ITGA2 monoclonal antibody and application thereof

Abstract

The invention discloses an ITGA2 monoclonal antibody and application thereof, and further discloses a fragment of the antibody, a DNA molecule, a carrier, a preparation method, application and a medicine for treating cancer. The antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID No: 1, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No: 2, or the amino acid sequence of the light chain variable region is shown as SEQ ID No: 5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No: 6. The ITGA2 monoclonal antibody disclosed by the invention has high specificity, high affinity and stability, can be used for effectively recognizing a target antigen ITGA2 on a gastric cancer cell line mkn-45 and effectively inhibiting proliferation of mkn-45 gastric cancer cells, and has good biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody bioengineering, and in particular to an ITGA2 monoclonal antibody or a fragment thereof, a DNA molecule, a vector, a preparation method, an application and a drug for treating cancer. Background Art

[0002] Cellular immunotherapy is an emerging therapy that has attracted much attention in recent years in the treatment of gastric cancer, pancreatic cancer, and colorectal cancer. It mainly stimulates or mobilizes the body's immune system to enhance the anti-tumor immunity of the tumor microenvironment, thereby inhibiting and killing tumor cells. Specific immune cell therapy for gastric cancer: such as CAR-T cell therapy, which transforms T cells through genetic engineering technology so that they can specifically recognize and attack specific antigens on the surface of tumor cells. This therapy has achieved remarkable results in the treatment of hematological tumors, and is currently being explored for its application in solid tumors (including gastric cancer).

[0003] CAR-T cell immunotherapy is a personalized treatment method, which involves extracting T cells from the patient's blood and modifying them in the laboratory through genetic engineering technology to express chimeric antigen receptors (CAR) on their surface. This CAR can specifically recognize and bind to antigens on the surface of tumor cells, thereby activating T cells and killing tumor cells. After the modified CAR-T cells are amplified in large quantities, they are then infused back into the patient's body to continue to proliferate and exert anti-tumor effects.

[0004] CT041 (also known as satri-cel or satricabtagene autoleucel) is an autologous CAR-T cell therapy targeting Claudin18.2 (CLDN18.2) for the treatment of CLDN18.2-positive advanced gastrointestinal tumors, especially gastric cancer and pancreatic cancer. In addition to CT041, there are other CAR-T therapies for gastric cancer in clinical trials, such as C-CAR031 targeting GPC3, which have also shown certain efficacy and safety.

[0005] In conclusion, CAR-T therapy for gastric cancer and pancreatic cancer has made encouraging progress in some clinical trials, providing new treatment options and hope for patients with solid tumors such as gastric cancer and pancreatic cancer. However, more research and clinical data are still needed to verify its efficacy and safety.

[0006] Integrin 2 (also known as CD18 or LFA-1β) is an important member of the integrin family. It is widely involved in the connection between the extracellular matrix and the cytoskeleton and has a profound impact on various physiological and pathological processes of cells. In recent years, with the rapid development of molecular biology, cell biology and clinical medicine, integrin 2 has achieved remarkable research results in cell function regulation, tissue engineering applications, angiogenesis regulation, disease relevance, drug development, gene polymorphism, structure-function relationship and clinical trial progress.

[0007] Integrin 2 mediates tight junctions and signal transduction between cells by binding to a variety of extracellular matrix ligands, such as intercellular adhesion molecules (ICAMs), thereby regulating cell adhesion, migration, proliferation and apoptosis. Studies have shown that the activation of integrin 2 can promote cytoskeleton remodeling and enhance cell migration ability, which is particularly important in the homing of immune cells, inflammatory response and wound healing. In addition, integrin 2 also participates in the regulation of cell proliferation by affecting the expression of cell cycle regulatory factors; under certain conditions, it can also induce cell apoptosis and maintain tissue homeostasis.

[0008] Integrin 2 is closely related to the occurrence and development of many diseases. In the inflammatory response, the adhesion and migration of leukocytes mediated by integrin 2 are important steps in the inflammatory response. In the field of tumors, integrin 2 is not only involved in the adhesion and migration of tumor cells, but also affects tumor progression by regulating the behavior of immune cells in the tumor microenvironment. In addition, the abnormal expression of integrin 2 is also closely related to the occurrence and development of cardiovascular diseases, such as atherosclerosis, myocardial infarction, etc.

