Use of btn2a2 as an immune checkpoint in the preparation of an anti-tumor product

Abstract

The application 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 application takes BTN2A2 as an immune checkpoint, finds that BTN2A2 is up-regulated in various tumor cells (including pancreatic cancer and glioma) and APC cells, combines with a corresponding receptor on a T cell, inhibits T cell proliferation and activation, makes the T cell in an inactivation state, and finally induces immune escape. By preparing an anti-BTN2A2 monoclonal antibody, combination of BTN2A2 and the receptor can be blocked, growth and proliferation of the T cell are up-regulated, recognition of the T cell to tumor cells is enhanced, attack and killing functions of the T cell are activated, and an anti-tumor effect is exerted.

Description

TECHNICAL FIELD

[0001] The application 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. BACKGROUND

[0002] In recent years, the number of cancer patients and deaths has shown an increasing trend year by year, and cancer has become one of the diseases that seriously affect human health. Surgical treatment, radiotherapy and chemotherapy are traditional treatment modes for tumors, but they have certain limitations due to high recurrence rate, serious complications and the like. In recent years, tumor immunotherapy has achieved remarkable achievements, and has the advantages of low recurrence, low toxicity, high specificity and the like.

[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 an inhibitory or stimulatory role on immune responses. Immune checkpoints play an important immunoregulatory role in maintaining immune homeostasis and preventing autoimmunity. Most of the reported immune checkpoint proteins are expressed on 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, thereby 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 found, and the most in-depth research is still PD-L1 / PD-1, but there are still many defects in its clinical application. Because PD-L1 / PD-1 is expressed differently in different disease states and different individuals, the therapeutic effect is not uniform, and not all tumor patients can benefit from it. The highest anti-tumor immune effect is only about 30%, in addition, there is a lack of evaluation of tumor prognosis-related biomarkers. Although the only FDA-approved PD-L1 expression has relatively high accuracy, it is also limited in its application due to different definitions. Therefore, it is particularly important to find new immune checkpoints and develop immune checkpoint inhibitors.

[0004] Butyrophilin (BTN) 2A2 belongs to the extended B7 family of molecules, has similar structural characteristics to PD-L1, and can inhibit the activation and proliferation of T cell function, thereby regulating the development of autoimmune diseases and inflammatory diseases. At present, there have been reports of mBTN2A2-Ig fusion proteins for improving collagen-induced arthritis (CIA) and autoimmune encephalomyelitis (EAE) in mice, but there have been no related reports on the application of BTN2A2 in the preparation of anti-tumor drugs and tumor treatment. SUMMARY

[0005] The application aims to make up for the deficiency of the prior art, provide the application of BTN2A2 as an immune checkpoint in the preparation of an anti-tumor product, enrich the tumor treatment target and the application direction of BTN2A2, and provide a new direction for the treatment of tumors.

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

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

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

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

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

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

[0012] The application also provides a DNA molecule coding the light chain variable region and the heavy chain variable region.

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

[0014] The application also provides an anti-BTN2A2 antibody, wherein the anti-BTN2A2 antibody comprises the light chain variable region and the heavy chain variable region.

[0015] The application also provides a preparation method of the anti-BTN2A2 antibody, comprising the following steps:

[0016] The nucleotide sequence coding the light chain variable region and the heavy chain variable region is connected to a basic vector respectively to obtain a light chain recombinant vector and a heavy chain recombinant vector;

[0017] The light chain recombinant vector and the heavy chain recombinant vector are co-expressed to obtain the anti-BTN2A2 antibody.

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

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

[0020] The application also provides an anti-tumor drug, which comprises a excipient and an immune checkpoint inhibitor.

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

[0022] Beneficial effects:

[0023] The application takes BTN2A2 as an immune checkpoint, finds that BTN2A2 is up-regulated in various tumor cells (including pancreatic cancer and glioma) and APC cells, combines with the corresponding receptor on the T cell, inhibits the proliferation and activation of the T cell, makes the T cell in an inactivated state, and finally induces immune escape. By preparing an anti-BTN2A2 monoclonal antibody, the combination of BTN2A2 and the receptor can be blocked, the growth and proliferation of the T cell are up-regulated, the recognition of the T cell to the tumor cell is enhanced, the attack and killing functions of the T cell are activated, and the anti-tumor effect is exerted. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0025] Figure 1 The TGGA and GTEs database are analyzed to obtain the expression difference results of BTN2A2 in human tumors and normal tissues;

[0026] Figure 2 The TGGA and GTEs database are analyzed to obtain the expression difference results of BTN2A2 in human pancreatic cancer (PADD) and glioma (GBM) and normal tissues;

[0027] Figure 3 The expression of BTN2A2 in human pancreatic cancer (PADD) is analyzed in relation to survival;

[0028] Figure 4 The expression of BTN2A2 in human glioma (GBM) is analyzed in relation to survival;

[0029] Figure 5 The results of the immunohistochemical analysis of the pan-cancer in Example 2 are shown in Table 2;

[0030] Figure 6 The results of the flow cytometry detection of the membrane and intracellular expression of BTN2A2 in the pan-cancer cell lines in Example 2 are shown in Table 3;

[0031] Figure 7 The statistical results of the membrane and intracellular expression of BTN2A2 in the pan-cancer cell lines in Example 2 are shown in Table 4; wherein, C is the statistical results of the membrane expression; and D is the statistical results of the intracellular expression of BTN2A2;

