Tumor antigen peptides and vaccines targeting CD73 and preparation methods and applications thereof

Abstract

The present invention discloses tumor antigen peptides targeting CD73, vaccines, and preparation methods and applications thereof. Based on the amino acid sequence of CD73 protein, the present invention combines MHC typing to synthesize antigen peptides for targeted and immunotherapy of solid tumors, including drugs and vaccines for the preparation of new antigen-reactive T cell therapy and engineered T cell therapy. The tumor peptide vaccine targeting CD73 provided by the present invention has good immunogenicity and strong targeted killing ability of induced antigen-specific T cells.

Description

Technical Field

[0001] The present invention relates to the field of targeted drugs and immunotherapy, and specifically to tumor antigen peptides and vaccines targeting CD73, and preparation methods and applications thereof. Background Art

[0002] Malignant tumors have become the second leading cause of death worldwide and are also a factor that seriously threatens the health of the Chinese population. Currently, traditional methods such as surgery, chemotherapy, and targeted therapy have limited efficacy in treating malignant tumors. In recent years, immunotherapy that activates or rebuilds the immune system has attracted much attention. Immunotherapy, including therapeutic tumor vaccines, has achieved certain efficacy in solid tumors such as melanoma, non-small cell lung cancer, and brain glioma.

[0003] Select antigens highly expressed on the surface of tumor cells and inhibitory immune cells in the tumor microenvironment (TME) (such as M2 macrophages, MDSC, Treg, etc.) as targets, design and screen peptide vaccines targeting such microenvironment antigens (TMA), and induce endogenous specific T cell responses through vaccination, which can kill tumors and inhibitory immune cells in TME at the same time, thereby exerting a dual anti-tumor effect of directly inhibiting tumors and regulating the immune microenvironment. CD73 (also known as extracellular-5'-nucleotidase) is an important enzyme in the adenosine metabolic pathway, encoded by the NT5E gene, and highly expressed on the surface of inhibitory immune cells in a variety of tumor cells and TME.

[0004] The preparation process of personalized neoantigen vaccines, which are currently being studied more, is complicated and has a limited number of patients with targetable mutations, which greatly limits the application of neoantigen vaccines. At the same time, due to the complexity and inhibitory nature of the tumor immune microenvironment, the efficacy of therapeutic vaccines in solid tumors is poor. Therefore, it is necessary to explore vaccine development methods that can expand the beneficiary population and overcome the tumor immunosuppressive microenvironment. Summary of the invention

[0005] The purpose of the present invention is to provide tumor antigen peptides, vaccines targeting CD73, and preparation methods and applications thereof, for targeted and immunotherapeutic treatment of solid tumors, including drugs and vaccines for antigen-reactive T cell therapy, engineered T cell therapy, etc.

[0006] To achieve the above purpose, the technical solution provided by the present invention is:

[0007] A tumor antigen peptide targeting CD73 is synthesized based on the amino acid sequence of CD73 protein and combined with MHC typing, and the amino acid sequence is at least one of the following sequences:

[0008] SEQ ID NO 1: GTFDLVQLK;

[0009] SEQ ID NO 2: PFLSNPGTNL;

[0010] SEQ ID NO 3: HYQGSFPLV.

[0011] The CD73 protein is human CD73 protein or mouse CD73 protein; when the CD73 protein is human CD73 protein, the sequence of the antigenic peptide is SEQ ID NO 1; when the CD73 protein is mouse CD73 protein, the sequence of the antigenic peptide is SEQID NO 2 or SEQ ID NO 3.

[0012] The present invention also protects a method for preparing the tumor antigen peptide targeting CD73, comprising the following steps:

[0013] Analyze the amino acid sequence of CD73 protein, combine with MHC typing, use T cell epitope prediction tools to predict CD8 T cell epitopes for the full-length polypeptide sequence, and synthesize polypeptide fragments based on the predicted amino acid polypeptide sequence;

[0014] Evaluate the antigen-specific killing ability of reactive T cells induced by the synthesized polypeptide fragments, screen out immunogenic polypeptide fragments, and obtain tumor antigen peptides targeting CD73.

[0015] Furthermore, the T cell epitope prediction tool is at least one of SYFPEITHI, RANKPEP, and NetMHCpan4.1.