[0009] In the field of tumors, integrin 2 has become one of the potential targets for anti-tumor therapy. By inhibiting integrin 2-mediated tumor cell adhesion and migration, the invasion and metastasis of tumor cells can be blocked. In addition, integrin 2 is also involved in the regulation of the tumor immune microenvironment, so immunotherapy targeting integrin 2 is also being actively explored.

[0010] Through previous studies, we found that integrin 2 (ITGA2) is a membrane protein with high expression in gastric cancer and pancreatic cancer tissues, making it an ideal target for CAR-T cell therapy. CAR-T cells targeting integrin 2 can effectively kill tumor cells and inhibit the proliferation of gastric cancer cells, providing a new strategy for CAR-T cell therapy of gastric cancer and pancreatic cancer. Summary of the invention

[0011] In previous studies, the inventors found that integrin 2 (ITGA2) is a membrane protein that is highly expressed in gastric cancer and pancreatic cancer tissues. It is an ideal target for CAR-T cell therapy of gastric cancer and pancreatic cancer. Therefore, constructing an antibody against ITGA2 is the primary technical goal of CAR-T cell immunotherapy.

[0012] The present invention is made to achieve the above purpose. The present invention provides ITGA2 monoclonal antibodies or fragments thereof, DNA molecules encoding the antibodies or fragments thereof, vectors, preparation methods and applications thereof, and drugs for treating cancer comprising ITGA2 monoclonal antibodies or fragments thereof, which can improve their specificity, affinity and stability by optimizing the antibody sequence, thereby enhancing the therapeutic effect.

[0013] The first object of the present invention is to provide an ITGA2 monoclonal antibody or a fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, characterized in that the amino acid sequence of the light chain variable region is as shown in SEQ ID No: 1, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No: 2.

[0014] Preferably, the nucleotide sequence of the light chain variable region is shown as SEQ ID No: 3, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID No: 4.

[0015] The second object of the present invention is to provide an ITGA2 monoclonal antibody or a fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID No: 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 6.

[0016] Preferably, the nucleotide sequence of the light chain variable region is shown as SEQ ID No: 7, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID No: 8.

[0017] The third object of the present invention is to provide a DNA molecule encoding an ITGA2 monoclonal antibody or a fragment thereof as described in any one of the above items.

[0018] The fourth object of the present invention is to provide a vector comprising a DNA molecule as described above.

[0019] The fifth object of the present invention is to provide a method for preparing an ITGA2 monoclonal antibody or a fragment thereof as described in any one of the above items, which is obtained by screening through hybridoma cell technology.

[0020] The sixth object of the present invention is to provide a use of the antibody or fragment thereof described in any one of the above items in the preparation of a drug for treating cancer, wherein the cancer is a cancer related to high expression of ITGA2.

[0021] Preferably, the cancer includes at least gastric cancer, pancreatic cancer and colorectal cancer.

[0022] The present invention also protects a drug for treating cancer, which comprises the antibody or fragment thereof as described in any one of the above items.

[0023] Furthermore, the present invention also provides a pharmaceutical composition, which comprises the antibody or fragment thereof as described above.

[0024] The medicine of the present invention also includes pharmaceutically acceptable excipients. The medicine is an oral preparation or an injection. The injection is an injection powder or injection liquid.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] 1. In the present invention, the ITGA2 monoclonal antibody is obtained by the hybridoma fusion method, especially the sequence of its variable region (V region), which determines the specificity of the ITGA2 antibody in recognizing and binding to the antigen. The ITGA2 monoclonal antibody of the present invention has high specificity, high affinity and stability, can effectively recognize the target antigen ITGA2 on the gastric cancer cell line MKN-45, and can effectively inhibit the proliferation of MKN-45 gastric cancer cells; and has good biocompatibility, which provides an important basis for the development of antibody drugs targeting ITGA2 and CAR-T cell immunotherapy methods.