[0032] Figure 8Results of preparation and in vitro functional verification of hBTN2A2-Ig fusion protein in Example 3; wherein A is the verification results of the purified hBTN2A2-Ig fusion protein by agarose gel electrophoresis, Coomassie brilliant blue and Western blot; B is the flow cytometry detection results of Control Ig and hBTN2A2-Ig protein on mouse CD4, CD8 T memory cell differentiation; C is the quantitative detection results of Control Ig and hBTN2A2-Ig protein on mouse CD4, CD8 T memory cell differentiation; D is the flow cytometry detection results of different concentrations of Control Ig and hBTN2A2-Ig protein on mouse CD4, CD8 T memory cell differentiation; E is the quantitative detection results of different concentrations of Control Ig and hBTN2A2-Ig protein on mouse CD4, CD8 T memory cell differentiation; F is the flow cytometry detection results of Control Ig and hBTN2A2-Ig protein on mouse T cell CFSE and Ki67 proliferation index; G is the quantitative results of Control Ig and hBTN2A2-Ig protein on mouse T cell CFSE and Ki67 proliferation index;

[0033] Figure 9 Results of preparation and functional verification of hBTN2A2 monoclonal antibody in Example 4; wherein, A is the results of PCR amplification of antibody light and heavy chain variable region of B cells of hBTN2A2-Ig immunized mice, and the red box is the B cells that simultaneously PCR out anti-hBTN2A2 light and heavy chains; B is the Anti-hBTN2A2 Ab typing, Western Blot identification combined with specificity of hBTN2A2-Ig protein, Ig protein, and Coomassie brilliant blue staining results; C is the flow cytometry detection results of step 3; D-E are the flow cytometry detection results of step 4; F-G are the flow cytometry detection results of step 5;

[0034] Figure 10 Effects of Anti-hBTN2A2 Ab on mouse pancreatic cancer symptoms and immune cell infiltration; wherein, A is the pancreatic cancer tumor size; B is the pancreatic cancer tumor volume change; C-D are flow cytometry detection results;

[0035] Figure 11 Effects of Anti-hBTN2A2 Ab on mouse pancreatic cancer immune cell infiltration; wherein, A is the HE staining result; B-C are CD4, CD8 immunofluorescence staining results; D-E are the flow cytometry detection results of step 5 (tumor);

[0036] Figure 12For Example 5 Step 6 Flow Cytometry Results (tumor); Wherein, A and B are mouse pancreatic cancer memory T cell typing results; C and D are mouse pancreatic cancer macrophage M1 / M2 typing results; E and F are mouse pancreatic cancer MDSC ratio; G and H are mouse pancreatic cancer Treg ratio;

[0037] Figure 13 For Example 5 Step 7 Flow Cytometry Results (tumor); Wherein, A and B are mouse pancreatic cancer CD4, CD8 T cell cytokine TNF-a ratio; C and D are IL-17 and IFN-g ratio; E and F are IL-4 and IL-2 ratio;

[0038] Figure 14 For Example 5 Step 8 Flow Cytometry Results (spleen); Wherein, A and B are macrophage M1 / M2 typing results; C is MDSC ratio; D is Treg ratio; E is CD4, CD8 T cell CD69+ activation; F is ki67+ proliferation; G is cytokine TNF-a ratio; H is cytokine IFN-g ratio; I is F is IL-4 ratio; J is IL-2 ratio;

[0039] Figure 15 For Example 6 Anti-hBTN2A2 Ab Effect on Mouse Glioma Symptoms and Immune Cell Infiltration; Wherein, A is glioma tumor size; B is glioma tumor volume change; C is Step 2 Flow Cytometry Results; D is HE Staining Results; E-F are Immunofluorescence Detection of Glioma CD4, CD8 T Cell Infiltration Results;

[0040] Figure 16 For Example 6 Step 5 Flow Cytometry Results (tumor); Wherein, A and B are mouse tumor CD4, CD8 T cell Ki67 proliferation; C and D are glioma B, DC, macrophage infiltration ratio; E and F are T cell ratio; G and H are MDSC ratio;

[0041] Figure 17 For Example 6 Step 6 Flow Cytometry Results (spleen); Wherein, A is memory T cell; B is Treg; C and D are macrophage M1 / M2 typing ratio; E and F are CD4, CD8 T cell CD69+ activation, B, DC, macrophage infiltration, TNF-a secretion ratio; G-J are CD4, CD8 T cell ki67+ and CFSE proliferation; K-L are CD4, CD8 T cell cytokine IFN-g secretion ratio. DETAILED DESCRIPTION

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

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

[0044] SEQ ID NO. 1: QFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFV YKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLV VAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTT AVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFMPSASP;

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

[0046] In the present application, the product preferably comprises a medicament; the medicament preferably comprises an antibody against-BTN2A2. The tumor according to the present application preferably comprises a glioma and / or a pancreatic cancer.