[0016] The present invention also protects a method for preparing a tumor peptide vaccine targeting CD73, wherein an adjuvant is added to the tumor antigen peptide targeting CD73 to prepare a tumor peptide vaccine targeting CD73.

[0017] Furthermore, the adjuvant is CpG ODN.

[0018] The present invention also protects the prepared tumor peptide vaccine targeting CD73.

[0019] The present invention also protects the use of the tumor antigen peptide targeting CD73 in the preparation of solid tumor targeted therapy or immunotherapy drugs.

[0020] Furthermore, it includes antigen-reactive T cell drugs and engineered T cell drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention analyzes various public databases of solid tumors and finds that the expression of NT5E in various solid tumor tissues such as gastric cancer, pancreatic cancer, and lung adenocarcinoma is significantly higher than that in normal tissues, and NT5E is expressed on the surface of various inhibitory immune cells including Treg, MDSC, and M2 macrophages in the tumor microenvironment. The present invention selects CD73, a target that is highly expressed on the surface of various immune regulatory cells and various tumor cell lines, to design and screen tumor vaccines, and evaluates the potential value of peptide vaccines targeting CD73 as a solid tumor immunotherapy strategy, in order to remove the key restrictive factors of the solid tumor microenvironment and provide a new treatment model for the field of solid tumor immunotherapy.

[0023] The present invention expands the feasible targets of tumor vaccines, and predicts high-scoring antigen peptides targeting human and mouse CD73 proteins through T cell epitope prediction tools SYFPEITHI, RANKPEP, and NetMHCpan4.1, and synthesizes three polypeptide fragments by chemical synthesis. Flow cytometry, CBA, ELISPOT, cell killing experiments and other methods verify that they can significantly activate T cells in vitro and significantly increase the killing ability of T cells against tumors; next, mouse 4T1 subcutaneous tumor, in situ tumor model and CT26 subcutaneous tumor model were established to verify that the peptide vaccine targeting CD73 can effectively inhibit tumor growth and improve the tumor microenvironment.

[0024] The CD73-targeting tumor peptide vaccine provided by the present invention has good immunogenicity and strong targeted killing ability of the induced specific T cells, and can be used for T cell immunotherapy of patients with solid tumors, preparation of engineered T cells, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Flow cytometry was used to analyze T cell activation in peripheral blood mononuclear cells from gastric cancer patients after stimulation with CD73 antigen peptide; negative control: no peptide was added.

[0026] Figure 2 : In vitro reactivity of peripheral blood mononuclear cells from gastric cancer patients to CD73 antigen peptide was analyzed by IFN-γ CBA assay; negative control: no peptide was added.

[0027] Figure 3 : Flow cytometry was used to analyze the T cell activation of Balb / c mouse lymph node cells after stimulation with CD73 antigen peptide; negative control: no peptide was added.

[0028] Figure 4 : The in vitro reactivity of Balb / c mouse lymph node cells to CD73 antigen peptide was analyzed by IFN-γCBA and ELISPOT assays; negative control: no peptide was added.

[0029] Figure 5: After Balb / c mouse lymph node cells were stimulated and activated by P2 and P3 peptides in vitro, the lymph node cells were used to kill 4T1 tumor cells at different effector-target ratios; negative control: mouse lymph node cells without peptide induction.

[0030] Figure 6 : Schematic diagram of the treatment mode of P2 and P3 vaccines given to mice bearing mouse breast cancer 4T1 homologous transplant tumor model, pictures of tumor size in each group at the experimental endpoint, curves of tumor volume change over time, and tumor weight in each group at the experimental endpoint; negative control: normal saline + CpG ODN.

[0031] Figure 7 : Schematic diagram of the treatment mode of P2 and P3 vaccines given to mice bearing the mouse colorectal cancer CT26 homologous transplant tumor model, pictures of tumor size in each group at the experimental endpoint, curves of tumor volume change over time, and tumor weight in each group at the experimental endpoint; negative control: normal saline + CpG ODN. DETAILED DESCRIPTION

[0032] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0033] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0034] Some of the terms in the present invention are explained and described as follows:

[0035] The CpG involved in the embodiments of the present invention refers to the abbreviation of CpG ODN.