[0027] 2. The ITGA2 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 immunology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0029] Figure 1 The figure is a non-reducing SDS-PAGE electrophoresis detection diagram of the purified antibody in the example.

[0030] Figure 2 The figure is a reduction SDS-PAGE electrophoresis detection diagram of the purified antibody in the example.

[0031] Figure 3 This is a schematic diagram of the high-definition magnification results of the purified ITGA2 antibody M05 after fluorescent labeling.

[0032] Figure 4This is a schematic diagram of the high-definition magnification results of the purified ITGA2 antibody M06 after fluorescent labeling.

[0033] Figure 5 This is a schematic diagram of the results of flow cytometry detection of the obtained antibody molecules.

[0034] Figure 6 Schematic representation of the cell proliferation results obtained from the CCK-8 assay for antibody molecules M05 and M06.

[0035] Figure 7 Schematic diagram of the cell adhesion experiment results of ITGA2 antibodies M05 and M06.

[0036] Figure 8 The diagram is a schematic diagram of the principle of using a drug containing the ITGA2 antibody obtained in the present invention to treat cancer. DETAILED DESCRIPTION

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0038] The present invention provides ITGA2 monoclonal antibodies, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art.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 common L-amino acids.

[0040] "Antibody" refers to a protein composed of one or more polypeptides that can specifically bind to an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer, which is composed of two identical pairs of antibody chains, each pair has a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light chain and the heavy chain are jointly responsible for binding to the antigen, while the constant region is responsible for the effector function of the antibody.

[0041] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0042] The present invention will be further described below in conjunction with embodiments:

[0043] 1. Embodiment 1:

[0044] This example provides a method for preparing an ITGA2 monoclonal antibody, which is a monoclonal antibody specifically binding to human integrin protein ITGA2 obtained by screening using hybridoma cell technology. The antibody has high specificity, high affinity and stability.

[0045] The preparation method is as follows:

[0046] 1. Animal immunization

[0047] Five female Balb / c mice aged 6-8 weeks were immunized, with a single immunization dose of 50 μg for each mouse. For the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to make an emulsion, and multiple subcutaneous injections were made in the abdomen. After an interval of 2 weeks, the same dose of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to make an emulsion, and multiple subcutaneous injections were made in the abdomen. Blood was collected one week after the three immunizations, and the serum titer was determined by indirect ELISA. When the protein was diluted 1:16000 times with mouse serum, OD450-Blank greater than 1.0 was the qualified titer standard. Among the animals with qualified titers, animals with high ElISA titers were preferably used for shock immunization. After 3-4 days of shock immunization, the mouse spleen was taken for hybridoma fusion. Mice with unqualified titers continued to be immunized until the titer was qualified, and a maximum of five immunizations were performed.

[0048] 2. Blood collection and titer testing

[0049] Serum collection: One week after the last immunization, 50-60 μL of blood was collected from the mouse orbital venous plexus. After standing at 4°C overnight, the blood was centrifuged at 4000 rpm and 4°C for 15 minutes to separate the upper serum for testing.

[0050] 3. ELISA binding titer detection

[0051] The serum titer was tested by indirect ELISA method, and the binding of antiserum to immunogen was tested at the same time. The protein titer was qualified when OD450-Blank was greater than 1.0 when the mouse serum was diluted 1:16000. Mice with high titer were preferred for hybridoma fusion.

[0052] The detection steps are as follows.

[0053] (1) Coating: Take an appropriate amount of the detection protein, dissolve the immunogen with coating buffer and dilute to 5 μg / mL, then add 100 μL to each well of a 96-well plate with a pipette, tap the plate to mix the sample, seal it with plastic wrap, and coat overnight at 4°C;

[0054] (2) Washing: Wash the plate once with washing solution at 300 μL / well and dry the ELISA plate;

[0055] (3) Blocking: Block the ELISA plate with blocking solution at 300 μL / well for 1 h at room temperature;

[0056] (4) Washing: Wash the plate twice with washing solution at 300 μL / well and dry the ELISA plate;

[0057] (5) Sample loading: Add the serially diluted serum sample and sample diluent at 100 μL / well;

[0058] (6) Adding secondary antibody: Add detection antibody at 100 μL / well to a 96-well plate and incubate at room temperature for 2 h;

[0059] (7) Washing: Wash the plate five times with washing solution at 300 μL / well and dry the ELISA plate;

[0060] (8) Color development: Add 200 μL / well of color development solution and incubate at room temperature for 12 min;

[0061] (9) Termination and detection: 50 μL / well of stop solution was added to terminate the reaction, and then the reaction was detected using an ELISA reader at a wavelength of 450 nm.

