Application of BTN2A2 as immune checkpoint in preparation of anti-tumor product

Abstract

The invention belongs to the technical field of biomedical technology, and particularly relates to application of BTN2A2 as an immune checkpoint in preparation of an anti-tumor product. The BTN2A2 is taken as an immune checkpoint, the BTN2A2 is found to have up-regulated expression in various tumor cells (including pancreatic cancer and glioma) and APC cells, and the BTN2A2 is combined with corresponding receptors on T cells to inhibit proliferation and activation of the T cells, so that the T cells are in an inactivated state, and finally immune escape is induced. By preparing the anti-BTN2A2 monoclonal antibody, the combination of BTN2A2 and a receptor can be blocked, the growth and proliferation of T cells can be up-regulated, the recognition of the T cells on tumor cells can be enhanced, the attacking and killing functions of the T cells can be activated, and the anti-tumor effect can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical technologies, and particularly relates to the application of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products. Background Art

[0002] In recent years, the incidence and death toll of cancer have been on the rise year by year, and it has increasingly become one of the diseases seriously affecting human health. Surgical treatment, radiotherapy and chemotherapy are traditional treatment modalities for tumors, but they have certain limitations due to high recurrence rates and serious complications. In recent years, tumor immunotherapy has achieved remarkable achievements, with advantages such as low recurrence, low toxicity and high specificity.

[0003] The development of immune checkpoint agonists or inhibitors is becoming a promising method for treating tumors. Immune checkpoint (ICP) molecules refer to ligand-receptor pairs that play inhibitory or stimulatory roles in immune responses. Immune checkpoints play an important immunomodulatory role in maintaining immune homeostasis and preventing autoimmunity. Most of the reported immune checkpoint proteins are expressed in cells of the adaptive immune system, especially T cells and the innate immune system. Some tumor cells limit normal anti-tumor immune responses by binding to co-inhibitory receptor molecules, thus helping immune escape. The immune checkpoint treatment strategy for tumors includes targeting these regulatory pathways to restore the anti-tumor function of immune cells. Many new therapeutic targets have been discovered, and among them, the more deeply studied one is still PD-L1 / PD-1, but there are still many deficiencies in its clinical application. Due to the expression differences of PD-L1 / PD-1 in different disease states and different individuals, the curative effects are not uniform, and not all tumor patients can benefit from it. The highest anti-tumor immune curative effect is only about 30%. In addition, there is a lack of biomarkers related to tumor prognosis evaluation. Although the only FDA-approved PD-L1 expression has relatively high accuracy, its application is also limited due to different definition methods. Therefore, it is particularly important to search for new immune checkpoints and develop immune checkpoint inhibitors.

[0004] Butyrophilin (BTN) 2A2 belongs to the extended B7 family of molecules and has similar structural characteristics to PD-L1. It can inhibit the activation and proliferation of T cell functions, thereby regulating the development of autoimmune diseases and inflammatory diseases. Currently, there are reports on the use of mBTN2A2-Ig fusion protein to improve murine collagen-induced arthritis (CIA) and autoimmune encephalomyelitis (EAE), but there are no related reports on the application of BTN2A2 in the preparation of anti-tumor drugs and tumor treatment. Summary of the Invention

[0005] The object of the present invention is to make up for the deficiencies of the prior art, and provide the application of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products, enrich the treatment targets of tumors and the application directions of BTN2A2, and provide a new direction for the treatment of tumors.

[0006] The present invention provides the application of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products.

[0007] Preferably, the amino acid sequence encoded by the BTN2A2 is as shown in SEQ ID NO.1.

[0008] Preferably, the product includes a drug; the drug includes an anti-BTN2A2 antibody.

[0009] Preferably, the tumor includes glioma and / or pancreatic cancer.

[0010] The present invention also provides the light chain variable region and the heavy chain variable region of an anti-BTN2A2 antibody, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3;

[0011] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.4.

[0012] The present invention also provides a DNA molecule encoding the light chain variable region and the heavy chain variable region described in the above technical solution.

[0013] The present invention also provides the application of the light chain variable region, the heavy chain variable region or the DNA molecule in the preparation of an anti-BTN2A2 antibody.

[0014] The present invention also provides an anti-BTN2A2 antibody, and the anti-BTN2A2 antibody includes the light chain variable region and the heavy chain variable region described in the above technical solution.

[0015] The present invention also provides a preparation method of the anti-BTN2A2 antibody described in the above technical solution, including the following steps:

[0016] Connect the nucleotide sequences encoding the light chain variable region and the heavy chain variable region to a basic vector respectively to obtain a light chain recombinant vector and a heavy chain recombinant vector;

[0017] Co-express the light chain recombinant vector and the heavy chain recombinant vector to obtain the anti-BTN2A2 antibody;

[0018] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3;

[0019] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.4.

[0020] The present invention also provides an anti-tumor drug, which comprises an excipient and an immune checkpoint inhibitor;

[0021] The immune checkpoint inhibitor is the anti-BTN2A2 antibody described in the above technical solution.

[0022] Beneficial effects:

[0023] In the present invention, BTN2A2 is used as an immune checkpoint, and it is found that BTN2A2 is up-regulated in various tumor cells (including pancreatic cancer and glioma) and APC cells. It binds to the corresponding receptor on T cells, inhibits the proliferation and activation of T cells, makes T cells in an inactivated state, and finally induces immune escape. By preparing an anti-BTN2A2 monoclonal antibody, the binding of BTN2A2 to the receptor can be blocked, the growth and proliferation of T cells can be up-regulated, the recognition of tumor cells by T cells can be enhanced, and its attack and killing functions can be activated, thereby exerting an anti-tumor effect. Description of the drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0025] Figure 1 Results of analyzing the expression differences of BTN2A2 in human tumors and normal tissues by TGGA and GTEs databases;

[0026] Figure 2 Results of analyzing the expression differences of BTN2A2 in human pancreatic cancer (PADD), glioma (GBM) and normal tissues by TGGA and GTEs databases;

[0027] Figure 3 Expression and survival analysis of BTN2A2 in human pancreatic cancer (PADD);

[0028] Figure 4 Expression and survival analysis of BTN2A2 in human glioma (GBM);

[0029] Figure 5 Results of pan-cancer immunohistochemical analysis in Example 2;

[0030] Figure 6 Results of detecting the expression of BTN2A2 by flow cytometry on the cell membrane and intracellularly in pan-cancer cells in Example 2;