[0036] The present invention synthesizes antigen peptides based on human and mouse CD73 protein sequences, respectively, combined with HLA typing HLA-A*1101 and Balb / c mouse MHC typing H2Kd.

[0037] 1. CD73 antigen peptide prediction

[0038] Based on the amino acid sequences of human and mouse CD73 proteins, combined with the most common HLA typing in the Chinese population (HLA-A*1101) and the Balb / c mouse MHC typing H2d, the T cell epitope prediction tools SYFPEITHI, RANKPEP, and NetMHCpan4.1 were used to synthesize epitope peptides with a length of 9-11 amino acids, and a total of one human CD73 antigen peptide and two mouse CD73 antigen peptides were synthesized.

[0039] 2. Patient HLA typing test

[0040] 2 ml of peripheral blood was drawn from the patient, anticoagulated with EDTA or sodium citrate, and the four-digit high-resolution typing of the HLA-A locus was detected using PCR-SBT technology to select HLA-A*1101 patients.

[0041] 3. Obtaining PBMC from Peripheral Blood

[0042] (1) After disinfecting the cell bag obtained by COBE SpectraTM MNC system (or the heparin anticoagulated blood drawn by syringe), place it in the clean bench, transfer the cells to a 50ml centrifuge tube using a 20ml syringe, and dilute with saline in a 1:1 ratio;

[0043] (2) Add 20 ml of human lymphocyte separation medium into the centrifuge tube;

[0044] (3) Using a Pasteur pipette, slowly superimpose the cell solution diluted with physiological saline onto the human lymphocyte separation solution along the tube wall;

[0045] (4) Horizontal centrifugation at 800 g for 20 min;

[0046] (5) After centrifugation, the tube is divided into three layers: the upper layer is plasma, the lower layer is mainly red blood cells and granulocytes, and the middle layer is lymphocyte separation fluid. At the interface between the upper and middle layers, there is a white film layer mainly composed of mononuclear cells, which is the peripheral blood mononuclear cell layer;

[0047] (6) Use a pipette to insert into the buffy coat layer, aspirate the mononuclear cells, place them into another 50 ml centrifuge tube, add physiological saline, and centrifuge horizontally at 300 g for 10 min.

[0048] (7) Discard the supernatant, add physiological saline, mix well, and centrifuge horizontally at 300g for 10 min;

[0049] (8) Discard the supernatant, add cell culture medium, mix well, and centrifuge horizontally at 300g for 5 min;

[0050] (9) Discard the supernatant, dilute with AIM-V culture medium, count the cells, and take an appropriate amount of cells to freeze as a specimen.

[0051] 4. Preparation of Single Cell Suspension from Mouse Draining Lymph Nodes

[0052] (1) Freshly dissect the intact mouse draining lymph nodes and immerse them in ice-cold saline;

[0053] (2) Moisten a 70 μm cell strainer with saline, place the lymph node in it, grind it gently, then rinse it with saline and collect the grinding liquid;

[0054] (3) The lymph node grinding solution was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and the lymph node was resuspended in physiological saline to a concentration of 1×106 Transfer the single cell suspension of about 100 μg / ml to a flow cytometry tube, mark the grouping information, and set aside.

[0055] 5. In vitro induction of PBMC and mouse lymph node cell activation

[0056] (1) PBMCs or lymph node single cells were isolated by the above method and suspended in AIM-V medium (Gibico, USA) and counted;

[0057] (2) Use a 96-well plate with a U-shaped bottom and add 1 × 10 5 Each cell was diluted in 200ul culture medium and incubated with 10μM antigen peptide. The U-shaped bottom is used to promote better contact between cells. The culture medium consists of AIM-V medium (Gibco) containing a small amount of interleukin-2 (IL-2, 100U / mL, Peprotech) and 10% fetal calf serum (FCS, Gibco).

