A tumor T cell antigen epitope peptide, pMHC and its preparation and application

Abstract

The present invention discloses a tumor T cell antigen epitope peptide, the amino acid sequence of the tumor T cell antigen epitope peptide is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3. The tumor T cell antigen epitope peptide of the present invention can be used for in vitro T cell activation experiments, and can be applied to the research and development of universal tumor vaccines for tumors with high expression of MAGE-A3 and MAGE-A10 antigens such as melanoma, non-small cell lung cancer, liver cancer, head and neck tumors, tumor diagnosis, and TCR-T therapy. The tumor T cell antigen epitope peptide of the present invention is prepared into a pMHC complex or directly loaded into antigen presenting cells to activate T cells, which can be applied to: the research and development and preparation of tumor vaccines, detection of whether the body has anti-tumor cellular immune function, auxiliary diagnosis and TCR-T cell immunotherapy development.

Description

[0001] This application is a divisional application of the invention patent application with application date of August 30, 2022, application number 202211049861X, and invention name “A tumor T cell antigen epitope peptide, pMHC and its preparation and application”. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a tumor T cell antigen epitope peptide, pMHC and the preparation and application thereof. Background Art

[0003] The incidence and mortality of tumors are increasing year by year around the world. The main problem currently is the lack of effective treatment methods. Tumor immunotherapy refers to exogenous intervention in the body's immune system, restarting and maintaining the "tumor-immune" cycle, restoring and improving the body's anti-tumor immune response, and strengthening the ability to identify and kill tumor cells, thereby achieving the therapeutic effect of controlling or even specifically eliminating tumors. Compared with traditional treatments such as surgery, radiotherapy and chemotherapy, tumor immunotherapy has the advantages of strong specificity and few side effects. At present, the tumor immunotherapy methods that have been used in clinical practice mainly include immune checkpoint inhibitor therapy, adoptive cell immunotherapy, cancer vaccines and some emerging immunotherapy methods. Cancer immunotherapy was rated as the top ten scientific breakthroughs in 2013 by Science Magazine, and is also a new anti-tumor treatment method that is currently being widely researched and developed.

[0004] The cancer immune cycle includes several steps: antigens produced by cancer cells are released after cancer cells die and are captured by dendritic cells (DC). Next, DC presents the antigens captured on MHC molecules to T cells, thereby triggering and activating the response of effector T cells to cancer antigens. Under the guidance of chemokines, activated T cells enter and infiltrate the tumor site. T cells specifically recognize and bind to cancer cells and kill cancer cells through the interaction of TCR and antigen-MHC complex. Therefore, T cell immune response plays an important role in anti-tumor immune defense and occupies an important position in vaccine development. The first step of CD8+T cell immune response is that T cells specifically recognize antigen epitope peptides presented by cancer cells through antigen recognition receptors on their surface. Therefore, antigen epitope peptides are important key molecules for T cells to specifically recognize tumor cells and exert immune protection, and are key targeting molecules for immune detection, immunotherapy and vaccine development.

[0005] Chimeric antigen receptor T cell technology (CAR-T) and T cell receptor chimeric T cells (TCR-T) are the two latest immune cell technologies and have received extensive attention and research. CAR-T cell therapy has achieved good results in blood tumors, but the cytokine storm and neurotoxic side effects it causes may be life-threatening. TCR-T cell therapy is one of the effective immune cell treatments for solid tumors. The TCRα and β chain genes that can recognize tumor-specific antigens are transfected into T cells, and the T cells are modified to express tumor antigen-specific TCR antigen binding regions, so that they can specifically recognize the corresponding tumor antigens. Then, after in vitro amplification and re-infusion into the human body, T lymphocytes expressing tumor antigen-specific TCR can recognize the HLA-antigen peptide complex on the surface of tumor cells, thereby triggering the immune effect of T cells to achieve the purpose of killing tumor cells. Tumor vaccines use tumor-associated antigens (TAAs), tumor peptides or tumor cell lysis products to induce the body to produce tumor-specific immune responses, protect the body from tumor cell invasion, and achieve the prevention and treatment of tumors. It includes two major categories: preventive tumor vaccines and therapeutic tumor vaccines. Therapeutic tumor vaccines are mainly used for tumor patients, and they induce the production of specific antibodies, effector cells and specific immune memory cells in patients through immunization to achieve the purpose of treating tumors. Clinical trials have confirmed that whole-cell tumor vaccines, tumor-specific protein or peptide vaccines and tumor nucleic acid vaccines have shown great application potential in the process of tumor treatment.