[0031] Figure 7 Statistical results of the expression of BTN2A2 on the cell membrane and intracellularly in pan-cancer cells in Example 2; among them, C is the statistical result of the expression on the cell membrane; D is the statistical result of the intracellular expression of BTN2A2;

[0032] Figure 8Results of the preparation of hBTN2A2-Ig fusion protein and in vitro functional verification in Example 3; where A shows the verification results of the purified hBTN2A2-Ig fusion protein by agarose gel electrophoresis, Coomassie Brilliant Blue, and Western blot; B shows the flow cytometry detection results of the differentiation of mouse CD4 and CD8 T memory cells by Control Ig and hBTN2A2-Ig proteins; C shows the quantitative detection results of the differentiation of mouse CD4 and CD8 T memory cells by Control Ig and hBTN2A2-Ig proteins; D shows the flow cytometry detection results of the differentiation of mouse CD4 and CD8 T memory cells by different concentrations of Control Ig and hBTN2A2-Ig proteins; E shows the quantitative detection results of the differentiation of mouse CD4 and CD8 T memory cells by different concentrations of Control Ig and hBTN2A2-Ig proteins; F shows the flow cytometry detection results of the CFSE and Ki67 proliferation indexes of mouse T cells by Control Ig and hBTN2A2-Ig proteins; G shows the quantitative results of the CFSE and Ki67 proliferation indexes of mouse T cells by Control Ig and hBTN2A2-Ig proteins;

[0033] Figure 9 Results of the preparation and functional verification of the anti-hBTN2A2 monoclonal antibody in Example 4; where A shows the results of PCR amplification of the variable regions of the heavy and light chains of antibodies from mouse B cells immunized with hBTN2A2-Ig, and the red box indicates the B cells that simultaneously amplified the heavy and light chains of anti-hBTN2A2 by PCR; B shows the anti-hBTN2A2 Ab typing, the binding specificity identified by Western Blot with hBTN2A2-Ig protein and Ig protein, and the Coomassie Brilliant Blue staining results; C shows the flow cytometry detection results in Step 3; D-E show the flow cytometry detection results in Step 4; F-G show the flow cytometry detection results in Step 5;

[0034] Figure 10 Effects of anti-hBTN2A2 Ab on mouse pancreatic cancer symptoms and immune cell infiltration in Example 5; where A shows the size of pancreatic cancer tumors; B shows the change in the volume of pancreatic cancer tumors; C-D show the flow cytometry detection results;

[0035] Figure 11 Effects of anti-hBTN2A2 Ab on immune cell infiltration in mouse pancreatic cancer in Example 5; where A shows the HE staining results; B-C show the immunofluorescence staining results of CD4 and CD8; D-E show the flow cytometry detection results (tumors) in Step 5;

[0036] Figure 12Flow cytometry detection results (tumor) for step 6 of Example 5; wherein, A and B are the classification results of memory T cells in mouse pancreatic cancer; C and D are the M1 / M2 classification results of macrophages in mouse pancreatic cancer; E and F are the proportions of MDSCs in mouse pancreatic cancer; G and H are the proportions of Tregs in mouse pancreatic cancer;

[0037] Figure 13 Flow cytometry detection results (tumor) for step 7 of Example 5; wherein, A and B are the proportions of cytokines TNF-α secreted by CD4 and CD8 cells in mouse pancreatic cancer; C and D are the proportions of IL-17 and IFN-γ; E and F are the proportions of IL-4 and IL-2;

[0038] Figure 14 Flow cytometry detection results (spleen) for step 8 of Example 5; wherein, A and B are the M1 / M2 classification results of macrophages; C is the proportion of MDSCs; D is the proportion of Tregs; E is the CD69+ activation of CD4 and CD8 T cells; F is the ki67+ proliferation; G is the proportion of cytokine TNF-α; H is the proportion of cytokine IFN-γ; I is the proportion of IL-4; J is the proportion of IL-2;

[0039] Figure 15 Effect of Anti-hBTN2A2 Ab on the symptoms and immune cell infiltration of glioma in mice; wherein, A is the size of glioma tumor; B is the change in glioma tumor volume; C is the flow cytometry detection result of step 2; D is the HE staining result; E-F are the detection results of CD4 and CD8 T cell infiltration in two groups of gliomas by immunofluorescence detection;

[0040] Figure 16 Flow cytometry detection results (tumor) for step 5 of Example 6; wherein, A and B are the Ki67 proliferation of CD4 and CD8 T cells in mouse tumors; C and D are the infiltration proportions of B, DC, and macrophages in gliomas; E and F are the proportions of T cells; G and H are the proportions of MDSCs;

[0041] Figure 17 Flow cytometry detection results (spleen) for step 6 of Example 6; wherein, A is memory T cells; B is Tregs; C and D are the M1 / M2 classification proportions of macrophages; E and F are the CD69+ activation, infiltration of B, DC, macrophages, and TNF-α secretion proportions of CD4 and CD8 T cells; G-J are the ki67+ and CFSE proliferation of CD4 and CD8 T cells; K-L are the proportions of cytokines IFN-γ secreted by CD4 and CD8 T cells. Detailed implementation methods

[0042] The present invention provides the application of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products.

[0043] In the present invention, the amino acid sequence encoded by BTN2A2 is preferably as shown in SEQ ID NO.1; the nucleotide sequence of BTN2A2 is preferably as shown in SEQ ID NO.2. The specific sequence information of SEQ ID NOs.1-2 in the present invention is as follows:

[0044] SEQ ID NO.1: QFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFMPSASP;

[0045] SEQ ID NO.2: 5'-cagtttactgtcgtggggccagctaatcccatcctggccatggtgggagaaaacactacattacgctgccatct gtcacccgagaaaaatgctgaggacatggaggtgcggtggttccggtctcagttctcccccgcagtgtttgtgtataagggtgggagagagagaacagaggagcagatggaggagtaccggggaagaatcacctttgtgagcaaagacatcaacaggggcagcgtggccctggtcatacataacgtcacagcccaggagaatgggatctaccgctgttacttccaagaaggcaggtcctacgatgaggccatcctacgcctcgtggtggcaggccttgggtctaagcccctcattgaaatcaaggcccaagaggatgggagcatctggctggagtgcatatctggagggtggtacccagagcccctcacagtgtggagggacccctacggtgaggttgtgcccgccctgaaggaggtttccatcgctgatgctgacggcctcttcatggtcaccacagctgtgatcatcagagacaagtatgtgaggaatgtgtcctgctctgtcaacaacaccctgctcggccaggagaaggaaactgtcatttttattccagaatcctttatgcccagcgcatctccc-3'.