[0058] (3) Each round of stimulation culture lasted 3 days, with the medium replaced by half and supplemented with the corresponding antigen peptide (10uM) and IL-2 (100U / mL);

[0059] (4) After 2-3 cycles of peptide stimulation in vitro, fresh culture medium was replaced and 10 μM antigen peptide was added again for overnight stimulation. Within 24 h of the next day after the last round of stimulation (i.e., on the 7th and / or 10th day of culture), the expression of IFN-γ was detected by ELISPOT to evaluate the level of T cell immune response, and the expression level of T cell activation marker 4-1BB (CD137) was evaluated by flow cytometry to identify the immunogenicity of different antigen peptides. PBMCs stimulated with no peptide (culture medium) or irrelevant peptide were used as negative controls (NS).

[0060] 6. Enzyme-linked immunosorbent assay (ELISPOT)

[0061] (1) Activate the pre-coated plate by adding 200 μL of AIM-V serum-free medium, leaving it at room temperature for 10 minutes, and pouring it;

[0062] (2) According to the designed grouping, T cells and peptides were added at 10-50 μg / ml, with three replicates in each group;

[0063] (3) After adding all samples, cover the plate, mark it, and place it in a 37°C, 5% CO2 incubator for 20 hours;

[0064] (4) Pour the cells and culture medium in the well;

[0065] (5) Cell lysis: Add 200 μL of ice-cold deionized water to each well and place in a 4°C refrigerator for 10 min (hypotonic cell lysis);

[0066] (6) Washing: Wash each well six times with 260 μL 1X Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0067] (7) Detection antibody incubation: Add 100 μL of diluted biotin-labeled antibody to each well and incubate at 37°C, 5% CO2 incubator for 1 hour;

[0068] (8) Washing: Wash each well six times with 260 μL 1X Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0069] (9) Avidin incubation: Add 100 μL of diluted enzyme-labeled avidin to each well and incubate at 37°C for 1 hour;

[0070] (10) Washing: Wash each well six times with 260 μL 1X Washing buffer for 60 seconds each time. Dry on absorbent paper after each wash.

[0071] (11) Color development: Prepare AEC color development solution according to the reagent preparation instructions, add 100 μL of color development solution to each well, and incubate at 37°C, 5% CO2 for color development, checking every 5 minutes;

[0072] (12) After the spots grow to a suitable size, wash twice with deionized water to terminate the color development process; turn the plate upside down on absorbent paper and pat dry the tiny water droplets. Then remove the protective layer and place it in a ventilated place at room temperature to allow the membrane to dry naturally;

[0073] (13) Count the spots on the ELISPOT plate and record various parameters of the spots for analysis.

[0074] 7. IFN-γ determination by cytometric bead array (CBA)

[0075] (1) Preparation of human and mouse soluble protein free combination reagent standards

[0076] 1) Transfer one bottle of freeze-dried cytokine standard pellets to a 15 mL centrifuge tube and mark it as high concentration standard (2500 pg / mL);

[0077] 2) Dilute the standard with 4 mL of Assay Diluent and leave at room temperature for more than 15 minutes;

[0078] 3) Mix the standard by gently pipetting. Do not vortex or shake vigorously.

[0079] 4) Mark the gradient dilution ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 on the 9 flow-through tubes;

[0080] 5) Add 500 μL Assay Diluent to each tube;

[0081] 6) Starting from the standard with the highest concentration, add 500 μL of diluent one by one to dilute the standard sample in a gradient manner;

[0082] 7) Add 500 μL Assay Diluent into a new flow cytometry tube as a control tube (0 pg / mL).

[0083] (2) Mixed human and mouse soluble protein free combination reagent capture microspheres

[0084] 1) Determine the quantity of BD CBA human soluble protein free combination reagent (i.e., the types of free combination cytokines);

[0085] 2) Determine the number of test samples; Note: To avoid liquid loss during the sample addition process leading to insufficient reagents, three more samples can be mixed when mixing the capture microspheres;

[0086] 3) Before mixing the capture microspheres, each capture microsphere needs to be vortexed for at least 15 seconds to fully shake and mix;

[0087] 4) Determine the total amount of capture microspheres that need to be diluted in the test based on 50 μL of each sample;

[0088] 5) Determine the volume of each capture microsphere according to 1 μL of each sample; if the number of samples is 40, the volume of the capture microsphere is 40 μL;

[0089] 6) Determine the volume of capture microsphere dilution solution;

[0090] 7) Take a new tube, mark it as "mixed capture microspheres", and mix the capture microspheres and the capture microsphere dilution solution in the tube.

