as follows.

[0045] (1) Select CH0 cells to overexpress CLDN3. After successful overexpression, collect the cells, centrifuge and resuspend the cells to a concentration of 2×10⁻⁶. 4 Add 100 μL of 100 cells to a 96-well culture plate and incubate until the cells are confluent.

[0046] (2) Wash the plate twice with PBS, add 80 μL of 4% tissue fixative per well, fix for 15 min at room temperature, wash twice with PBS, air dry, store at 2-8 ℃, and use within one week.

[0047] (3) Add 250 μL of blocking buffer (containing 2% BSA) to each well and incubate at 37 °C for 1 h. Wash three times with PBS.

[0048] (4) Add serially diluted serum samples and sample diluent at 100 μL / well, incubate at 37 ℃ for 1 h, and wash the plate five times with PBST. Add 100 μL of HRP-labeled anti-mouse secondary antibody / well, incubate at 37 ℃ for 1 h, and wash the plate five times with PBST.

[0049] (5) Color development: Add TMB solution, 100 μL / well, and develop color for 20 min in the dark.

[0050] (6) Termination: Add 50 μL of ELISA termination solution per well.

[0051] (7) Reading: OD450 value of each well is measured by single wavelength at 450 nm.

[0052] 4. Mouse monoclonal antibody fusion and screening

[0053] Mouse spleen cells and myeloma cells were fused using the polyethylene glycol (PEG) method. Positive master clones binding to CLDN3-overexpressing CHO cells were obtained using indirect ELISA. The master clones underwent 2-3 rounds of limiting dilution and appropriate selection until stable monoclonal cell lines were obtained.

[0054] 4.1 Hybridoma cell fusion

[0055] Spleen cells from immunized mice were collected and mixed with mouse myeloma cells at a ratio of 10:1, and then fused using the PEG fusion method.

[0056] Hybridoma cells were obtained. On the 3rd day, the HAT selective medium was replaced with HT selective medium, and then the medium was changed every other day using the half-medium replacement method. On the 8th day, the presence of viable hybridoma cells was observed. When the cell count reached more than 70%, ELISA was performed to screen for positive cells in the wells.

[0057] 4.2 Hybridoma cell screening

[0058] ELISA binding screening: Indirect ELISA was used to detect binding in the supernatant of hybridoma cell culture, retaining clones that bound the antigen. The detection procedure is as follows:

[0059] 1) Select CHO cells that stably overexpress CLDN3, collect the cells from the culture flasks, centrifuge, and resuspend the cells to a concentration of 2×10⁻⁶. 4 Add 100 μL of 100 cells to a 96-well culture plate and incubate until the cells are confluent.

[0060] 2) Wash the plate twice with PBS, add 80 μL of 4% tissue fixative per well, fix at room temperature for 15 min, wash twice with PBS, air dry, store at 2-8 ℃, and use within one week.

[0061] 3) Add 250 μL of blocking buffer (containing 2% BSA) to each well and incubate at 37 °C for 1 h. Wash three times with PBS.

[0062] 4) Add 50 μL of hybridoma cell supernatant to each well, incubate at 37 ℃ for 1 h, and wash the plate five times with PBST. Add 100 μL of HRP-labeled anti-mouse secondary antibody per well, incubate at 37 ℃ for 1 h, and wash the plate five times with PBST.

[0063] 5) Color development: Add TMB solution, 100 μL / well, and develop color for 20 min in the dark.

[0064] 6) Termination: Add 50 μL of ELISA termination solution per well.

[0065] 7) Reading: OD450 value of each well is measured at a single wavelength of 450 nm.

[0066] 4.3 Limiting dilution and subcloning screening

[0067] Using HT selective medium, each selected positive hybridoma cell line was seeded at a density of 0.75 cells per well in half of a 96-well cell culture plate and incubated statically at 37°C with 8% CO2. Monoclonal clusters were observed on day 10. When the cell count in each well reached over 70%, the supernatant was collected for indirect ELISA testing to confirm positive binding of the antibody to the antigen. The specific testing procedure is described in section 4.2 of the master clone indirect ELISA testing procedure. ELISA-specific positive monoclonal cells were selected and subjected to 1-2 rounds of limiting dilution until stable, positive monoclonal cells were obtained.