[0062] 4. Mouse monoclonal antibody fusion and screening

[0063] The electrofusion method was used to fuse mouse spleen cells and myeloma cells. The indirect ELISA method was used to screen and obtain

[0064] The positive main clone cells that bind to the antigen protein. The main clone undergoes 2-3 consecutive rounds of limiting dilution and corresponding screening until a stable monoclonal cell line is obtained.

[0065] 5. Hybridoma cell fusion

[0066] Take the spleen cells of immunized mice, mix them with mouse myeloma cells at a ratio of 1:1, and fuse them using the electrofusion method to obtain hybridoma cells. Use HAT selection medium to change the cell medium once on the 6th and 8th day after fusion; take the supernatant of the main clone stage cells for screening and detection on the 10th day after cell fusion.

[0067] 6. Hybridoma cell screening

[0068] ELISA binding screening: The hybridoma cell culture supernatant is tested by indirect ELISA, and the clones that bind to the antigen are retained. The detection process is as follows.

[0069] (1) Coating: Take an appropriate amount of the detection protein, dissolve it in coating buffer and dilute it to 1 μg / mL, then add 100 μL to each well of a 96-well plate with a pipette, tap the plate to mix the sample, seal it with plastic wrap, and coat it overnight at 4°C;

[0070] (2) Blocking: Shake off the solution and block the ELISA plate with blocking solution at 300 μL / well for 1 h at room temperature;

[0071] (3) Sample loading: Shake off the liquid and dry the ELISA plate; add 50 μL / well of hybridoma cell supernatant and leave at room temperature for 1 h;

[0072] (4) Washing: Wash the plate once with washing solution at 300 μL / well and dry the ELISA plate;

[0073] (5) Adding secondary antibody: Add detection antibody at 100 μL / well to a 96-well plate and incubate at room temperature for 2 h;

[0074] (6) Washing: Wash the plate 5 times with washing solution at 300 μL / well and dry the plate;

[0075] (7) Color development: Add 200 μL / well of color development solution and leave at room temperature for 16 min for the first screening and 8 min for the second screening and subcloning;

[0076] (8) Termination and detection: 50 μL / well of stop solution was added to terminate the reaction, and then the reaction was detected using an ELISA reader at a wavelength of 450 nm.

[0077] 7. Limiting dilution and subclone screening

[0078] Use HT selection medium to inoculate each positive hybridoma cell obtained by screening in half a 96-well cell culture plate at a seeding density of 0.75 cells per well, and culture statically under 8% CO2 and 37°C. Take the supernatant for indirect ELISA test about the 7th day after culture to confirm that the antibody to be tested binds positively to the antigen. For the specific detection process, refer to the main clone indirect ELISA detection process 3.2.2. Select ELISA specific binding positive monoclonal cells and perform 1-2 rounds of limiting dilution again until stable, positive monoclonal cells are obtained.

[0079] 8. Hybridoma cell culture

[0080] Transfer 1 mL of hybridoma cells into a 100 mL culture flask, add a certain amount of culture medium regularly to expand the cells until the cells reach the required production volume. Then add hybridoma feed solution, feed every other day. Collect the feed after 6-8 days of culture.

[0081] 9. Monoclonal antibody purification

[0082] Sample preparation: The collected hybridoma cell supernatant was centrifuged using a desktop centrifuge at 4000 g for 30 min, and the cell supernatant after centrifugation was collected; the cell supernatant after centrifugation was filtered using a 0.45 μM filter membrane.