[0046] In the present invention, the product preferably includes a drug; the drug preferably includes an antibody against BTN2A2. The tumor in the present invention preferably includes glioma and / or pancreatic cancer.

[0047] BTN2A2 belongs to the extended B7 family of molecules, has similar structural characteristics to PD-L1, can inhibit the activation and proliferation of T cell functions, thereby regulating the development of autoimmune and inflammatory diseases. Existing technologies usually use it to improve collagen-induced arthritis (CIA) and autoimmune encephalomyelitis (EAE). The present invention creatively uses BTN2A2 as an immune checkpoint. Experiments have found that BTN2A2 is upregulated in a variety of tumor cells and APC cells. It binds to the corresponding receptor on T cells, inhibits T cell proliferation and activation, renders T cells in an inactivated state, enabling tumor cells to achieve immune escape. Preparing anti-BTN2A2 monoclonal antibodies can block the binding of BTN2A2 to the receptor, upregulate the growth and proliferation of T cells, enhance the recognition of tumor cells by T cells, activate their attack and killing functions, and play an anti-tumor role, enriching the treatment targets for tumors and the application directions of BTN2A2, providing a new direction for the treatment of tumors.

[0048] The present invention provides a light chain variable region and a heavy chain variable region of an anti-BTN2A2 antibody, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3;

[0049] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.4.

[0050] In the present invention, the nucleotide sequence encoding the light chain variable region is preferably as shown in SEQ ID NO.5; the nucleotide sequence encoding the heavy chain variable region is preferably as shown in SEQ ID NO.6.

[0051] The specific sequence information of SEQ ID NOs.3 - 6 in the present invention is as follows:

[0052] SEQ ID NO.3: MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQN VGTYVAWYQQKPGQSPEALIYSASYRYSGVPYRFAGSGSGTEFTLTISNVQSEDLAEYFCQQYI SYPYTFGGGTKLEIK;

[0053] SEQ ID NO.4: MNFGLSLIFLVLVLKGVQCEVQLVESGGVLVKPGGSLKLSCTASGFIFSD

[0054] YYIHWVRQTPEKRLEWVATISDGGGYTHYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCVRDSTRSWGQGTLVTVSA;

[0055] SEQ ID NO.5: 5'-ATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTC TGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTTATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTGAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTTATCGCTTCGCAGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAACAATATATCAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA-3';

[0056] SEQ ID NO.6: 5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGG TGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGTCTTAGTGAAGCCTGGAGGGTCCCTAAAACTGTCCTGTACAGCCTCTGGATTCATTTTCAGTGACTATTACATACATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTGGTTACACCCACTATCCAGACAGTGTGAAGGGGCGATTTACCATCTCCAGAGACAATGCCAAGAACAACCTATACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGTAAGAGATAGTACCCGCAGTTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA-3'.

[0057] The present invention also provides a DNA molecule encoding the light chain variable region and the heavy chain variable region described in the above technical solution.

[0058] The present invention also provides the use of the light chain variable region and the heavy chain variable region or the DNA molecule in the preparation of an anti-BTN2A2 antibody.

[0059] The present invention also provides an anti-BTN2A2 antibody, comprising the light chain variable region and the heavy chain variable region described in the above technical solution. The preparation method of the anti-BTN2A2 antibody of the present invention preferably comprises the following steps:

[0060] Connecting the nucleotide sequences encoding the light chain variable region and the heavy chain variable region to the basic vector respectively to obtain a light chain recombinant vector and a heavy chain recombinant vector;

[0061] The light chain recombinant vector and the heavy chain recombinant vector are co-expressed to obtain the anti-BTN2A2 antibody.

[0062] The present invention connects the nucleotide sequences of the light chain variable region and the heavy chain variable region to the basic vector respectively to obtain a light chain recombinant vector and a heavy chain recombinant vector. In the present invention, the basic vector preferably includes pCDNA3.4 (with an Fc segment, i.e., an Ig segment). The present invention has no strict requirements on the method of connection, and the conventional method in the art can be used.

[0063] After obtaining the light chain recombinant vector and the heavy chain recombinant vector, the present invention co-expresses the light chain recombinant vector and the heavy chain recombinant vector to obtain the anti-BTN2A2 antibody. In the present invention, the co-expression method includes: co-transfecting the light chain recombinant vector and the heavy chain recombinant vector into cells and then culturing, using cells to simultaneously express heavy chains and light chains, and assembling them into a complete antibody structure and releasing them into the culture medium. The cells of the present invention preferably include HEK-293F cells. The present invention preferably purifies the antibodies released into the culture medium to obtain the anti-BTN2A2 antibody.

[0064] The anti-BTN2A2 antibody provided by the present invention can block the binding of BTN2A2 to the receptor, thereby causing T cells to proliferate in large quantities and attack tumor cells, thereby playing an anti-tumor role, and is particularly capable of treating gliomas and pancreatic cancer. The results of the embodiment show that after pancreatic cancer is treated with anti-BTN2A2 antibodies, the growth rate of pancreatic cancer can be reduced, and the infiltration of lymphocytes in the tumor microenvironment is increased, and the secretion of cytokines TNF-α, IFN-γ, IL-17, IL-4, and IL-2 by B cells, macrophages, CD8 effector T cells, M1 macrophages, and T cells in the tumor microenvironment is upregulated; M2 macrophages, Treg, and MDSC are downregulated; after glioma is treated with anti-BTN2A2 antibodies, the growth rate of glioma can be reduced, and the infiltration of lymphocytes in the tumor microenvironment is increased, and the secretion of cytokines TNF-α, IFN-γ, IL-17, IL-4, and IL-2 by B cells, macrophages, CD8 effector T cells, and T cells in the tumor microenvironment is upregulated; MDSC cells are downregulated.

[0065] The present invention also provides an anti-tumor drug, which comprises an excipient and an immune checkpoint inhibitor; the immune checkpoint inhibitor is an anti-BTN2A2 antibody obtained by the preparation method. The present invention has no strict requirements on the type of the excipient, and it can be conventionally selected according to the dosage form of the drug.

[0066] To further illustrate the present invention, the application of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1

[0068] Download the expression data of BTN2A2 in various human tumors and normal tissues from the official websites of TGGA and GTEs databases, and analyze the expression differences of BTN2A2 in human tumors and normal tissues. The results are as Figures 1 - 4 shown, Figures 1 - 4 The specific tumor names are shown in Table 1.