[0006] The MAGE family is a proto-cancer antigen and a member of the cancer / testis antigen superfamily. There are more than 60 members of the MAGE family, all of which exist in the same MAGE homology domain. It is mainly divided into two subclasses: MAGE-I class antigens and MAGE-II class antigens. The expression of MAGE-II class antigens is commonly found in normal cells, while MAGE-I class antigens are TAAs, that is, they are not expressed in human tissues other than testicular germ cells and placental trophoblastic tissues, but are highly expressed in tumors, making them a hot topic in tumor research. Since germ cells lack HLA, cancer / testis antigens cannot be recognized by T cells, but in malignant tumors, cancer / testis antigens and HLA molecules are expressed simultaneously and can be recognized by T cells. MAGE-I class antigens can be divided into three subclasses: MAGE-A, MAGE-B, and MAGE-C. The MAGE-A subfamily has a strict expression pattern, among which MAGE-A3, MAGE-A4, MAGE-A10 and MAGE-A12 are antigens with unique characteristics in the MAGE family, and are specifically highly expressed in patients with melanoma, liver cancer, lung cancer and esophageal cancer. In recent years, two methods are commonly used to target MAGE-A family genes for anti-cancer effects: (1) vaccination with MAGE epitope antigen vaccines; (2) TCR-T immune cell therapy. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a tumor T cell epitope peptide, pMHC and their preparation and application.

[0008] The first object of the present invention is to provide a tumor T cell antigen epitope peptide.

[0009] The second object of the present invention is to provide a tumor T cell antigen epitope peptide composition.

[0010] The third object of the present invention is to provide a pMHC complex.

[0011] The fourth object of the present invention is to provide an antigen peptide-antigen presenting cell complex.

[0012] The fifth object of the present invention is to provide a gene encoding the above tumor T cell antigen epitope peptide.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned tumor T cell antigen epitope peptide, tumor T cell antigen epitope peptide composition, pMHC complex, antigen peptide-antigen presenting cell complex and / or gene in the preparation of tumor vaccines and / or tumor drugs.

[0014] In order to achieve the above object, the present invention is implemented by the following scheme:

[0015] T2-A2 is an antigen presenting cell line that expresses the human MHC class I molecule HLA-A2 through recombinant genetic engineering technology. Only effective antigen epitope peptides can be presented by it, thereby forming a stable pMHC complex on its cell surface, so it can be used as an artificial antigen presenting cell to stimulate T cells.

[0016] T cell antigen epitope peptides cannot work alone, and T cell activation must be performed in the form of pMHC complexes or antigen peptide-antigen presenting cell complexes. The present invention uses MHC monomers and identified novel tumor T cell epitopes for joint renaturation to prepare pMHC complexes. The identified novel tumor T cell antigen epitope peptides are loaded on the surface of antigen presenting cells (T2-A2 cells) to prepare antigen peptide-antigen presenting cell complexes, and then the prepared pMHC complexes are used to label T cells. It is found that the antigen epitope peptides can effectively activate T cells in the peripheral blood of healthy people to produce killer cytokines, and can also effectively kill target cells carrying tumor MAGE-A3 and MAGE-A10 antigens. The T cell antigen epitope peptides are assembled into pMHC complexes and can be detected in the peripheral blood of patients with non-small cell lung cancer. It is proved that the newly discovered MAGE-A3 and MAGE-A10 T cell antigen epitope peptides can effectively induce T cell immunity and can be applied to the development of universal tumor vaccines and immune cell treatment methods, such as TCR-T.