[0047] BTN2A2 belongs to the extended B7 family molecules, has similar structural characteristics with PD-L1, can inhibit the activation and proliferation of T cell function, thereby regulating the development of autoimmune diseases and inflammatory diseases, and the prior art usually uses it to improve collagen-induced arthritis (CIA) and autoimmune encephalomyelitis (EAE), and the application creatively uses BTN2A2 as an immune checkpoint, experiments find that BTN2A2 is up-regulated in various tumor cells and APC cells, it combines with the corresponding receptor on the T cell, inhibits the proliferation and activation of the T cell, makes the T cell in an inactivated state, the tumor cell realizes immune escape, the preparation of anti-BTN2A2 monoclonal antibody can block the combination of BTN2A2 and the receptor, up-regulate the growth and proliferation of the T cell, enhance the recognition of the T cell to the tumor cell, activate the attack and killing function thereof, and play an anti-tumor effect, enrich the tumor treatment target and the application direction of BTN2A2, and provide a new direction for the treatment of tumors.

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

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

[0050] In the application, the nucleotide sequence for coding the light chain variable region is preferably shown in SEQ ID NO. 5, and the nucleotide sequence for coding the heavy chain variable region is preferably shown in SEQ ID NO. 6.

[0051] The specific sequence information of SEQ ID NO. 3-6 in the application is as follows:

[0052] SEQ ID NO. 3: MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVGT YVAWYQQKPGQSPEALIYSASYRYSGVPYRFAGSGSGTEFTLTISNVQSEDLAEYFCQQ YISYPYTFGGGTKLEIK.

[0053] SEQ ID NO. 4: MNFGLSLIFLVLVLKGVQCEVQLVESGGVLVKPGGSLKLSCTASGFIFSD

[0054] YYIHWVRQTPEKRLEWVATISDGGGYTHYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDT AMYYCVRDSTRSWGQGTLVTVSA.

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

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

[0057] The application further provides a DNA molecule encoding the light chain variable region and the heavy chain variable region according to the above technical solution.

[0058] The application further provides use of the light chain variable region and the heavy chain variable region or the DNA molecule in preparation of an antibody against BTN2A2.

[0059] The application 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 described in the application preferably comprises the following steps:

[0060] The nucleotide sequences encoding the light chain variable region and the heavy chain variable region are respectively connected with a basic vector 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 nucleotide sequences of the light chain variable region and the heavy chain variable region are respectively connected with a basic vector to obtain a light chain recombinant vector and a heavy chain recombinant vector. In the application, the basic vector preferably comprises pCDNA3.4 (with an Fc segment, i.e., an Ig segment). The application does not have strict requirements for the connection mode, and a conventional mode in the art can be adopted.

[0063] After obtaining the light chain recombinant vector and the heavy chain recombinant vector, the light chain recombinant vector and the heavy chain recombinant vector are co-expressed to obtain the anti-BTN2A2 antibody. In the application, the co-expression mode comprises the following steps: the light chain recombinant vector and the heavy chain recombinant vector are co-transfected into cells, the cells are cultured, the heavy chain and the light chain are simultaneously expressed by the cells, and the complete antibody structure is assembled and released into the culture medium. The cells preferably comprise HEK-293F cells. The application preferably purifies the antibody released into the culture medium to obtain the anti-BTN2A2 antibody.

[0064] The anti-BTN2A2 antibody provided by the application can make T cells proliferate in large quantities and attack tumor cells by blocking the binding of BTN2A2 to the receptor, thereby playing an anti-tumor role, especially in treating glioma and pancreatic cancer. The results of the examples show that after the pancreatic cancer is treated by the anti-BTN2A2 antibody, the growth rate of the pancreatic cancer can be reduced, the lymphocyte infiltration in the tumor microenvironment can be increased, the B cells, macrophages, CD8 effector T cells, M1 type macrophages and T cell secreted cytokines TNF-α, IFN-γ, IL-17, IL-4 and IL-2 in the tumor microenvironment can be up-regulated, and M2 type macrophages and Treg, MDSC can be down-regulated. After the glioma is treated by the anti-BTN2A2 antibody, the growth rate of the glioma can be reduced, the lymphocyte infiltration in the tumor microenvironment can be increased, the B cells, macrophages, CD8 effector T cells and T cell secreted cytokines TNF-α, IFN-γ, IL-17, IL-4 and IL-2 in the tumor microenvironment can be up-regulated, and MDSC cells can be down-regulated.

[0065] The application also provides an anti-tumor drug comprising an excipient and an immune checkpoint inhibitor; the immune checkpoint inhibitor is an anti-BTN2A2 antibody obtained by the preparation method. The application does not have strict requirements for the type of the excipient, and the excipient can be selected according to the conventional selection of the dosage form of the drug.

[0066] In order to further illustrate the application, the application of BTN2A2 as an immune checkpoint in the preparation of an anti-tumor product is described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the application.

[0067] Example 1

[0068] The expression data of BTN2A2 in various human tumors and normal tissues were downloaded from the TGGA and GTEs database websites, and the expression differences of BTN2A2 in human tumors and normal tissues were analyzed, and the results are shown in Figures 1-4 Figures 1-4 The tumor names are specifically shown in Table 1.

[0069] Table 1: Correspondence table of tumor names

[0070]

[0071]

[0072] According to Figures 1-4 It can be seen that the expression of BTN2A2 on human glioma and pancreatic cancer is higher than that in normal tissues, and the survival analysis shows that the low expression of BTN2A2 in glioma patients has a relatively longer survival period than the high expression; the low expression of BTN2A2 in pancreatic cancer patients has a relatively longer survival period of more than 2 years. BTN2A2 (milk fat protein 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] The expression of BTN2A2 in skin (Skin), colon (Colon), pancreas (Pancrea), brain cancer (Brain) and cancer-adjacent tissues was analyzed by immunohistochemistry, and the specific steps are as follows:

[0076] (1) Waxing: Place the tumor tissue section in xylene for the first time (about 30 min), and in xylene for the second time (about 30 min).