[0069] Table 1 Corresponding table of tumor names

[0070]

[0071]

[0072] According to Figures 1 - 4 It can be seen that the expressions of BTN2A2 in human glioma and pancreatic cancer are both higher than those in normal tissues. Survival analysis shows that in glioma patients, those with low expression of BTN2A2 have a relatively longer survival time than those with high expression; in pancreatic cancer patients, those with low expression of BTN2A2 have a relatively longer survival time of more than 2 years than those with high expression. BTN2A2 (butyrophilin 2A2) is highly expressed in glioma and pancreatic cancer, and is related to the poor prognosis of glioma and pancreatic cancer patients.

[0073] Example 2

[0074] 1. Immunohistochemical detection

[0075] Use immunohistochemistry to analyze the expressions of BTN2A2 in skin, colon, pancreas, brain cancer and adjacent tissues respectively. The specific steps are as follows:

[0076] (1) Dewaxing: Place the tumor tissue sections in xylene for the first time (about 30 minutes) and the second time (about 30 minutes).

[0077] (2) Descend the alcohol with concentration gradient to water: 100% alcohol 1, 100% alcohol 2, 90% alcohol 1, 90% alcohol 2, 80% alcohol, 70% alcohol for 3 - 5 minutes each, and distill and rinse the slides.

[0078] (3) Eliminate the activity of endogenous peroxidase: Immerse the sections in 3% hydrogen peroxide (20 minutes), and distill and rinse the slides.

[0079] (4) Antigen repair of the tissue: Drop protease K (1:2000) onto the tissue of the sections and incubate for 10 minutes.

[0080] (5) Block non - specific binding sites: Drop the working solution of goat serum onto the tissue and incubate in an oven at 37°C for 45 minutes.

[0081] (6) Primary antibody: Remove the working solution of goat serum, drop rabbit anti - human BTN2A2, and drop rabbit - derived serum IgG for the isotype control, and incubate overnight at 4°C.

[0082] (7) Secondary antibody: Drop goat anti - rabbit secondary antibody labeled with horseradish peroxidase onto the sections and incubate in an oven at 37°C for about 35 minutes.

[0083] (8) DAB color development: Drop the DAB color - developing solution and start timing. A positive result shows yellow - brown or tan under the microscope, and rinse repeatedly with running water to terminate the color development.

[0084] (9) Counter - stain with hematoxylin: Immerse the sections in hematoxylin dye (2 - 4 minutes), rinse with running water, then immerse in acid alcohol for color separation, rinse with running water, blue with ammonia alcohol, rinse with running water, and dry the excess water.

[0085] (10) Clearing: Fresh xylene 1 (30 minutes), xylene 2 (30 minutes).

[0086] (11) Sealing the slides: Drop a small amount of neutral gum on the tissue, cover the tissue on the glass slide, and take images under an upright microscope. The results show that the expression of BTN2A2 in human glioma and pancreatic cancer is higher than that in the adjacent (i.e., normal) tissues ( Figure 5 ).

[0087] 2. Flow cytometry detection

[0088] Using human breast cancer cells (4T1), human glioblastoma cells (A172), human non-small cell lung cancer cells (A549), human melanoma cells (B16), mouse colon cancer cells (GL261), mouse glioma cells (GL261), mouse lung cancer cells (LLC), human hepatoma cells (Hep3B), human hepatoma cells (HepG2), mouse pancreatic cancer cells (Pan02), mouse renal cancer cells (Renca), and human glioma cells (U251) as the research objects, flow cytometry detection was performed. The steps were as follows:

[0089] (1) Digest various tumor cells from the culture dish with trypsin, centrifuge at 3000 rpm for 5 min, discard the supernatant, and retain the precipitate.

[0090] (2) Add rabbit anti-human BTN2A2 antibody and the corresponding isotype control to the tumor cells, incubate at 4°C for 45 min, wash once with 1×PBS, add goat anti-rabbit-FITC secondary antibody, incubate at 4°C for 45 min, and wash once with PBS.

[0091] (3) Suspend with PBS, transfer to a flow tube, load onto a flow cytometer, analyze the results using Flowjo software, and statistically analyze the expression levels of BTN2A2 on the cell membrane and intracellularly in various tumor cell lines. The results showed that BTN2A2 was highly expressed in pancreatic cancer and glioma cell lines.

[0092] It can be seen from steps 1 and 2 that BTN2A2 was highly expressed in pancreatic cancer and glioma tissues and cell lines.

[0093] Example 3

[0094] Preparation of hBTN2A2-Ig fusion protein and in vitro functional verification

[0095] 1. Preparation and verification of hBTN2A2-Ig fusion protein

[0096] (1) Design upstream and downstream primers according to the full-length gene sequence of the extracellular region of human BTN2A2 retrieved from NCBI (accession number NM_001197237.2). The upstream primer sequence is 5'-GTCACGAATTCGGCGATCGCCAGTTTACTGTCGTGGGGCC-3' (SEQ ID NO.7), and the downstream primer sequence is 5'-GCGGCCGCGTACGCGTGGGAGATGCGCTGGGCATAA-3' (SEQ ID NO.8); using human cDNA as a template, perform PCR amplification with the above upstream and downstream primers to obtain the extracellular region sequence of hBTN2A2, and cut and recover the hBTN2A2 target gene;

[0097] (2) The pCMV6-AC-FC-S expression vector (purchased from Shanghai Kelei Co., Ltd.) was digested with MIU 1 and Sigf 1 enzymes. After obtaining the linearized vector, the hBTN2A2 target gene in step (2) was ligated to the linearized vector using In-Fusion HD Enzyme Premix DNA ligase to obtain a recombinant vector;

[0098] (3) The recombinant vector in step (2) was transfected into HEK-293F cells, and stable expression cell lines were screened with G418 geneticin. The cells were cultured in FreeStyle TM 293 expression medium for 2 weeks. The supernatant was collected and the protein was purified using a Protein G protein purification column. hBTN2A2 could be attached to the purification column through the Fc tag (i.e., Ig), and the miscellaneous proteins flowed through. Eluting the column yielded the hBTN2A2-Ig fusion protein;

[0099] (4) The hBTN2A2-Ig fusion protein obtained in step (3) was identified and verified using gel electrophoresis (SDS-PAGE) and Western Blot. A single band of protein could be seen, which was the purified hBTN2A2-Ig fusion protein, indicating the successful preparation of the hBTN2A2-Ig fusion protein ( Figure 8 in A).