[0068] 5. Preparation of ascites

[0069] Prepare female 6-8 week old purebred BALB / c mice, inject 300 μL of sterile liquid paraffin into the fingertip via intraperitoneal injection, and gently massage the mouse abdomen. One week later, collect 2 × 10⁶ monoclonal hybridoma cells. 7 Each sample was resuspended in 800 μl of physiological saline and injected into the peritoneal cavity of mice using a syringe. The mice were observed and euthanized by cervical dislocation when their abdomens became noticeably distended (approximately 7-10 days). The mice were then placed in a beaker containing alcohol, their limbs fixed on a foam board, and the peritoneum was aspirated through a small incision in the peritoneum. This yielded a large amount of monoclonal antibody. The ascites fluid was collected into centrifuge tubes and centrifuged at 800 rpm for 30 minutes to remove tissues and cells. The supernatant was stored at -80°C for later use. The ascites fluid was then serially diluted with ELISA diluent, mixed thoroughly, and the antibody titer was determined by ELISA. Refer to Procedure 3 for the specific detection process.

[0070] 6. Antibody subtype identification

[0071] To confirm the obtained antibody subtype, it was identified using a mouse monoclonal antibody subtype identification kit from Wuhan Sanying Biotechnology Co., Ltd. The detection procedure is as follows:

[0072] 1) Material Preparation: Remove the mouse monoclonal antibody subtype identification kit from 4°C and allow it to equilibrate at room temperature for 30 minutes. Prepare the following reagents:

[0073] a. 1×PBST: 10 mL 20×PBST + 190 mL ultrapure water

[0074] b. ELISA strips: Select the number of ELISA strips according to the number of samples (one ELISA strip is needed to test one sample). Place the remaining strips in a sealed bag and store at 4°C.

[0075] c. 1× Goat anti-mouse IgM+IgG-HRP: 10 uL 100× Goat anti-mouse IgM+IgG-HRP + 990 uL 1×PBST

[0076] 2) Sample preparation: Dilute the ascites fluid with 1×PBST at a ratio of 1:100000, and then take 5 uL of the above mixture + 495 uL of 1×PBST.

[0077] 3) Monoclonal antibody subtype identification:

[0078] a. Dilute the sample to be tested appropriately and add it to the well of the strip sample, 50 uL / well.

[0079] b. No incubation required. Add 1× goat anti-mouse IgM+IgG-HRP to each sample well (50 μL / well). Gently mix using a mixer or by gently tapping the sides of the plate holder for 1 minute.

[0080] c. Cover with sealing film and incubate at room temperature for 1 hour.

[0081] d. Discard the liquid in the wells, wash the plate 3 times with 1×PBST, and pat dry on absorbent paper.

[0082] e. Add the freshly prepared colorimetric solution to the wells, 100 μL / well.

[0083] f. Develop color at room temperature in the dark for 10-20 min. Add stop solution to each well, 100 μL / well.

[0084] g. Result interpretation: Read the OD450 using a microplate reader. The well with the darkest color or the highest OD value corresponds to the specific subtype.

[0085] 7. Monoclonal antibody purification

[0086] The obtained ascites fluid was purified using immunoaffinity chromatography to harvest mouse monoclonal antibodies.

[0087] 8. Mouse monoclonal antibody detection

[0088] (1) Perform antibody concentration and purity testing;

[0089] (2) ELISA binding detection: The purified mouse monoclonal antibody was detected by indirect ELISA method.

[0090] According to the circumstances.

[0091] 9. Screening and selection of single hybridoma cells

[0092] Because hybridoma cells are unstable heterokaryotic organisms, they are prone to losing antibody-secreting chromosomes (especially mouse chromosomes) during passage, leading to a decrease or even complete loss of antibody secretion capacity. Therefore, in order to avoid interference from mutant cells when obtaining sequences of positive monoclonal hybridoma cells, the CellCelector Flex instrument is used to precisely select single hybridoma cells capable of secreting antibodies.

[0093] 10. Antibody sequence sequencing

[0094] (1) Whole genome cDNA was obtained by lysing a single positive hybridoma cell and extracting total RNA using Thermo Fisher Scientific’s single-cell cDNA pre-amplification kit;

[0095] (2) Using the obtained cDNA as a template, nested PCR amplification of the antibody heavy and light chains was performed using Novizan's Phanta Max Super-Fidelity DNA Polymerase.

[0096] (3) Perform DNA gel electrophoresis on the obtained PCR products to confirm that the fragments of the heavy and light chains of the antibody exist and are in the correct position. Then insert the amplified fragments into the expression vector or commercial vector and sequence to obtain plasmids containing the correct sequence.