[0077] ​(2) Concentration gradient alcohol down to water: 100% alcohol 1, 100% alcohol 2, 90% alcohol 1, 90% alcohol 2, 80% alcohol, 70% alcohol each 3-5 min, distill the film.

[0078] (3) Eliminate endogenous peroxidase activity: 3% hydrogen peroxide soak the section (20 min), distill the film.

[0079] (4) Tissue antigen repair: add protease K (1:2000) to the tissue on the section, incubate for 10 min.

[0080] (5) Block non-specific binding sites: add goat serum working solution to the tissue, incubate in 37°C oven for 45 min.

[0081] (6) Primary antibody: remove the goat serum working solution, add rabbit anti-human BTN2A2, add rabbit-derived serum IgG as a control, incubate overnight at 4°C.

[0082] (7) Secondary antibody: add horseradish peroxidase-labeled goat anti-rabbit secondary antibody to the section, incubate in 37°C oven for about 35 min.

[0083] (8) DAB color development: add DAB color developing solution and time, brown or yellow-brown under the microscope is a positive result, repeated washing with running water to stop color development.

[0084] (9) Hematoxylin restain: soak the section in hematoxylin dye (2-4 min), rinse with running water, then soak in acid alcohol for color separation, rinse with running water, ammonia alcohol counterstain, rinse with running water, bake off excess moisture.

[0085] (10) Transparency: fresh xylene 1 (30 min), xylene 2 (30 min).

[0086] (11) Mounting: add a small amount of neutral gum to the tissue, cover the tissue on the slide, place the microscope to take the image, the results show that the expression of BTN2A2 in human glioma and pancreatic cancer is higher than that in the adjacent normal tissue (i.e. Figure 5 ).

[0087] 2. Flow cytometry detection

[0088] Take 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 hepatocellular carcinoma cells (Hep3B), human hepatocellular carcinoma cells (HepG2), mouse pancreatic cancer cells (Pan02), mouse renal cancer cells (Renca) and human glioma cells (U251) as the research object, and perform flow cytometry detection, the steps are:

[0089] (1) A plurality of tumor cells are digested from the culture dish with trypsin, centrifuged at 3000 rpm for 5 min, the supernatant is discarded, and the precipitate is retained.

[0090] (2) The tumor cells are added with rabbit anti-human BTN2A2 antibody and the corresponding isotype control, incubated at 4°C for 45 min, washed once with 1xPBS, added with goat anti-rabbit-FITC secondary antibody, and incubated at 4°C for 45 min, and washed once with PBS.

[0091] (3) Suspended with PBS, transferred to a flow tube, and subjected to flow cytometry on a machine, and the results are analyzed by Flowjo software, and the expression amounts of BTN2A2 on the cell membranes and in the cells of various tumor cell lines are counted, and the results show that the BTN2A2 on the pancreatic cancer and glioma cell lines has high expression.

[0092] It can be seen from steps 1 and 2 that the BTN2A2 on the pancreatic cancer and glioma tissues and cell lines has high expression.

[0093] Example 3

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

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

[0096] (1) The human BTN2A2 extracellular region full-length gene sequence (accession number NM_001197237.2) obtained according to NCBI is used to design an upstream primer and a downstream primer, wherein the sequence of the upstream primer is 5'-GTCACGAATTCGGCGATCGCCAGTTTACTGTCGTGGGGCC-3' (SEQ ID NO. 7), and the sequence of the downstream primer is 5'-GCGGCCGCGTACGCGTGGGAGATGCGCTGGGCATAA-3' (SEQ ID NO. 8); the human cDNA is used as a template, and the above-mentioned upstream primer and downstream primer are used for PCR amplification to obtain the hBTN2A2 extracellular region sequence, and the hBTN2A2 target gene is recovered by cutting gel;

[0097] (2) pCMV6-AC-FC-S expression vector (purchased from Shanghai Coyle Company) was linearized by MIU 1 and Sigf 1 enzyme, and then the hBTN2A2 gene obtained in step (2) was connected to the linearized vector by using In-Fusion HD Enzyme Premix DNA ligase to obtain a recombinant vector;

[0098] (3) The recombinant vector obtained in step (2) was transfected into HEK-293F cells, and stable expression cell lines were screened by G418 geneticin. The cells were cultured in FreeStyle 293 expression medium for 2 weeks, and the supernatant was collected. The hBTN2A2-Ig fusion protein was obtained by purifying the protein with a Protein G protein purification column. The hBTN2A2 can be hung on the purification column through the Fc tag (i.e. Ig), and the impure protein flows down. The hBTN2A2-Ig fusion protein was obtained by eluting the column. TM 293 expression medium for 2 weeks, and the supernatant was collected. The hBTN2A2-Ig fusion protein was obtained by purifying the protein with a Protein G protein purification column. The hBTN2A2 can be hung on the purification column through the Fc tag (i.e. Ig), and the impure protein flows down. The hBTN2A2-Ig fusion protein was obtained by eluting the column.