[0100] 2. Flow cytometry detection of the effect of hBTN2A2-Ig protein on T cell activation and effector T cell differentiation

[0101] (1) Experimental group (hBTN2A2 Ig): 96-well plates were coated with anti-CD3 plus hBTN2A2-Ig protein and incubated overnight at 4°C;

[0102] Control group (control Ig): 96-well plates were coated with anti-CD3 plus Ig protein (i.e., Fc protein) with the same molar mass as the experimental group and incubated overnight at 4°C.

[0103] (2) The single cell suspension of the spleen of normal C57BL / 6 mice was seeded into 96-well plates. After culturing in an incubator for 16 - 18 hours, the supernatant was discarded by centrifugation.

[0104] (3) Add antibodies APC CD4, PerCP / Cy5.5 CD8a, and FITC CD69 to each tube of cells to detect the activation of splenic T cells; stain with PE CD44 and FITC CD62L to detect the ratio of effector T cells and naive T cells in the spleen; incubate for 45 min, wash once with PBS, resuspend and analyze by flow cytometry. The results showed that compared with the control group (control Ig), the percentage of effector T cells with CD44hiCD62Llo in the experimental group (hBTN2A2 Ig) decreased, the percentage of naive T cells with CD44loCD62Lhi increased, and the activation of CD69 decreased ( Figure 8 as shown in B-E in the figure), and the hBTN2A2-Ig fusion protein could inhibit T cell activation and the differentiation of effector T cells in vitro.

[0105] 3. Detection of the effect of hBTN2A2-Ig protein on T cell proliferation by flow cytometry

[0106] (1) Experimental group (hBTN2A2 Ig): Coat a 96-well plate with anti-CD3 plus hBTN2A2-Ig protein;

[0107] Control group (control Ig): Coat a 96-well plate with anti-CD3 plus Ig protein with the same molar mass as the experimental group.

[0108] (2) The experimental group and the control group were each divided into a Ki67 group and a CFSE group. The Ki67 group was directly seeded with a single-cell suspension of the spleen of normal C57BL / 6 mice. The CFSE group incubated the single-cell suspension of the spleen with CFSE for 15 min for staining, and then seeded it into a 96-well plate. After culturing in an incubator for 5 days, the cells were collected and centrifuged to remove the supernatant.

[0109] (3) Add antibodies APC CD4 and PerCP / Cy5.5 CD8a to each tube of cells and incubate for 45 min.

[0110] (4) After resuspension, the CFSE group was analyzed by flow cytometry for the proliferation ratio of CD4 and CD8 T cells. The Ki67 group was fixed with 4% paraformaldehyde for 10 min in each tube, then punched on ice with 100 μl of 0.2% Triton X-100 for 10 min. PE Ki67 antibody was added to each tube and incubated on ice for 45 min, and then analyzed by flow cytometry to detect cell proliferation. The results showed that compared with the control group (control Ig), the proliferation of CFSE and Ki67 in the experimental group (hBTN2A2 Ig) decreased ( Figure 8 as shown in F-G in the figure), and the hBTN2A2-Ig fusion protein could inhibit T cell proliferation in vitro.

[0111] Example 4

[0112] Preparation and Functional Verification of Anti-hBTN2A2 Monoclonal Antibody Ig

[0113] 1. Obtaining the Light Chain Gene and Heavy Chain Gene of hBTN2A Monoclonal Antibody

[0114] (1) Immunize mice with the hBTN2A2-Ig fusion protein obtained in step 1 of Example 3. For the primary immunization, inject subcutaneously the hBTN2A2-Ig fusion protein emulsified with complete Freund's adjuvant (CFA). The booster immunization is prepared by mixing the hBTN2A2-protein with incomplete Freund's adjuvant (IFA). Boost the immunization every two weeks after the primary immunization, for a total of four times. The last boost immunization is 3 days before spleen collection. Collect mouse serum samples before the primary immunization as a control for evaluating the titer of the antiserum later. Collect serum samples 1 week after each boost immunization and test the level and function of the polyclonal antibody through ELISA assays. Select the mice with better determination results (strong specific signals) for B cell sorting. Collect mouse spleen cells and sort CD19+ B cells by MACS (magnetic bead sorting method) to make a single B cell suspension (containing plasma cells and memory cells). Culture the single B cells in a 96-well plate and take the supernatant of the cell culture for ELISA detection to screen for positive cell lines against hBTN2A2;

[0115] (2) Lyse the B cells of the positive cell line against hBTN2A2 in step (1), extract total RNA and mRNA. During reverse transcription polymerase chain reaction (RT-PCR), use random hexamer primers, oligo-dT, or gene-specific primers to reverse transcribe mRNA to synthesize cDNA; using cDNA as a template, perform PCR amplification with the light chain upstream primer and light chain downstream primer, and perform PCR amplification with the heavy chain upstream primer and heavy chain downstream primer. Cut the gel and sequence to obtain the heavy chain (VH) and light chain (VL) gene sequence information of anti-hBTN2A2. The results are as Figure 9 shown in A, where the heavy chain (VH) gene sequence is as SEQ ID NO.6 and the light chain (VL) gene sequence is as SEQ ID NO.5.

[0116] Among them, the light chain upstream primer sequence is: 5'-ATGGAGTCACAGACTCAGGT-3' (SEQ ID NO.9), the light chain downstream primer sequence is: 5'-TTTTATTTCCAGCTTGGTCC-3' (SEQ ID NO.10); the heavy chain upstream primer sequence is: 5'-ATGAACTTCGGGCTCAGCTT-3' (SEQ ID NO.11), and the heavy chain downstream primer sequence is: 5'-ACGTCTCTGTCACTGGTCTC-3' (SEQ ID NO.12);

[0117] The PCR amplification system is as follows: 12.5 μL of Go Taq Green Master mix 2x; 10 μM μL of the upstream primer, 10 μL of the downstream primer, 1 μL of the template, and 9.5 μL of DEPC water; The PCR amplification program is: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, for 30 cycles; 72°C for 7 min, 4°C ∞;

[0118] 2. Preparation of Anti-hBTN2A2 monoclonal antibody

[0119] (1) Use BamHI and HindIII endonucleases to shear the pcDNA3.4 plasmid (carrying the mouse Fc segment, i.e., the Ig segment) into linearized fragments; Recover the fragments containing the anti-hBTN2A2 heavy chain gene (Fab segment) and light chain gene (Fab segment) in step 1 respectively, and ligate them to the linearized fragments through T4 DNA ligase to obtain the heavy chain recombinant vector and light chain recombinant vector;

[0120] (2) Co-transfect the heavy chain recombinant vector and light chain recombinant vector into HEK-293F cells. The heavy chain and light chain are expressed by the cells and assembled into a complete antibody structure and released into the culture medium. Screen the stable expression cell line, collect the culture supernatant after amplification, purify the fusion protein by Protein G, and identify it by gel electrophoresis (SDS-PAGE) and Western Blot. The fusion protein is typed as IgG1. Western Blot verifies that it can bind to the BTN2A2-Ig protein but not to the Ig protein, indicating that the fusion protein is anti-BTN2A2 but not anti-Ig. Coomassie Brilliant Blue shows that the antibody has two heavy and light chains and is an antibody against BTN2A2, denoted as Anti-hBTN2A2 Ab( Figure 9 in B).