[0017] Therefore, the present invention claims protection for a tumor T cell antigen epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0018] MAGE-A3-Mp4 (SEQ ID NO: 1): LVFGIELMEV;

[0019] MAGE-A10-Mp30 (SEQ ID NO: 2): VIWEALNMM;

[0020] MAGE-A10-Mp32 (SEQ ID NO: 3): SLLKFLAKV.

[0021] The present invention also claims protection for a tumor T cell antigen epitope peptide composition, wherein the tumor T cell antigen epitope peptide contains one or more of the tumor T cell antigen epitope peptides with amino acid sequences such as SEQ ID NO: 1 to SEQ ID NO: 3.

[0022] The present invention also claims a pMHC complex, wherein the pMHC complex contains the above-mentioned tumor T cell antigen epitope peptide and / or the above-mentioned tumor T cell antigen epitope peptide composition.

[0023] Preferably, the pMHC complex is prepared by renaturing the HLA-A2 heavy chain, the HLA-A2 light chain β2m and the above-mentioned tumor T cell antigen epitope peptide.

[0024] The present invention also claims protection for an antigen peptide-antigen presenting cell complex, wherein the antigen peptide-antigen presenting cell complex is an antigen presenting cell having the above-mentioned tumor T cell antigen epitope peptide and / or the above-mentioned tumor T cell antigen epitope peptide composition on its surface.

[0025] Preferably, the antigen presenting cells are T2-A2 cells.

[0026] More preferably, the T2-A2 cells are T2 cells that overexpress HLA-A2.

[0027] More preferably, the method for preparing the antigen peptide-antigen presenting cell complex is: mixing and incubating the above tumor T cell antigen epitope peptide with antigen presenting cells to obtain the antigen peptide-antigen presenting cell complex.

[0028] The present invention also claims protection for a gene encoding the above tumor T cell antigen epitope peptide.

[0029] The present invention also seeks to protect the use of the above-mentioned tumor T cell antigen epitope peptide, tumor T cell antigen epitope peptide composition, pMHC complex, antigen peptide-antigen presenting cell complex and / or gene in the preparation of tumor vaccines and / or tumor drugs; the tumor is lung cancer.

[0030] Preferably, the tumor also includes a tumor with high expression of MAGE-A3 and / or MAGE-A10.

[0031] More preferably, the tumor with high expression of MAGE-A3 and / or MAGE-A10 is melanoma, liver cancer and / or esophageal cancer.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention has discovered three tumor T cell antigen epitope peptides that can be used for in vitro T cell activation experiments. The tumor T cell antigen epitope peptides can be applied to the development of universal tumor vaccines for tumors with high expression of MAGE-A3 and MAGE-A10 antigens, such as melanoma, non-small cell lung cancer, liver cancer, head and neck tumors, tumor diagnosis, and TCR-T therapy research.

[0034] The tumor T cell antigen epitope peptide of the present invention is prepared into a pMHC complex or directly loaded into antigen presenting cells to activate T cells, and can be applied to:

[0035] (1) Research and development and preparation of tumor vaccines: Tumor antigens are released after tumor cells die. DCs present the antigens captured on MHC molecules to T cells, thereby initiating and activating the response of effector T cells to cancer antigens. The tumor T cell antigen epitope peptides of the present invention can induce the body to produce an immune response to generate antigen-specific T cells. Therefore, the tumor T cell antigen epitope peptides of the present invention are candidate antigen epitope peptides for universal tumor vaccines.

[0036] (2) Detecting whether the body has anti-tumor cellular immune function: The detection of tumor antigen-specific T cells in the subject indicates that the body has developed T cell immune function. The ratio of antigen-specific CD8T labeled with pMHC complex prepared from the antigen epitope peptide reflects the strength of the subject's anti-tumor immune response.