[0099] (4) The hBTN2A2-Ig fusion protein obtained in step (3) was identified and verified by gel electrophoresis (SDS-PAGE) and Western Blot. A single band of protein was observed, which was the purified hBTN2A2-Ig fusion protein. The hBTN2A2-Ig fusion protein was successfully prepared. Figure 8

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

[0101] (1) Experimental group (hBTN2A2 Ig): anti-CD3 plus hBTN2A2-Ig protein coated 96-well plate, 4°C overnight;

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

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

[0104] ​(3) Each tube of cells was added with antibodies APC CD4, PerCP / Cy5.5CD8a, and incubated for 45 min. Figure 8 In the middle of B-E), the hBTN2A2-Ig fusion protein can inhibit T cell activation and the differentiation of effector T cells in vitro.

[0105] 3. Flow detection of the effect of hBTN2A2-Ig protein on T cell proliferation

[0106] (1) Experimental group (hBTN2A2 Ig): anti-CD3 plus hBTN2A2-Ig protein was used to package 96-well plates.

[0107] Control group (control Ig): anti-CD3 plus Ig protein with the same molar mass as the experimental group was used to package 96-well plates.

[0108] (2) The experimental group and the control group were divided into Ki67 group and CFSE group, respectively. The Ki67 group was directly plated into the spleen single cell suspension of normal C57BL / 6 mice, and the CFSE group was stained by incubating the spleen single cell suspension with CFSE for 15 min, and then plated into 96-well plates. After 5 days of incubation in the incubator, the cells were collected and centrifuged to remove the supernatant.

[0109] (3) Each tube of cells was added with antibodies APC CD4, PerCP / Cy5.5CD8a, and incubated for 45 min.

[0110] (4) After resuspension, the CFSE group was analyzed by flow cytometry for CD4 and CD8 T cell proliferation ratio, and the Ki67 group was added with 4% paraformaldehyde per tube for 10 min, then 100 μl 0.2% Trion X-100 was punched for 10 min on ice, and then PE Ki67 antibody was added per tube, and incubated for 45 min on ice. Flow cytometry was used to detect cell proliferation. The results showed that compared with the control group (control Ig), the CFSE and ki67 proliferation in the experimental group (hBTN2A2 Ig) were reduced. Figure 8 In the middle of F-G), the hBTN2A2-Ig fusion protein can inhibit T cell proliferation in vitro.

[0111] Example 4

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

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

[0114] (1) Immunize mice with the hBTN2A2-Ig fusion protein obtained in step 1 of Example 3, subcutaneously inject the hBTN2A2-Ig fusion protein emulsified with complete Freund's adjuvant (CFA) for the first immunization, and prepare the hBTN2A2 protein mixed with incomplete Freund's adjuvant (IFA) for the booster immunization. The booster immunization is performed every two weeks after the first immunization, a total of four times, and the last impact immunization is performed three days before the spleen is taken. Collect mouse serum samples before the first immunization as a control for evaluating the titer of the antiserum. Collect serum samples one week after each booster immunization, and test the level and function of polyclonal antibodies by ELISA. Select mice with better determination results (strong specific signal) for B cell sorting. Collect mouse spleen cells, MACS (magnetic bead sorting) sort CD19+ B cells, prepare single B cell suspension (containing plasma cells and memory cells), and culture single B cells in a 96-well plate. Take the supernatant of the cell culture for ELISA detection to screen positive cell strains against hBTN2A2;

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

[0116] wherein 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] ​PCR amplification system: Go Taq Green Master mix 2x 12.5μL; upstream primer 10uMμL, downstream primer 10μL, template 1μL, DEPC water 9.5μL; PCR amplification procedure: 95℃ 5min; 95℃ 30s, 56℃ 30s, 72℃ 30s, 30 cycles; 72℃ 7min, 4℃ ∞;

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

[0119] (1) The pcDNA3.4 plasmid (with mouse Fc segment, i.e. Ig segment) is cut into a linearized fragment by using BamHI and HindIII endonucleases; the anti-hBTN2A2 heavy chain gene (Fab segment) and light chain gene (Fab segment) contained in step 1 are recovered respectively and connected to the linearized fragment by using T4 DNA ligase to obtain a heavy chain recombinant vector and a light chain recombinant vector;

[0120] (2) The heavy chain recombinant vector and the light chain recombinant vector are co-transfected into HEK-293F cells, the heavy chain and the light chain are expressed by the cells and assembled into a complete antibody structure to be released into the culture medium. A stable expression cell strain is screened, the culture supernatant is collected after amplification, the fusion protein is obtained by Protein G purification, gel electrophoresis (SDS-PAGE) and Western Blot identification, the fusion protein is typed as IgG1, Western Blot verifies that it can bind to BTN2A2-Ig protein but not to Ig protein, indicating that the fusion protein is anti-BTN2A2 but not anti-Ig, and the antibody is shown by Coomassie blue to have light and heavy two chains, which is an antibody against BTN2A2, denoted as Anti-hBTN2A2 Ab. Figure 9 B).

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

[0122] (1) Anti-hBTN2A2 Ab (10μg / ml) and isotype control antibody (mouse anti-human IgG1) are coated on a 6-well plate overnight, and glioma cells and pancreatic cancer cells are stained with CFSE. Macrophage Raw264.7 and tumor cells are placed in a 6-well plate for co-culture for 2 hours, and the cells are collected by centrifugation and the supernatant is discarded.