[0121] 3. Effect of Anti-hBTN2A2 Ab on macrophage phagocytosis of tumor cells

[0122] (1) Coat a 6-well plate overnight with Anti-hBTN2A2 Ab (10 μg / ml) and isotype control antibody (mouse anti-human IgG1). Stain glioma cells and pancreatic cancer cells with CFSE. Seed macrophages Raw264.7 and tumor cells into the 6-well plate and co-culture for 2 hours. Collect the cells and centrifuge to discard the supernatant.

[0123] (2) Add antibody F4 / 80-Percpcy5.5 to each tube and stain for 45 min, then analyze by flow cytometry. The proportion of tumor cells phagocytosed by macrophages is detected as double positive for CFSE and F4 / 80. The results show that Anti-hBTN2A2 Ab can promote macrophage phagocytosis of mouse pancreatic cancer and glioma cells( Figure 9 in C).

[0124] 4. Expression of CD69 activation in T cells after anti-hBTN2A2 Ab neutralizes BTN2A2-Ig

[0125] (1) Coat a 96-well plate overnight in four groups for CD69 activation: Ig protein, BTN2A2-Ig, BTN2A2-Ig plus anti-BTN2A2 Ab, and BTN2A2-Ig plus isotype control (mouse anti-human IgG1). Stimulate all four groups with anti-CD3.

[0126] (2) Seed C57 mouse splenocytes, culture for 16 - 18 hours, then collect the cells and centrifuge to remove the supernatant.

[0127] (3) Add antibodies APC CD4, PerCP / Cy5.5 CD8a, and FITC CD69 to each tube of cells and incubate for 45 min. Wash once with PBS and detect CD69 activation by flow cytometry. The results show that anti-BTN2A2 Ab can neutralize the inhibitory effect of hBTN2A2-Ig fusion protein on T cell activation ( Figure 9 in D - E).

[0128] 5. Expression of Ki67+ proliferation in T cells after anti-hBTN2A2 Ab neutralizes BTN2A2-Ig

[0129] (1) Coat a 96-well plate overnight in five groups for Ki67 proliferation: Ig protein, BTN2A2-Ig, BTN2A2-Ig + 6.25 μg / ml anti-BTN2A2 Ab, BTN2A2-Ig + 12.5 μg / ml anti-BTN2A2 Ab, and BTN2A2-Ig + 25 μg / ml anti-BTN2A2 Ab. Stimulate all five groups with anti-CD3.

[0130] (2) Collect the cells from the 5 groups, add antibodies APC CD4 and PerCP / Cy5.5 CD8a to each tube of cells, and incubate for 45 min.

[0131] (3) Wash once with PBS, add 4% paraformaldehyde to each tube to fix for 10 min, then punch holes on ice with 100 μl of 0.2% Triton X-100 for 10 min. Add PE Ki67 antibody to each tube and incubate on ice for 45 min, then detect the Ki67 proliferation of the cells by flow cytometry. The results show that anti-BTN2A2 Ab can neutralize the inhibitory effect of hBTN2A2-Ig fusion protein on T cell proliferation ( Figure 9 in F - G).

[0132] Example 5

[0133] Effect of Anti-hBTN2A2 Ab on Symptoms and Immune Cell Infiltration in Mouse Pancreatic Cancer

[0134] 1. Pan02 mouse pancreatic cancer cells in the logarithmic growth phase were collected and inoculated subcutaneously under the left rib of mice at 2×10 6 cells / mouse to establish a mouse pancreatic cancer model (Zhou Ling, Yang Chen, Gao Yan, et al. Experimental study on gemcitabine enhancing the anti-pancreatic cancer effect of HUVEC vaccine [J]. Chinese Pharmacological Bulletin, 2021, 37(07).). The mice were randomly divided into a Control Ab group and an anti-hBTN2A2 Ab group. The Control Ab group was administered (mouse anti-human IgG1); the anti-hBTN2A2 Ab group was administered anti-hBTN2A2 Ab. Each group was administered twice a week, 200 μg each time, by intratumoral injection. The changes in the size of the tumors in the mice were observed, and a tumor change curve was plotted. The results showed that anti-hBTN2A2 Ab had an anti-pancreatic cancer effect ( Figure 10 A and B in the figure).

[0135] 2. Tumors from the mice in the Control Ab group and the anti-hBTN2A2 Ab group were taken respectively, minced and ground into single-cell suspensions. After resuspension, CD45-APCCy7, CD4-APC, and CD8-Percpcy5.5 were added to each tube and incubated for 45 min. The infiltration of T cells in the tumors was detected by flow cytometry. The results showed that anti-hBTN2A2 Ab promoted the infiltration of CD45+ cells, CD3+, CD4+, and CD8+ T cells in mouse pancreatic cancer, thus promoting the killing effect of T cells on tumors ( Figure 10 C and D in the figure).

[0136] 3. HE staining

[0137] Glioma tissues from the mice in the Control Ab group and the anti-hBTN2A2 Ab group were taken respectively, paraffin-embedded and sectioned. The sections were dewaxed to water, the cell nuclei were stained with hematoxylin, the cytoplasm was stained with eosin, dehydrated and mounted, and observed under a microscope. The results showed that the mouse glioma tissue model was successfully constructed ( Figure 11 C in the figure).

[0138] 4. CD4 and CD8 immunofluorescence staining

[0139] The pancreatic cancer sections of the Control Ab group and the anti-hBTN2A2 Ab group were dewaxed to water, the sections were repaired with antigen repair solution, blocked with goat serum, and then incubated with CD4 and CD8 (1:1000) primary antibodies overnight at 4°C. The next day, after washing the slides with PBS, secondary antibodies goat anti-rabbit FITC and APC were added respectively, and incubated at room temperature in the dark for 1 h. After washing the slides with PBS, the nuclei were stained with DAPI for 10 min. After mounting, the slides were observed and images were collected under a fluorescence microscope. The results showed that anti-hBTN2A2 Ab promoted the infiltration of CD4 and CD8 T cells in murine pancreatic cancer, thus promoting the killing effect of T cells on tumors ( Figure 11 in B and C).