[0037] (3) Auxiliary diagnosis: The tumor T cell antigen epitope peptide or tumor T cell antigen epitope peptide composition of the present invention can detect specific immune response components, thereby improving the specificity of diagnosis and quickly screening out tumor patients who are positive for MAGE-A3 or MAGE-A10 antigen.

[0038] (4) Development of TCR-T cell immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 An example diagram of the assembly of a pMHC complex;

[0040] Figure 2 Identification of T2-A2 antigen presentation of 31 candidate tumor T cell antigen epitope peptides; A: Identification test results of T2-A2 antigen presentation of 31 candidate tumor T cell antigen epitope peptides; B: Statistical chart of Figure A;

[0041] Figure 3 Detection diagram of pMHC complex formation for 31 candidate tumor T cell antigen epitope peptides;

[0042] Figure 4 The activation diagram of CD8+T cells by 31 candidate tumor T cell antigen epitope peptides; A: flow cytometry detection diagram; B: CD8+T cell activation bar graph corresponding to MAGE-A3-Mp4, MAGE-A10-Mp30 and MAGE-A10-Mp32;

[0043] Figure 5 The release detection diagram of IFN-γ and GZMB released by CD8+T cells; A: The ratio diagram of GZMB released by CD8+T cells detected by flow cytometry; B: The ratio diagram of IFN-γ released by CD8+T cells detected by flow cytometry;

[0044] Figure 6This is the test result diagram of Annexin V-APC;

[0045] Figure 7 The graph shows the proportion of specific CD8+T cells in non-small cell carcinoma patients and healthy subjects; A: Flow cytometry detection of MAGE-A3-Mp4, MAGE-A10-Mp30 and MAGE-A10-Mp32 antigen epitope-specific CD8+T cells in HLA-A2 positive NSCLC patients and healthy subjects; B: Detection of CD8+T cells in NSCLC patients and healthy subjects using pMHC complexes prepared with MAGE-A3-Mp4, MAGE-A10-Mp30 and MAGE-A10-Mp32. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0047] Example 1 Prediction and identification of tumor T cell antigen epitope peptides

[0048] Figure 1: Example of pMHC complex assembly Figure 1 shown.

[0049] 1. Experimental methods

[0050] (1) Prediction of tumor T cell antigen epitope peptides

[0051] CD8 T cell epitope prediction was performed on the tumor antigen MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10 and MAGE-A12 protein sequences using the “MHC IBinding” tool in the IEDB Recommended 2.22 (http: / / www.iedb.org / ) online website.

[0052] The MHC allele was selected as HLA-A*02:01, and 31 candidate tumor T cell antigen epitope peptides were obtained, as shown in Table 1.

[0053] (2) Identification of tumor T cell antigen epitope peptides

[0054] According to the sequences in Table 1, the 31 candidate tumor T cell antigen epitope peptides obtained in the artificial synthesis step (1) were each prepared into a stock solution with a concentration of 10 mM using DMSO.

[0055] T2-A2 cells in the logarithmic growth phase were inoculated into 96-well plates (2×10 5 / well), set up blank group, negative control group, positive control group and each tumor T cell antigen epitope peptide group, each group had 3 replicate wells, and the final volume of each well was 200 μL.

[0056] The blank group consisted of wells with only T2-A2 cells;

[0057] The negative control group was co-incubated with T2-A2 cells and Negative peptide (Epstein-Barr virus peptide, amino acid sequence: IVTDFSVIK, as shown in SEQ ID NO: 4);

[0058] The positive control group was co-incubated with T2-A2 cells and Positive peptide (influenza A M1 peptide, amino acid sequence: GILGFVEFTL, as shown in SEQ ID NO: 5);

[0059] 31 tumor T cell antigen epitope peptide groups were set up, and 31 different artificially synthesized candidate tumor T cell antigen epitope peptides were co-incubated with T2-A2 cells.