[0097] II. Test Results:

[0098] 1. The obtained ascites antibodies were subtyped, and the results are shown in Table 1. The results in Table 1 indicate that all three antibodies obtained were of the IgM heavy chain type and the Kappa light chain type.

[0099] Table 1

[0100]

[0101] 2. The binding of the three ascites-type monoclonal antibodies was detected by ELISA, and the results are shown in Table 2.

[0102] Table 2

[0103]

[0104] 3. Flow cytometry was used to detect the binding of the three ascites-type monoclonal antibodies, and the results are as follows: Figure 1 As shown in the image.

[0105] As shown in Table 2 and Figure 1, after four immunizations with LNP-CLDN3 mRNA, both mice reached the fusion standard for CHO-CLDN3 binding titer via ELISA. The mouse with the best binding titer was selected for hybridoma fusion. After successful hybridoma fusion, three hybridoma cell lines binding to CHO-CLDN3 were obtained through two rounds of subcloning screening. Three monoclonal antibodies were obtained by preparing ascites fluid. ELISA showed that all three antibodies bound to CHO-CLDN3, with 1H1F3E10 exhibiting the strongest binding ability. This monoclonal antibody was selected for purification and further validation.

[0106] 4. The monoclonal antibody-1H1F3E10 mentioned above was purified. The antibodies before and after purification were detected by reducing SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown. By Figure 2 The SDS-PAGE analysis results showed that the corresponding lanes had a band at 70kDa and 25kDa, which were divided into heavy and light chains, and the purity reached over 90%.

[0107] 5. Antibody sequence sequencing

[0108] The inventors selected single cells and amplified the light and heavy chains of the 1H1F3E10 hybridoma cell line, such as... Figure 3 , Figure 4 As shown, the antibody light and heavy chain gene sequences were obtained after sequencing, and the sequencing results are as follows:

[0109] (1) CLDN3-1H1F3E10 light chain: The full-length amino acid sequence of CLDN3-1H1F3E10-PL (L20250615-1-2, as the code number) is shown in SEQ ID No: 1:

[0110] MGWSCIILFLVATATGVHSDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIREL TRSEGGPSWKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0111] (2) CLDN3-1H1F3E10 heavy chain: CLDN3-1H1F3E10-PH (L20250615-1-2, as the code name) The full-length amino acid sequence is shown in SEQ ID No: 2:

[0112] MGWSCIILFLVATATGVHSEVKLVESGGGLVQPGGSRLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARDYYGSSYWYFDVWGAGTTVTVSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLY NVSLIMSDTGGTCY。

[0113] (3)CLDN3 - 1H1F3E10 light chain: The full-length nucleotide sequence of CLDN3 - 1H1F3E10 - PL (L20250615 - 1 - 2, as the number) is shown in SEQ ID No: 3:

[0114] ATGGGCTGGAGCTGCATCATCCTGTTCCTCGTGGCTACAGCTACCGGAGTGCACAGCGACATTGTGCTGACCCAAAGCCCTGCTAGCCTGGCTGTGAGCCTGGGTCAACGTGCTACCATTAGCTATCGTGCTAGCAAAAGCGTGAGCACCAGCGGTTATAGCTATATGCATTGGAATCAACAAAAACCTGGTCAACCTCCTCGTCTGCTGATTTATCTGGTGAGCAATCTGGAGAGCGGTGTGCCTGCTCGTTTTAGCGGTAGCGGTAGCGGTACCGATTTTACCCTGAATATTCATCCTGTGGAGGAGGAGGATGCTGCTACCTATTATTGTCAACATATTCGTGAGCTGACCCGTAGCGAGGGTGGTCCTAGCTGGAAAAGGGCCGACGCAGCTCCTACCGTGTCTATCTTCCCCCCTAGCAGCGAGCAGCTGACATCAGGAGGAGCTAGCGTGGTCTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACTCTTGGACCGACCAGGATAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCTACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGGAACGAGTGCTGATAA。

[0115] (4) CLDN3-1H1F3E10 heavy chain: The full-length nucleotide sequence of CLDN3-1H1F3E10-PH (L20250615-1-2, as the number) is shown in SEQ ID No: 4:

[0116]

[0117] Example 2: Application

[0118] 1. The inventors fluorescently labeled the obtained CLDN3 antibody 1H1F3E10, as shown in Figure 5. Confocal localization effectively verified the antibody's recognition of the CLDN3 target on tumor cells.