[0140] 5. Tumors of mice in the Control Ab group and the anti-hBTN2A2 Ab group were taken respectively, minced and ground into single-cell suspensions. After resuspension, CD45-APCCy7, CD4-APC, and CD8-Percpcy5.5 were added to each tube and incubated for 45 min. Flow cytometry was used to detect the infiltration of T cells in the tumor. The results showed that anti-hBTN2A2 Ab promoted the infiltration of B cells and macrophages into pancreatic cancer, strengthening the killing effect on cancer cells ( Figure 11 in D and E).

[0141] 6. Tumors of mice in the Control Ab group and the anti-hBTN2A2 Ab group were taken respectively, minced and ground into single-cell suspensions. After resuspension, antibodies CD45-APCCy7, CD62L-FITC, CD44-PE, F4 / 80-FITC, CD86-BV421, CD206-AF647, CD11b-PE, Gr-1-FITC, CD4-PE, CD25-APC, and Foxp3-FITC were added and incubated on ice for 45 min. Flow cytometry was used to detect the infiltration of immune cells in the tumor. The results showed that compared with Control Ab, Anti-hBTN2A2 Ab promoted the differentiation of effector T cells with CD44 hiCD62Llo ( Figure 12 in A and B); Anti-hBTN2A2 Ab promoted the polarization of macrophages in murine pancreatic cancer to M1 ( Figure 12 in C and D); down-regulated the infiltration ratio of MDSC cells in murine pancreatic cancer ( Figure 12 in E and F) and the infiltration ratio of Treg cells ( Figure 12 in G and H). Anti-hBTN2A2 Ab enhanced the anti-tumor effect by up-regulating immune cells (T cells, M1 macrophages) and down-regulating immunosuppressive cells (MDSC, M2 macrophages, Treg) infiltrating into pancreatic cancer.

[0142] 7. Tumors were taken from the mice in the Control Ab group and anti-hBTN2A2 Ab group respectively, minced and ground into single-cell suspensions. After resuspension, antibodies CD45-APCCy7, CD4-APC, and CD8-FITC were added and incubated on ice for 45 min. After washing once with PBS, antibodies TNF-α-PE, IL-4-PE, IFN-γ-FITC, IL-2-PE, and IL-17-Percpcy5.5 were added after fixation and permeabilization and incubated on ice for 45 min. Flow cytometry was used to detect the cytokine secretion of tumor-infiltrating lymphocytes. The results showed that compared with Control Ab, Anti-hBTN2A2Ab promoted the secretion of cytokines TNF-α ( Figure 13 in A and B); IL-17 and IFN-γ ( Figure 13 in C and D); IL-4 and IL-2 ( Figure 13 in E and F) by CD4 and CD8 T cells in murine pancreatic cancer, thus enhancing the anti-tumor effect. Anti-hBTN2A2 Ab promoted the secretion of multiple inflammatory cytokines (TNF-α, IL-17, IFN-γ, IL-2) by pancreatic cancer-infiltrating lymphocytes, thus enhancing the tumor-killing effect.

[0143] 8. Spleens were taken from the mice in the Control Ab group and anti-hBTN2A2 Ab group respectively, minced and ground into single-cell suspensions. After resuspension, antibodies CD4-APC, CD8-Percpcy5.5, CD69-FITC, F4 / 80-FITC, CD86-BV421, CD206-AF647, CD11b-PE, Gr-1-FITC, CD25-APC, and Foxp3-FITC were added and incubated on ice for 45 min. After washing once with PBS, antibodies Ki67-PE, TNF-α-PE, IL-4-PE, IFN-γ-FITC, IL-2-PE were added after fixation and permeabilization and incubated on ice for 45 min. Flow cytometry was used to detect the infiltration of immune cells in the tumor. The results showed that Anti-hBTN2A2 Ab promoted the polarization of macrophages in the spleen of pancreatic cancer mice to M1 ( Figure 14 in A and B); down-regulated the proportions of immunosuppressive cells Treg and MDSC cells ( Figure 14 in C and D); promoted the activation and proliferation of CD4 and CD8 T cells ( Figure 14 in E and F); promoted the secretion of cytokines TNF-α, IFN-γ, IL-4, and IL-2 by CD4 and CD8 T cells ( Figure 14 in G and J). Anti-hBTN2A2 Ab up-regulated the secretion of immune cells (T cells, M1 macrophages) and inflammatory cytokines in the spleen of pancreatic cancer mice, and down-regulated immunosuppressive cells (Treg and MDSC), enhancing the anti-tumor effect.

[0144] Example 6

[0145] Effect of Anti-hBTN2A2 Ab on Glioma Symptoms and Immune Cell Infiltration in Mice

[0146] 1. Collect Gl261 mouse glioma cells in the logarithmic growth phase and inoculate 5×10 6 cells / mouse subcutaneously under the left rib of the mouse to establish a mouse glioma model (Xu Qiongguan, Ouyang Yibin, Xie Zhenming. Anti-tumor activity study of oncolytic virus co-expressing BiTE against glioma [J]. Journal of Modern Oncology, 2022, 30(08): 1354-1359.). Randomly divide them into the Control Ab group and the anti-hBTN2A2 Ab group. The Control Ab group is administered (murine anti-human IgG1); the anti-hBTN2A2 Ab group is administered anti-hBTN2A2 Ab. Each group is administered twice a week, 200 μg each time, by intratumoral injection. Observe the size change of the tumor in the mice and draw a tumor change curve. The results show that anti-hBTN2A2 Ab has an anti-pancreatic cancer effect ( Figure 15 in A and B).

[0147] 2. Take the tumors of the mice in the Control Ab group and the anti-hBTN2A2 Ab group respectively, cut them into pieces and grind them into single-cell suspensions. After resuspension, add CD45-APCCy7, CD4-APC, and CD8-Percpcy5.5 to each tube and incubate for 45 min. Flow cytometry is used to detect the infiltration of T cells in the tumor. The results show that it promotes the infiltration of CD45 cells, CD4, and CD8 T cells in the glioma of mice turning white, thus promoting the killing effect of T cells on the tumor ( Figure 15 in C).