[0123] (2) Each tube is added with antibody F4 / 80-Percpcy5.5 for staining for 45min, and flow cytometry is performed to detect the proportion of CFSE and F4 / 80 double positive cells, which are tumor cells phagocytosed by macrophages. The results show that Anti-hBTN2A2 Ab can promote the phagocytosis of mouse pancreatic cancer and glioma cells by macrophages Figure 9 C).

[0124] 4. Expression of T cell CD69 activation after neutralization of BTN2A2-Ig by Anti-hBTN2A2 Ab

[0125] (1) CD69 activation was divided into four groups to coat 96-well plates overnight: Ig protein, BTN2A2-Ig, BTN2A2-Ig plus anti-BTN2A2 Ab, BTN2A2-Ig plus isotype control (mouse anti-human IgG1), and the above four groups plus anti-CD3 stimulation.

[0126] (2) C57 mouse spleen cells were plated and cultured for 16-18 hours, after which the cells were collected and centrifuged to remove the supernatant.

[0127] (3) Antibodies APC CD4, PerCP / Cy5.5 CD8a, and FITC CD69 were added to each tube of cells and incubated for 45 min, followed by PBS washing once and flow cytometry detection of CD69 activation. The results showed that anti-BTN2A2 Ab can neutralize the inhibitory effect of hBTN2A2-Ig fusion protein on T cell activation Figure 9 MID-E)

[0128] 5. Expression of T cell Ki67+ proliferation after neutralization of BTN2A2-Ig by Anti-hBTN2A2 Ab

[0129] (1) Ki67 proliferation was divided into five groups to coat 96-well plates overnight: Ig protein, BTN2A2-Ig, BTN2A2-Ig + 6.25 μg / ml anti-BTN2A2 Ab, BTN2A2-Ig + 12.5 μg / ml anti-BTN2A2 Ab, BTN2A2-Ig + 25 μg / ml anti-BTN2A2 Ab, and the above five groups plus anti-CD3 stimulation.

[0130] (2) The cells of the five groups were collected, and antibodies APC CD4 and PerCP / Cy5.5 CD8a were added to each tube of cells and incubated for 45 min.

[0131] (3) PBS was washed once, 4% paraformaldehyde was added to each tube and fixed for 10 min, and then 100 μl of 0.2% Trion X-100 was added to each tube and punched on ice for 10 min. PE Ki67 antibody was added to each tube and incubated on ice for 45 min, and then flow cytometry was performed to detect cell ki67 proliferation. The results showed that anti-BTN2A2 Ab can neutralize the inhibitory effect of hBTN2A2-Ig fusion protein on T cell proliferation Figure 9 MID-F-G).

[0132] Example 5

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

[0134] 1. Collect Pan02 mouse pancreatic cancer cells in logarithmic growth phase, inoculate 2×10 6 cells / mouse subcutaneously in the left flank of mice to establish a mouse pancreatic cancer model (Zhou L, Yang C, Gao Y, et al. Experimental study on the effect of gemcitabine on HUVEC vaccine against pancreatic cancer [J]. Chinese Bulletin of Pharmacology, 2021, 37(07).), and randomly divide them into Control Ab and anti-hBTN2A2 Ab groups. The Control Ab group is given mouse anti-human IgG1, and the anti-hBTN2A2 Ab group is given anti-hBTN2A2 Ab, 200 μg per time, twice a week, intratumorally. Observe the size of the tumor in mice, draw the tumor change curve, and the results show that anti-hBTN2A2 Ab has an anti-pancreatic cancer effect (Figures 1A and B). Figure 10

[0135] 2. Take the tumors of the Control Ab and anti-hBTN2A2 Ab groups, cut and grind them into single-cell suspensions, and 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 intratumoral T cell infiltration, and the results show that anti-hBTN2A2 Ab promotes the infiltration of CD45 cells, CD3, CD4, and CD8 T cells in mouse pancreatic cancer, thereby promoting the killing effect of T cells on the tumor (Figures 1C and D). Figure 10

[0136] 3. HE staining

[0137] Take the glioma tissues of the Control Ab and anti-hBTN2A2 Ab groups, paraffin-embed the sections, deparaffinize the sections to water, stain the cell nucleus with hematoxylin, and stain the cytoplasm with eosin. After dehydration and mounting, observe under a microscope, and the results show that the mouse glioma tissue model is successfully constructed (Figure 2C). Figure 11

[0138] 4. CD4 and CD8 immunofluorescence staining

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

[0140] 5. The tumors of the Control Ab group and the anti-hBTN2A2 Ab group mice were taken respectively, cut and ground to prepare a single cell suspension, then CD45-APCCy7, CD4-APC and CD8-Percpcy5.5 were added to each tube and incubated for 45 min, and the intratumoral T cell infiltration was detected by flow cytometry. The results showed that anti-hBTN2A2 Ab promoted the infiltration of B cells and macrophages into pancreatic cancer, thereby enhancing the killing effect on cancer cells Figure 11 D and E).