[0119] 2. The inventors conducted tests using flow cytometry, and the results are shown in Figure 1. This further verifies that the CLDN3 antibody 1H1F3E10 prepared via hybridoma is the most effective antibody molecule for recognizing the CLDN3 target.

[0120] 3. The inventors conducted a CCK-8 assay using the antibody molecule 1H1F3E10 to study the proliferation activity of the gastric cancer cell line mkn-45. The results are shown in Figure 6. Figure 6 shows that the CLDN3 antibody molecule 1H1F3E10 at the cellular level can inhibit the proliferation of the gastric cancer cell line mkn-45, with a statistically significant difference compared to control cells (P<0.05). This indicates that the CLDN3 antibody of the present invention can effectively recognize the target antigen CLDN3 on the gastric cancer cell line mkn-45 and effectively inhibit the proliferation of mkn-45 gastric cancer cells. Since mkn-45 highly expresses CLDN3, this indicates that the CLDN3 antibody of the present invention can efficiently recognize tumor cells that highly express the CLDN3 antigen and effectively inhibit the proliferation of tumor cells.

[0121] like Figure 7 As shown, the principle of antibody action of CLDN3 obtained in this invention is as follows:

[0122] The CLDN3 antibody of this invention can utilize the strong multivalent binding ability (high affinity) of IgM to recognize a specific antigen (CLDN3) highly expressed on the surface of gastric cancer cells, blocking the key growth signaling pathway where CLDN3 is located, thereby inhibiting the growth of gastric cancer cells. Furthermore, as the antibody with the highest efficiency in activating the classical complement pathway, the binding of a single IgM molecule to the antigen surface can effectively initiate the complement cascade reaction, playing a role in inhibiting and killing tumors.

[0123] In summary, this invention utilizes a hybridoma fusion combined with single B-cell screening technology to develop an antibody sequence for CLDN3, particularly the sequence of its variable region (V region), which determines the specificity of CLDN3 antibody recognition and binding to antigens. This means that different antibody sequences can recognize and bind to different antigens, which is fundamental to the crucial role of antibodies in immune responses. Antibody sequence analysis can predict the three-dimensional structure and function of CLDN3 antibodies, such as their antigen-binding ability and the way they mediate immune responses. This contributes to a deeper understanding of the antibody's mechanism of action and allows for optimization of its performance.

[0124] As monoclonal antibody drugs have become an indispensable part of modern medicine, accurate information on the CLDN3 antibody sequence is crucial for the design and production of these drugs. Optimizing the antibody sequence can improve its specificity, affinity, and stability, thereby enhancing therapeutic efficacy and reducing side effects.

[0125] Therefore, the CLDN3 antibody sequence obtained in this invention has important significance and value in antibody structure and function, disease diagnosis and treatment, biotechnology and vaccine design, and immunological research.

[0126] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CLDN3 monoclonal antibody or fragment thereof, the antibody comprising a light chain and a heavy chain, characterized in that, The amino acid sequence of the light chain is shown as SEQ ID No: 1, and the amino acid sequence of the heavy chain is shown as SEQ ID No:

2. 2.The CLDN3 monoclonal antibody or fragment thereof of claim 1, characterized in that, The nucleotide sequence of the light chain is shown as SEQ ID No: 3, and the nucleotide sequence of the heavy chain is shown as SEQ ID No:

4.

3. A DNA molecule encoding the CLDN3 monoclonal antibody or fragment thereof according to any one of claims 1-2.

4. A vector, characterized by, A DNA molecule according to claim 3.

5. A recombinant, characterized in that, A DNA molecule according to claim 3 or a vector according to claim 4.

6. Use of the CLDN3 monoclonal antibody or fragment thereof according to any one of claims 1-2 in the preparation of a medicament for treating cancer, wherein the cancer is a cancer that overexpresses CLDN3.

7. Use according to claim 6, characterized in that, The cancer is at least one of gastric cancer, pancreatic cancer, and colorectal cancer.

8. Use according to claim 6, characterized in that, The cancer is gastric cancer.

9. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. It comprises the CLDN3 monoclonal antibody or fragment thereof according to any one of claims 1-2.

10. A pharmaceutical composition, characterized by, A medicament according to claim 9.

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