[0148] 3. HE staining

[0149] Take the pancreatic cancer tissues of the mice in the Control Ab group and the anti-hBTN2A2 Ab group respectively, embed them in paraffin and section them. Dewax the sections to water, stain the cell nuclei with hematoxylin, stain the cytoplasm with eosin, dehydrate and mount the slides, and observe them under a microscope. The results show that the mouse pancreatic cancer model is successfully constructed ( Figure 15 in D).

[0150] 4. Immunofluorescence staining

[0151] Take the glioma sections of mice in the Control Ab group and the anti-hBTN2A2 Ab group respectively, dewax to water, repair the sections with antigen repair solution, block with goat serum, incubate with primary antibodies CD4 and CD8 (1:1000) at 4°C overnight, add secondary antibodies goat anti-rabbit FITC and APC respectively after washing the sections with PBS the next day, incubate in the dark at room temperature for 1 h, stain the nuclei with DAPI for 10 min after washing the sections with PBS, observe and collect images under a fluorescence microscope after mounting. The results show that the anti-hBTN2A2 monoclonal antibody promotes the infiltration of CD45 cells, CD4, and CD8 T cells with white appearance in mouse glioma, thus promoting the killing effect of T cells on tumors. Anti-hBTN2A2 Ab can play an anti-glioma role by promoting the infiltration of T cells into the tumor.

[0152] 5. Take the gliomas of mice in the Control Ab and Anti-hBTN2A2 Ab groups, cut them into pieces and grind them into single-cell suspensions. After resuspension, add antibodies CD45-APCCy7, CD62L-FITC, CD44-PE, CD19-FITC, CD11c-PE, F4 / 80-Percpcy5.5, CD4-APC, CD8-Percpcy5.5, CD11b-PE, Gr-1-FITC, incubate on ice for 45 min, wash once with PBS, add antibody Ki67-PE after fixation and permeabilization, incubate on ice for 45 min, and detect the infiltration of immune cells in the tumor by flow cytometry. The results show that compared with Control Ab, Anti-hBTN2A2 Ab promotes the proliferation of CD4 and CD8 T lymphocytes infiltrating into mouse glioma ( Figure 16 in A and B); and enhances the anti-tumor effect by upregulating the infiltration of immune cells (effector T cells, B cells, macrophages with CD44 hiCD62Llo) and downregulating the infiltration of immunosuppressive cells MDSC ( Figure 16 in C-H). Anti-hBTN2A2 Ab enhances the anti-glioma effect by upregulating the infiltration of immune cells (T cells, B cells, macrophages) and downregulating the infiltration of immunosuppressive cells MDSC into the tumor.

[0153] 6. Take the spleens of glioma mice in the two groups of ControlAb and Anti-hBTN2A2 Ab, cut them into pieces and grind them into single-cell suspensions. After resuspension, add the antibodies CD4-APC, CD8-Percpcy5.5, CD69-FITC, F4 / 80-FITC, CD86-BV421, CD206-AF647, CD25-APC, Foxp3-FITC and incubate on ice for 45 min. Wash once with PBS. After fixation and permeabilization, add the antibodies Ki67-PE, TNF-α-PE, IFN-γ-FITC and incubate on ice for 45 min. Flow cytometry was used to detect the infiltration of immune cells in the tumor. For CFSE proliferation assay, take the spleen single-cell suspension, stain it with CFSE and culture it in an incubator for 5 days. Then stain it with CD4-APC and CD8-Percpcy5.5 and perform flow cytometry to detect T cell proliferation. The results showed that compared with ControlAb, Anti-hBTN2A2 Ab upregulated the infiltration of immune cells (effector T cells, B cells, M1 macrophages with CD44 hiCD62Llo) and downregulated the infiltration of immunosuppressive cells (Tregs and M2 macrophages) in the spleens of glioma mice ( Figure 17 in A-F), and promoted the proliferation and activation of CD4 and CD8 T cells ( Figure 17 in E-J) and the secretion of TNF-α and IFN-γ ( Figure 17 in K-L), further enhancing the killing effect on glioma. Anti-hBTN2A2 Ab upregulated the infiltration of immune cells (T cells, B cells, M1 macrophages) and the secretion of inflammatory cytokines in the spleens of glioma mice, downregulated the infiltration of immunosuppressive cells (Tregs and M2 macrophages) into the tumor, and enhanced the anti-tumor effect.

[0154] It can be seen from the above content that BTN2A2 can inhibit T cell proliferation and activation, render T cells in an inactivated state, and ultimately induce immune escape. BTN2A2 is upregulated in a variety of tumor cells. It binds to the corresponding receptor on T cells, inhibits T cell proliferation and activation, renders T cells in an inactivated state, and ultimately induces immune escape. Anti-BTN2A2 monoclonal antibody can block the binding of BTN2A2 to the receptor, promote the activation and proliferation of T cells, enhance the recognition of tumor cells by T cells, and exert an anti-tumor effect. It has been verified in mouse pancreatic cancer and glioma models that anti-BTN2A2 monoclonal antibody has the effects of upregulating the immune response, improving the tumor microenvironment of mice, and reducing the tumor growth rate of mice.

[0155] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to this embodiment without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of BTN2A2 as an immune checkpoint in the preparation of anti-tumor products.

2. The application according to claim 1, wherein The amino acid sequence encoded by BTN2A2 is as shown in SEQ ID NO.

1.

3. The application according to claim 1, wherein The product includes a drug; the drug includes an anti-BTN2A2 antibody.

4. The application according to any one of claims 1 to 3, characterized in that The tumor includes glioma and / or pancreatic cancer.

5. The light chain variable region and heavy chain variable region of an anti-BTN2A2 antibody, characterized in that, The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.

4.

6. A DNA molecule, characterized in that, Encoding the light chain variable region and heavy chain variable region of claim 5.

7. Use of the light chain variable region and heavy chain variable region of claim 6 or the DNA molecule of claim 7 in the preparation of an anti-BTN2A2 antibody.

8. An antibody against BTN2A2, characterized in that, The anti-BTN2A2 antibody includes the light chain variable region and heavy chain variable region of claim 6.

9. The method for preparing the anti-BTN2A2 antibody according to claim 8, characterized in that, Comprising the following steps: Connecting the nucleotide sequences encoding the light chain variable region and heavy chain variable region to a basic vector respectively to obtain a light chain recombinant vector and a heavy chain recombinant vector; Co-expressing the light chain recombinant vector and the heavy chain recombinant vector to obtain the anti-BTN2A2 antibody; The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.

4.

10. An anti-tumor drug, characterized in that, The anti-tumor drug includes excipients and an immune checkpoint inhibitor; The immune checkpoint inhibitor is the anti-BTN2A2 antibody of claim 8.

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