[0060] The 96-well plate was incubated at 37°C for 4 hours, then the supernatant was removed by centrifugation and washed twice. The cell pellet was incubated with FITC anti-human HLA-A2 (β2m) antibody at 4°C in the dark for 30 minutes, and the cells in each group were detected by flow cytometry.

[0061] Repeat the above steps for three parallel tests.

[0062] 2. Experimental results

[0063] The detailed information of the 31 predicted candidate tumor T cell antigen epitope peptides is shown in Table 1.

[0064] Table 1: 31 candidate tumor T cell antigen epitope peptides

[0065]

[0066]

[0067] The identification results of tumor T cell antigen epitope peptides are as follows Figure 2 As shown, the results show that 29 of the 31 tumor T cell antigen epitope peptides predicted in step (1) (except MAGE-A4-Mp14 and MAGE-A6-Mp24) can be effectively presented to T cells by antigen presenting cells.

[0068] The results show that these 29 tumor T cell antigen epitope peptides are T cell antigen epitope peptides.

[0069] Example 2 Detection of pMHC tetramer complexes formed by tumor T cell antigen epitope peptides

[0070] 1. Experimental methods

[0071] (1) Preparation of pMHC complex monomers of tumor T cell antigen epitope peptides

[0072] The mother solution (10 mM) obtained in step (2) of Example 1 was diluted to 400 μM with PBS to obtain a diluted mother solution, which was placed on ice for later use.

[0073] Add 20 μL of the diluted mother solution and 20 μL of Flex-T monomer (200 μg / mL) to a 96-well U-shaped plate, mix well by blowing, and remove the sealing plate; place the 96-well U-shaped plate on ice and irradiate with a UV lamp for 30 min (the distance between the UV lamp and the sample is maintained at 2 to 5 cm); cover the sealing plate and incubate in a dark place at 37°C for 30 min to obtain 31 pMHC complex monomers formed by 31 candidate tumor T cell antigen epitope peptides and Flex-T monomers, which are set as experimental groups.

[0074] The pMHC complex formed by influenza A M1 peptide (amino acid sequence: GILGFVEFTL, as shown in SEQ ID NO: 5) and Flex-T monomer was set as the positive control group Pos;

[0075] The pMHC complex formed by Epstein-Barr virus peptide (amino acid sequence: IVTDFSVIK, as shown in SEQ ID NO: 4) and Flex-T monomer was set as the negative control group Neg;

[0076] The pMHC monomer formed by PBS and Flex-T monomer was set as the UV control group;

[0077] (2) ELISA to detect the ability of epitope peptides to form pMHC tetramer complexes

[0078] The ELISA method was used to detect whether the 31 candidate tumor T cell antigen epitope peptides synthesized in step (2) of Example 1 could form pMHC tetramers. The specific method was as follows:

[0079] At room temperature, add 100 μL of 0.5 μg / mL streptavidin (Streptavidin solution) to a 96-well plate and incubate overnight (16-18 h), then wash three times with 300 μL of 1× Wash Buffer, and add 1× Dilution Buffer (1 M NaCl, 0.5 M Tris, 1% BSA (w / v), 0.2% Tween 20 (w / v), pH = 8.0) to block at room temperature for 30 min.

[0080] The pMHC complex monomers (positive control group Pos, negative control group Neg, UV control group and experimental group) in step (1) were diluted 1200 times with 1×Dilution Buffer.

[0081] At the same time, set up the Monomer group: dilute the Flex-T monomer equally; Blank group: only add the same amount of 1× Dilution Buffer as other groups.

[0082] Take the positive control group Pos as an example:

[0083] The liquid in the 96-well plate was discarded, and the wells were patted dry on filter paper. 100 μL of the diluted pMHC complex monomers were added to the 96-well plate.

[0084] The negative control group Neg, UV control group, experimental group, Monomer group and Blank group were treated identically and added into a 96-well plate.