[0083] Antibody purification: Protein A affinity purification was used to purify the hybridoma cell culture supernatant after centrifugation to harvest mouse monoclonal antibodies.

[0084] 10. Mouse monoclonal antibody detection

[0085] (1) Conduct antibody concentration and purity testing;

[0086] (2) ELISA binding detection: Use the indirect ELISA method to detect the binding of purified mouse monoclonal antibodies to the immunogen.

[0087] 2. Test results:

[0088] 1. Purify the monoclonal antibody, and perform non-reducing SDS-PAGE electrophoresis on the purified antibody. The results are shown in Table 1 and Figure 1 As shown in . Figure 1 The SDS-PAGE test results in the figure show that there is a band in the lane corresponding to each product, and there are no other miscellaneous bands, which can indicate that the total molecular weight of the purified antibody product is around 160 and 180 kDa, and the purity is relatively high.

[0089] Table 1

[0090] Serial number Product Name Molecular weight MW(kDa) 1 Marker (molecular weight marker) 2 LZE2-2-MM05 HB18JA2405 182.21 3 LZE2-2-MM06 HB18JA2406 184.22 4 Marker 5 LZE2-2-MM04 HB18JA2505 168.00 6 Marker 7 LZE2-2-MM03 HB18JA3009 164.63 8 Marker 9 LZE2-2-MM01 HB18JA3119 146.85

[0091] 2. The monoclonal antibody was purified and the purified antibody was subjected to reducing SDS-PAGE electrophoresis detection. The results are shown in Table 2 and Figure 2 As shown in . Figure 2 The SDS-PAGE test results in the results showed that the corresponding lanes had a band at 50kDa and 25kDa respectively, which were divided into heavy chain and light chain, and the purity of both reached more than 95%.

[0092] Table 2

[0093]

[0094]

[0095] 3. The purified antibodies were subjected to ELISA binding test. The results are shown in Table 3.

[0096] Table 3

[0097]

[0098] From Tables 1, 2, 3 and Figure 1 , 2It can be seen that the protein Human ITGA2 was used to immunize 5 mice. After five immunizations, the ELISA binding titer of 2 mice with the immunogen reached the fusion standard. After one hybridoma fusion, a total of 6 hybridoma cells that bound to the antigen protein were obtained. Among them, the total amount of antibodies after purification of 5 mouse monoclonal antibodies met the requirements, with a purity of >90%; ELISA detection showed that all 5 purified antibodies bound to the antigen protein.

[0099] 4. Antibody Sequencing:

[0100] The inventors extracted and sequenced the antibody genes from the above two hybridoma cells to obtain the antibody heavy and light chain gene sequences.

[0101] (1) Extract RNA after hybridoma cell lysis;

[0102] (2) Reverse transcription: Using the extracted RNA as a template, the cDNA was obtained in two steps using the Sino-Biotech reverse transcription kit (by first opening the RNA secondary structure and then allowing the primer to bind to the RNA and polymerize the cDNA to achieve the purpose of obtaining cDNA);

[0103] (3) Using the cDNA obtained by reverse transcription as a template, amplify the fragment by PCR, insert it into an expression vector or commercial vector, and sequence it to obtain a plasmid containing the correct sequence.

[0104] The sequencing results are as follows:

[0105] (1) ITGA2-MM05 light chain: ITGA2-MM05-PL (L20240313-1-2, as numbering) full-length amino acid sequence is shown in SEQ ID No: 1:

[0106] MNLPVHLLVLLLFWIPGSRGDVVVTQTPLSLPVSFGDQVSISCRSSQSLAKSSGKTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGSHQP WTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0107] (2) ITGA2-MM05 heavy chain: ITGA2-MM05-PH (L20240313-2-1, as numbering) full-length amino acid sequence is shown in SEQ ID No: 2:

[0108] MYLGLSCVFIVFLLKGVQCEVKLDETGGGLVQPGRPMKLSCVASGFTFSAYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRAEDMGIYYCTTMDYWGQGTSVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK。

[0109] (3) The full-length nucleotide sequence of ITGA2-MM05 light chain ITGA2-MM05-PL (L20240313-1-2, as the number) is shown in SEQ ID No: 3:

[0110] ATGAATTTGCCTGTTCATCTCTTGGTGCTTCTGTTGTTCTGGATTCCTGGTTCCAGAGGTGATGTTGTGGTGACTCAAACTCCACTCTCCCTGCCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGCAAAAAGTTCTGGGAAGACCTATTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCACAGCTCCTCATCTATGGGATTTCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTTCA CATCAGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

[0111] (4) The full-length nucleotide sequence of the ITGA2-MM05 heavy chain ITGA2-MM05-PH (L20240313-2-1, as the serial number) is shown in SEQ ID No: 4:

[0112] ATGTACTTGGGACTGAGCTGTGTATTCATTGTTTTTCTCTTAAAAGGTGTCCAGTGTGAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTTAGTGCCTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGAAACAAACCTTATAATTATGAAACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACATGGGTATCTATTACTGTACAACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCCGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATATAGCAAGCTCAATATGAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTAC CTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATAA。

[0113] (5) The full-length amino acid sequence of the ITGA2-MM06 light chain ITGA2-MM06-PL (L20240313-3-2, as the number) is shown in SEQ ID No: 5:

[0114] MNLPVHLLVLLLFWIPGSRGDVVVTQTPLSLPVSFGDQVSISCRSSQSLAKSYGKTYLSWYLHKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGSHQPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0115] (6) The full-length amino acid sequence of the ITGA2-MM06 heavy chain ITGA2-MM06-PH (L20240313-4-1, as the number) is shown in SEQ ID No: 6:

[0116] MYLGLSCVFIVVLLKGVQCEVKLDETGGGLVQPGRPMKLSCVASGLTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRAEDMGIYYCTTMDYWGQGTSVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK.

[0117] (7) The full-length nucleotide sequence of the ITGA2-MM06 light chain ITGA2-MM06-PL (L20240313-3-2, as the serial number) is shown in SEQ ID No: 7:

[0118] ATGAATTTGCCTGTTCATCTCTTGGTGCTTCTGTTGTTCTGGATTCCTGGTTCCAGAGGTGATGTTGTGGTGACTCAAACTCCACTCTCCCTGCCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGCAAAGAGTTATGGGAAGACCTATTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCCCCTCAGCTCCTCATCTATGGGATTTCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGTTTACAAGGTTCACATCAGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

[0119] (8) The full-length nucleotide sequence of the ITGA2-MM06 heavy chain ITGA2-MM06-PH (L20240313-4-1, as the serial number) is shown in SEQ ID No: 8:

[0120] ATGTACTTGGGACTGAGCTGTGTATTCATTGTTGTTCTCTTAAAAGGTGTCCAGTGTGAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTGTGTTGCCTCTGGACTCACTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGAAACAAACCTTATAATTATGAAACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACATGGGTATCTATTACTGTACAACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCCGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATATAGCAAGCTCAATATGAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTAC CTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATAA。

[0121] III. Application

[0122] 1. The inventors fluorescently labeled the ITGA2 antibodies M05 and M06 obtained, and the results were as follows: Figure 3 , 4 As shown in . And confocal localization was well verified that the antibody recognized the target ITGA2 on tumor cells.

[0123] 2. The inventors conducted a flow cytometer test, and the results were as follows: Figure 5 As shown in , it was once again verified that ITGA2 antibody M05 and ITGA2 antibody M06 produced by hybridoma were the most effective antibody molecules for recognizing ITGA2 targets.

[0124] 3. The inventors conducted CCK-8 experiments using antibody molecules M05 and M06 to study the proliferation activity of gastric cancer cell line mkn-45. The results are as follows: Figure 6 As shown in Figure 6 It can be seen that at the cellular level, the two antibody molecules M05 and M06 of ITGA2 can inhibit the proliferation of the gastric cancer cell line MKN-45, which is statistically significant compared with the control cells, P<0.05. This shows that the ITGA2 antibody of the present invention can effectively recognize the target antigen ITGA2 on the gastric cancer cell line MKN-45, and can effectively inhibit the proliferation of MKN-45 gastric cancer cells. Since MKN45 highly expresses ITGA2, it shows that the ITGA2 antibody of the present invention can efficiently recognize tumor cells that highly express the ITGA2 antigen and effectively inhibit the proliferation of tumor cells.