[0141] 6. The tumors of the Control Ab group and the anti-hBTN2A2 Ab group mice were taken respectively, cut and ground to prepare a single cell suspension, then antibodies CD45-APCCy7, CD62L-FITC, CD44-PE, F4 / 80-FITC, CD86-BV421, CD206-AF647, CD11b-PE, Gr-1-FITC, CD4-PE and CD25-APC were added to each tube and incubated on ice for 45 min, and the intratumoral immune cell infiltration was detected by flow cytometry. The results showed that, compared with the Control Ab, Anti-hBTN2A2 Ab promoted the differentiation of CD44 hiCD62Llo effector T cells Figure 12 A and B); Anti-hBTN2A2 Ab promoted the polarization of mouse pancreatic cancer macrophages to M1 Figure 12 C and D); down-regulated the infiltration proportion of mouse pancreatic cancer MDSC cells Figure 12 E and F) and Treg cells Figure 12 G and H), Anti-hBTN2A2 Ab promoted the infiltration of immune cells (T cells, M1 type macrophages) and down-regulated the infiltration of immune suppressor cells (MDSC, M2 type macrophages, Treg) into pancreatic cancer, thereby enhancing the anti-tumor effect.

[0142] 7. Take the tumors of Control Ab group and anti-hBTN2A2 Ab group mice respectively, cut and grind into single cell suspension, add antibody CD45-APCCy7, CD4-APC, CD8-FITC after resuspension, incubate on ice for 45 min, wash once with PBS, add antibody TNF-a-PE, IL-4-PE, IFN-g-FITC, IL-2-PE, IL-17-Percpcy5.5 after fixation and membrane breaking, incubate on ice for 45 min, detect the secretion of cytokines of tumor infiltrating lymphocytes by flow cytometry. The results show that, compared with Control Ab, Anti-hBTN2A2 Ab promotes the secretion of cytokines TNF-a (Fig. 6A and 6B), IL-17, IFN-g (Fig. 6C and 6D), IL-4 and IL-2 (Fig. 6E and 6F) of CD4 and CD8 T cells of pancreatic cancer, thereby enhancing the anti-tumor effect. Anti-hBTN2A2 Ab promotes the secretion of multiple inflammatory cytokines (TNF-a, IL-17, IFN-g, IL-2) of pancreatic cancer infiltrating lymphocytes, thereby enhancing the tumor killing effect. Figure 13 Figure 13 Figure 13

[0143] 8. Take the spleen of Control Ab group and anti-hBTN2A2 Ab group mice respectively, cut and grind into single cell suspension, add antibody CD4-APC, CD8-Percpcy5.5, CD69-FITC, F4 / 80-FITC, CD86-BV421, CD206-AF647, CD11b-PE, Gr-1-FITC, CD25-APC, Foxp3-FITC after resuspension, incubate on ice for 45 min, wash once with PBS, add antibody Ki67-PE, TNF-a-PE, IL-4-PE, IFN-g-FITC, IL-2-PE after fixation and membrane breaking, incubate on ice for 45 min, detect the intratumoral immune cell infiltration by flow cytometry. The results show that, Anti-hBTN2A2 Ab promotes the M1 polarization of macrophages in the spleen of pancreatic cancer mice (Fig. 7A and 7B), down-regulates the proportion of immunosuppressive cells Treg and MDSC (Fig. 7C and 7D), promotes the activation and proliferation of CD4 and CD8 T cells (Fig. 7E and 7F), promotes the secretion of cytokines TNF-a, IFN-g, IL-4 and IL-2 of CD4 and CD8 T cells (Fig. 7G and 7J). Anti-hBTN2A2 Ab up-regulates the secretion of immune cells (T cells, M1 macrophages) and inflammatory cytokines in the spleen of pancreatic cancer mice, down-regulates the immunosuppressive cells (Treg and MDSC), and enhances the anti-tumor effect. Figure 14 Figure 14 Figure 14 Figure 14

[0144] Example 6​​​​​​​

[0145] Effects of Anti-hBTN2A2 Ab on the Symptoms and Immune Cell Infiltration of Mouse Glioma

[0146] 1. Collect Gl261 mouse glioma cells in the logarithmic growth phase, and inoculate 5 x 10 6 cells / mouse subcutaneously in the left flank of mice to establish a mouse glioma model (Xu Q G, Ouyang Y B, Xie Z M. Anti-tumor activity of oncolytic virus co-expressing BiTE against glioma [J]. Modern Oncology Medicine, 2022, 30(08): 1354-1359.), and randomly divide them into Control Ab and anti-hBTN2A2 Ab groups. The Control Ab group is given (murine anti-human IgG1), and the anti-hBTN2A2 Ab group is given anti-hBTN2A2 Ab, 200 μg each time, twice a week, intratumorally. Observe the size of the mouse tumor, draw the tumor change curve, and the results show that anti-hBTN2A2 Ab has an anti-pancreatic cancer effect (Fig. 2A and B). Figure 15

[0147] 2. Take the tumors of the Control Ab and anti-hBTN2A2 Ab groups, respectively, cut and grind them into a single cell suspension. 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 intratumoral T cell infiltration, and the results show that anti-hBTN2A2 Ab promotes the infiltration of CD45 cells, CD4, and CD8 T cells in mouse glioma, thereby promoting the killing effect of T cells on the tumor (Fig. 3C). Figure 15