[0085] Cover the plate with a sealer and incubate at 37°C for 1 hour. After incubation, wash the 96-well plate three times with washing buffer, then add 100 μL of diluted HRP-anti-β2M (antibody BioLegend, Cat#280303, US), continue incubation at 37°C for 1 hour, and wash after incubation.

[0086] Then, 100 μL of substrate solution (10.34 mL of deionized water, 1.2 mL of pH 4.0, 0.1 M citric acid monohydrate / trisodium citrate dihydrate, 240 μL of 40 nM ABTS, 120 μL of hydrogen peroxide solution) was added to each well, and the color was developed for 8 min at room temperature (18-25°C) with shaking (400-500 rpm) in the dark.

[0087] The reaction was terminated with 50 μL of Stop Solution (2% oxalic acid dihydrate, w / v), and the absorbance (OD value) was measured at a wavelength of 414 nm using an ELISA reader within 30 min.

[0088] 2. Experimental results

[0089] Taking the OD value of the Monomer group as 100%, the relative OD values ​​of the UV control group, the positive control group Pos, the effect control group Neg and the experimental group were calculated. The ratio represents the formation of pMHC tetramers by each tumor antigen epitope peptide. If the relative OD value is greater than that of the UV control group, it is judged that pMHC tetramers can be formed.

[0090] The results are as follows Figure 3 As shown, the results showed that compared with the UV control group, the relative OD values ​​of the MAGE-A4-Mp14 and MAGE-A4-Mp22 complex groups were significantly reduced, while the other groups were significantly increased (P < 0.001). The results showed that except for MAGE-A4-Mp14 and MAGE-A4-Mp22, the other 29 candidate tumor T cell antigen epitope peptides among the 31 candidate tumor T cell antigen epitope peptides can form pMHC tetramer complexes and are potential candidate antigen epitope peptides that can induce specific immune responses.

[0091] Example 3 Tumor T cell antigen epitope peptide-antigen presenting cell activation of T cells

[0092] The antigen peptide / MHC complex on the surface of APC or target cell binds to TCR, providing the first signal for T cell activation; the B7 molecule on the surface of APC binds to the CD28 molecule on the T cell, providing the second signal; IL-2 and other molecules provide co-stimulatory signals. After loading APC cells T2A2 with tumor antigen epitope peptides that can bind to HLA molecules and co-culturing with CD8T cells, the proportion of activated CD8T cells is detected by tetramer, and the proportion of antigen-specific T cells that produce gamma interferon (IFN-γ) and granzyme (GZMB) is analyzed by flow cytometry to analyze the ability of T2A2 loaded with tumor antigens to induce CD8T cell activation.

[0093] 1. Experimental methods

[0094] Mononuclear lymphocytes (PBMCs) from peripheral venous blood of healthy volunteers were isolated, and CD8+ T cells were further isolated. T2-A2 cells were labeled with CFSE, then treated with 20 μg / mL mitomycin for 30 min, and incubated with the 31 candidate tumor T cell antigen epitope peptides of step (1) of Example 1.

[0095] Take 30 μL of the pMHC complex monomer of the tumor T cell antigen epitope peptide obtained in step (1) of Example 2 into a 1.5 mL EP tube, add 3.3 μL of streptavidin (BioLegend Cat#405203, US), mix well with pipette tip, and incubate at 4°C in the dark for 30 min.

[0096] After the incubation, add 2.4 μL of blocking solution (1.6 ul, 50 mM D-biotin (ThermoFisher, Cat# B20656, US), 6 ul of 10% (w / v) NaN3 and 192.4 ul PBS) to the EP tube, and terminate the reaction by pipetting. Incubate overnight at 4-8°C to obtain the pMHC tetramer complex.

[0097] The Mixed Peps group was set to: 0.5×10 6 CD8+T cells and 0.5×10 6 T2-A2 cells loaded with 31 candidate tumor T cell antigen epitope peptides were co-cultured in culture medium.