[0125] 4. The inventors also conducted cell adhesion experiments to study the biocompatibility of ITGA2 antibodies M05 and M06. Figure 7 As shown, it can be seen that the ITGA2 antibodies M05 and M06 have good biocompatibility and can inhibit the spread of tumor cells, providing an important basis for the development of antibody drugs targeting ITGA2 and CAR-T cell immunotherapy methods.

[0126] like Figure 8 As shown in , the principle of the ITGA2 antibody obtained by the present invention is:

[0127] The ITGA2 antibody of the present invention can use genetic engineering technology to equip T cells with a special molecule called a chimeric antigen receptor (CAR). This CAR molecule can recognize specific antigens (ITGA2) on the surface of gastric cancer cells. The modified CAR-T cells are amplified in vitro and then re-injected into the patient. When the CAR-T cells are re-infused into the patient, they can quickly and specifically recognize and bind to gastric cancer cells. Once bound, the CAR-T cells will activate a series of immune responses, release cytotoxic substances, directly kill gastric cancer cells, and induce their apoptosis.

[0128] In summary, the antibody sequence of ITGA2 invented by the hybridoma fusion method in the present invention, especially the sequence of its variable region (V region), determines the specificity of ITGA2 antibody recognition and binding to antigen. This means that different antibody sequences can recognize and bind to different antigens, which is the basis for antibodies to play a key role in immune response. Through antibody sequence analysis, the three-dimensional structure and function of ITGA2 antibodies, such as the ability to bind to antigens, the way to mediate immune responses, etc., can be predicted. This helps to deeply understand the mechanism of action of antibodies and optimize their performance.

[0129] Since monoclonal antibody drugs have become an indispensable part of modern medicine, accurate information on the ITGA2 antibody sequence is essential for the design and production of these drugs. By optimizing the antibody sequence, its specificity, affinity, and stability can be improved, thereby enhancing the therapeutic effect and reducing side effects. By analyzing the response of ITGA2 antibodies to the target, the best antigen epitope can be selected as the target of CAR-T. This will help develop more effective and safe CAR-T treatments and improve people's immunity to diseases.

[0130] Therefore, the ITGA2 antibody sequence obtained by the present invention has important significance and value in antibody structure and function, disease diagnosis and treatment, biotechnology and vaccine design, and immunology research.

[0131] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods 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 in the scope of protection of the present invention.

Claims

1. An ITGA2 monoclonal antibody or a fragment thereof, the antibody comprising a light chain variable region and a heavy chain variable region, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID No: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

2.

2. An ITGA2 monoclonal antibody or a fragment thereof according to claim 1, characterized in that: The nucleotide sequence of the light chain variable region is shown in SEQ ID No: 3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

4.

3. An ITGA2 monoclonal antibody or a fragment thereof, the antibody comprising a light chain variable region and a heavy chain variable region, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID No: 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

6.

4. An ITGA2 monoclonal antibody or a fragment thereof according to claim 3, characterized in that: The nucleotide sequence of the light chain variable region is shown in SEQ ID No: 7, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No:

8.

5. A DNA molecule encoding an ITGA2 monoclonal antibody or a fragment thereof according to any one of claims 1 to 4.

6. A vector comprising a DNA molecule according to claim 5.

7. The method for preparing an ITGA2 monoclonal antibody or a fragment thereof according to any one of claims 1 to 4, characterized in that: Obtained through hybridoma cell screening.

8. Use of the antibody or fragment thereof according to any one of claims 1 to 4 in the preparation of a drug for treating cancer, wherein the cancer is a cancer related to high expression of ITGA2.

9. The use according to claim 8, wherein the cancer at least includes gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, and glioma.

10. A drug for treating cancer, characterized in that: The invention comprises the antibody or fragment thereof according to any one of claims 1 to 4.

Images