[0148] 3. HE staining

[0149] Take the pancreatic cancer tissues of the Control Ab and anti-hBTN2A2 Ab groups, respectively, paraffin-embed the sections, deparaffinize the sections to water, stain the cell nucleus with hematoxylin, and stain the cytoplasm with eosin. After dehydration and mounting, observe under a microscope, and the results show that the mouse pancreatic cancer model is successfully constructed (Fig. 4D). Figure 15

[0150] 4. Immunofluorescence staining

[0151] ​​​Take the Control Ab group and anti-hBTN2A2 Ab group mice glioma sections, respectively, to water, antigen repair solution repair section, goat serum blocking after CD4, CD8 (1:1000) primary antibody 4 ℃ incubation overnight, the next day PBS wash piece after adding secondary antibody goat anti-rabbit FITC, APC, avoid light incubation 1 h, PBS wash piece after DAPI dye 10 min, after mounting under the fluorescence microscope and collect images, the results show that anti-hBTN2A2 monoclonal antibody promotes the infiltration of mouse glioma pan white CD45 cells, CD4, CD8 T cells, thereby promoting the killing effect of T cells on tumor. Anti-hBTN2A2 Ab can play a role in anti-glioma by promoting T cells to infiltrate into the tumor.

[0152] 5. Take Control Ab and Anti-hBTN2A2 Ab two groups of mice glioma, cut and grind into single cell suspension, after resuspension, add antibody 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, PBS wash once, after fixation and membrane rupture, add antibody Ki67-PE, incubate on ice for 45 min, flow cytometry detection of intratumoral immune cell infiltration, the results show that compared with Control Ab, Anti-hBTN2A2 Ab promotes the proliferation of CD4, CD8 T lymphocytes in mouse glioma Figure 16 A and B); and by up-regulating immune cells (CD44 hiCD62Llo effector T cells, B cells, macrophages) and down-regulating immune suppressor cells MDSC infiltration Figure 16 C-H), enhance anti-tumor effect. Anti-hBTN2A2 Ab enhances the anti-glioma effect by up-regulating immune cells (T cells, B cells, macrophages) and down-regulating immune suppressor cells MDSC infiltration into the tumor.

[0153] 6. Take the spleen of the ControlAb and Anti-hBTN2A2 Ab groups of glioma mice, cut and grind into a single cell suspension, after resuspension, add 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 membrane rupture, add antibodies Ki67-PE, TNF-a-PE, IFN-g-FITC, and incubate on ice for 45 min, flow cytometry to detect intratumoral immune cell infiltration; CFSE proliferation takes the single cell suspension of the spleen, CFSE staining, and then flow cytometry to detect T cell proliferation after CD4-APC and CD8-Percpcy5.5 staining, the results show that, compared with ControlAb, Anti-hBTN2A2 Ab up-regulates the infiltration of immune cells (CD44 hiCD62Llo effector T cells, B cells, M1 macrophages) and down-regulates the infiltration of immunosuppressive cells (Tregs and M2 macrophages) in the spleen of glioma mice (Figs. 6A-F), and promotes the proliferation and activation of CD4 and CD8 T cells (Figs. 6E-J) and the secretion of TNF-a and IFN-g (Figs. 6K-L), further enhancing the killing effect on glioma. Anti-hBTN2A2 Ab up-regulates the infiltration of immune cells (T cells, B cells, M1 macrophages) and the secretion of inflammatory cytokines in the spleen of glioma mice, down-regulates the infiltration of immunosuppressive cells (Tregs and M2 macrophages) into the tumor, and enhances the anti-tumor effect. Figure 17 Figure 17 Figure 17

[0154] From the above, it can be seen that BTN2A2 can inhibit T cell proliferation and activation, make T cells in an inactive state, and ultimately induce immune escape. BTN2A2 is up-regulated in various tumor cells, it binds to the corresponding receptor on T cells, inhibits T cell proliferation and activation, makes T cells in an inactive 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 play an anti-tumor role. It is verified in mouse pancreatic cancer and glioma models that anti-BTN2A2 monoclonal antibody has the effects of up-regulating the immune response, improving the tumor microenvironment of mice, and reducing the growth rate of tumors in mice.

[0155] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity according to the present embodiments, which all belong to the protection scope of the present application.​​​

Claims

1. An antibody against-BTN2A2, characterized in that, The antibody against-BTN2A2 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. 3; and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.

4.

2. A DNA molecule, characterized in that, The antibody against-BTN2A2 of claim 1 is encoded.

3. The method of producing an antibody against-BTN2A2 according to claim 1, characterized in that, The method comprises the following steps: connecting the nucleotide sequence 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; co-expressing the light chain recombinant vector and the heavy chain recombinant vector to obtain the antibody against-BTN2A2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.

4.

4. Use of the antibody against-BTN2A2 of claim 1 or the antibody against-BTN2A2 encoded by the DNA molecule of claim 2 or the antibody against-BTN2A2 obtained by the preparation method of claim 3 in the preparation of an anti-tumor product; the product is a drug; the tumor is glioma and / or pancreatic cancer.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The anti-tumor drug comprises an adjuvant and an immune checkpoint inhibitor; the immune checkpoint inhibitor is the antibody against-BTN2A2 of claim 1 or the antibody against-BTN2A2 encoded by the DNA molecule of claim 2 or the antibody against-BTN2A2 obtained by the preparation method of claim 3.

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