[0098] The cells were co-stimulated with 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2. During the culture, the culture medium was supplemented with 50 IU / mL IL-2 and 20 μM candidate tumor T cell antigen epitope peptide every two days.

[0099] After 7 days of culture, specific CD8+T cells were labeled with pMHC tetramer complexes, and the specific CD8+T ratio and the release of IFN-γ and GZMB by antigen-specific CD8+T cells were detected. At the same time, the percentage of AnnexinV-APC, a T2-A2 cell apoptosis marker, was detected.

[0100] At the same time, a positive control group (T2-A2 cells loaded with influenza A M1 peptide), a negative control group (T2-A2 cells loaded with Epstein-Barr virus peptide) and a UV control group (pMHC complex formed by PBS and Flex-T monomer) were set up for the same treatment and detection.

[0101] 2. Experimental results

[0102] The results of activating CD8+T cells with different tumor T cell antigen epitope peptides are as follows Figure 4 As shown; the results showed that among the 31 candidate tumor T cell antigen epitope peptides, 3 tumor T cell antigen epitope peptides can activate T cells, namely MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3), and the proportions of activated CD8+T cells were 5.20%, 0.55% and 5.21%, respectively.

[0103] The release of IFN-γ and GZMB by CD8+ T cells Figure 5As shown; the results showed that compared with the negative control group Negctrl, the efficiency of activated CD8+T cells releasing IFN-γ and GZMB in the mixed peps group was significantly increased (P < 0.001); the results indicated that specific CD8+T cells activated by MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) antigen epitopes can release IFN-γ and GZMB.

[0104] Annexin V-APC test results are as follows Figure 6 As shown; the results showed that compared with the negative control group Neg ctrl, the proportion of Annexin V+CFSE+T2A2 cells in the mixed peps group was extremely significantly increased (P<0.001); the results indicated that specific CD8+T cells activated by MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) antigen epitopes can kill target cells T2-A2.

[0105] Example 4 Detection of Epitope Peptide-Specific Cytotoxic T Cells in Peripheral Blood of Clinical Patients

[0106] 1. Experimental methods

[0107] To isolate mononuclear lymphocytes (PBMCs) from peripheral venous blood of patients with non-small cell carcinoma (NSCLC) and healthy subjects, and identify their HLA subtypes.

[0108] The HLA-A2-positive PBMCs samples were stained with the pMHC tetramer complex obtained in Example 3 and CD8-APC antibody, and then observed by flow cytometry.

[0109] 2. Experimental results

[0110] The results of the flow cytometry observation are shown in the figure below. Figure 7As shown, the results show that the pMHC complexes of the three tumor T cell antigen epitope peptides MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) with amino acid sequences as shown in SEQ ID NOs 1 to 3 can recognize antigen-specific CD8+T cells produced in NSCLC patients, and are significantly increased compared with healthy people; The results show that the three antigen epitope peptides MAGE-A3-Mp4 (SEQ ID NO: 1), MAGE-A10-Mp30 (SEQ ID NO: 2) and MAGE-A10-Mp32 (SEQ ID NO: 3) exist in NSCLC patients to produce specific killer CD8+T cells of immune response

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of an antigen peptide-antigen presenting cell complex in the preparation of tumor vaccines and / or tumor drugs, characterized in that: The antigen peptide-antigen presenting cell complex is an antigen presenting cell having a tumor T cell antigen epitope peptide with an amino acid sequence such as SEQ ID NO: 1 or SEQ ID NO: 3 on its surface; and the tumor is lung cancer.

2. The use according to claim 1, characterized in that: The antigen presenting cells are T2-A2 cells.

3. The use according to claim 2, characterized in that: The T2-A2 cells are T2 cells that overexpress HLA-A2.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of the antigen peptide-antigen presenting cell complex is: mixing and incubating a tumor T cell antigen epitope peptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 with antigen presenting cells to obtain the antigen peptide-antigen presenting cell complex.

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