[0091] (3) Preparation of human and mouse soluble protein free combination reagent PE-labeled detection antibodies

[0092] 1) Determine the quantity of BD CBA human soluble protein free combination reagent (i.e., the types of free combination cytokines);

[0093] 2) Determine the number of test samples;

[0094] 3) Determine the total amount of PE-labeled detection antibody to be diluted in the test according to the volume of 50 μL per sample;

[0095] 4) Determine the amount of PE-labeled detection antibody reagent according to 1 μL of each sample;

[0096] 5) Determine the volume of PE-labeled detection antibody dilution solution;

[0097] 6) Dilute the PE detection antibody reagent with the detection antibody diluent, mix well in a tube, and store at 4°C in the dark before use.

[0098] (4) Incubation of human and mouse soluble protein free combination reagents with samples

[0099] 1) Standards and samples to be tested: Add 50 μL of the gradient diluted standard to each standard tube, and add 50 μL of the sample to be tested to each sample tube;

[0100] 2) Vortex the microspheres for at least 5 seconds, add 50 μL of mixed microspheres to each tube, and mix gently;

[0101] 3) Incubate at room temperature for 1 hour;

[0102] 4) Add 50 μL of PE-labeled detection antibody to each tube and mix gently;

[0103] 5) Incubate at room temperature for 2 hours.

[0104] (5) On-machine testing

[0105] 1) Add 1 mL of PBS solution to each tube to wash the samples and centrifuge at 200 g for 5 minutes;

[0106] 2) Carefully aspirate or gently pour off the supernatant, and add 300 μL PBS solution to each tube to resuspend the cells;

[0107] 3) After the instrument is calibrated, it should be tested as soon as possible.

[0108] (6) Data analysis

[0109] After the sample data were collected (FCS2.0 format), the CBA-specific analysis software FCAP Array V3.0 was used to draw the standard curve and analyze the data.

[0110] 8. Flow cytometry to measure markers of T cell activation

[0111] (1) Adjust the concentration of activated PBMCs or mouse lymph node cells to 1×10 6 / ml. After the cell suspension is evenly blown, centrifuge at 1500rpm for 5min, discard the supernatant, add PBS, centrifuge at 1500rpm for 5min, discard the supernatant, and resuspend the cells in 100μl PBS into the flow tube, and mark each group of cells on the tube wall;

[0112] (2) Add anti-human CD3, CD8, CD25, CD69, CD107a, CD137 and other fluorescent antibodies to human PBMCs to label the cells, and set up a negative control group, a single-label group, and an isotype control group; add anti-mouse CD3, CD8, CD25, CD69, CD107a, CD137 and other fluorescent antibodies to mouse lymph node cells to label the cells, and set up a negative control group, a single-label group, and an isotype control group;

[0113] (3) Stain at 4°C in the dark for 30 min;

[0114] (4) Add 1 ml of PBS and mix gently by pipetting;

[0115] (5) Centrifugation, 1500 rpm, 5 min, discard the supernatant;

[0116] (6) Add 100 μl PBS to resuspend the cells and test on the flow cytometer. Collect data using Beckman software and analyze flow cytometry results using Flowjo software.

[0117] 9. Antigen-specific T cells killing tumor cells in vitro experiment

[0118] CFSE / PI labeling method: The specific T cells induced by antigen peptide for one week were used as effector cells (E), and the mouse 4T1 breast cancer cell line was used as target cells (T) to evaluate the antigen-specific killing ability of specific T cells.

[0119] (1) Marking of target cells:

[0120] a) Collect mouse 4T1 cells in the logarithmic growth phase, place them in serum-free RPMI 1640 medium, and incubate them in a 37°C, 5% CO2 incubator for 2 to 4 hours;

[0121] b) Then, 4T1 cells were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes to precipitate the cells, and washed once with PBS. The target cell concentration was adjusted to 10 6 10 cells / ml, add CFSE (final concentration 4 nM), mix well and incubate at 37°C in the dark for 10 minutes, add 10 times the volume of PBS, centrifuge again at 1000 rpm for 5 minutes, discard the supernatant, wash twice; resuspend the target cells in culture medium, the cell concentration is 10 5 cells / ml; placed in a 37°C, CO2 incubator for later use.

[0122] (2) Preparation of effector cells

[0123] Collect different groups of effector cells into different centrifuge tubes, centrifuge at 1000 rpm for 5 minutes to precipitate the cells, and discard the supernatant. Resuspend the cells in culture medium at a cell concentration of 5×10 6cells / ml. Add effector cells and CFSE-labeled target cells according to the predetermined effector-target ratio. Incubate the cells at 37°C in a CO2 incubator for 4 to 16 hours.

[0124] (3) PI staining

[0125] The cell mixture was centrifuged to precipitate the cells and the supernatant was discarded; each group of cells was resuspended in 100 μl PBS, and PI was added to a final concentration of 50 μg / ml; incubated at 4°C in the dark for 15 minutes; the cell mixture was centrifuged to precipitate the cells, and then the cells were resuspended in 400 μl PBS; the results were detected and analyzed using a flow cytometer.

[0126] 10. Establishment of 4T1 and CT26 subcutaneous tumor models in mice

[0127] (1) Mouse 4T1 and CT26 cell lines in the logarithmic growth phase were selected, and the cells were collected after trypsin digestion and washing. The final cell concentration was adjusted to 2×10 6 / mL and 2×10 7 / mL; BALB / C female mice aged 4-5 weeks were selected, and the mixed tumor cell suspension was sucked into a 1mL sterile syringe that was pre-cooled on ice for 2 hours. Subcutaneous tumors were implanted in BALB / C female mice. The tumor cell mass was approximately 2×10 5 / only and 2×10 6 / mouse (100μL), implanted subcutaneously in the left groin, until a small bulge is seen at the implantation site, and the tumor formation is observed after 5-7 days;

[0128] (2) Evaluation of anti-tumor efficacy in 4T1 and CT26 subcutaneous tumor animal models

[0129] The above method was used to establish 4T1 and CT26 subcutaneous tumor models, respectively. The day of tumor inoculation was the starting time, marked as Day 0 (D0); the mice were randomly divided into three groups, 5 mice in each group, including a blank control group (Ctrl) injected subcutaneously with normal saline and an experimental group (P2, P3) injected subcutaneously with P2 and P3 vaccines, where the doses of P2 and P3 were 100 μg / mouse, and the dose of CpG was 20 μg / mouse. The 4T1 model was administered on D6, D13, and D19; the mice were weighed every other day, and the long diameter (a) and short diameter (b) of the tumor were measured. The tumor volume was calculated using the formula a×b 2 / 2; mice were killed on D22; CT26 models were given drugs on D3, D9, and D15; mice were weighed every other day, and the long diameter (a) and short diameter (b) of the tumor were measured. The tumor volume was calculated using the formula a×b 2 / 2; mice were killed on D17; tumors were completely removed and tumor weights were recorded.

[0130] Experimental results analysis:

[0131] 1. Prediction of highly immunogenic CD73 antigen

[0132] The higher the score of epitope prediction software SYFPEITHI and RANKPEP, the stronger the peptide is considered to be able to bind to MHC molecules; the positive interpretation of the prediction results of MHCpan4.1 software: IC50 < 500nM or %Rank < 2.0 is considered to be able to bind to MHC molecules; IC50 < 50nM or %Rank < 0.5 is considered to be able to bind strongly to MHC molecules.

[0133] The present invention selects peptide sequences with higher SYFPEITHI and RANKPEP scores and lower MHCpan4.1% Rank scores for synthesis; the table shows three synthesized short peptide sequences, including the short peptide sequence P1 targeting human CD73; and the short peptide sequences P2-3 targeting mouse CD73.

[0134]

[0135]

[0136] 2. P1, P2 and P3 were used to induce activation of PBMC and mouse lymph node cells in vitro, respectively. The IFN-γ secretion levels in the activated cells and their supernatants were detected by ELISPOT and CBA, respectively. The results showed that the short peptide P1 targeting human CD73 stimulated PBMC to secrete IFN-γ levels that were more than 3 times higher than those in the negative control group; the short peptides P2 and P3 targeting mouse CD73 stimulated mouse lymph node cells to secrete IFN-γ levels that were more than 4 times higher than those in the negative control group.

[0137] 3. After using P1 peptide and P2, P3 peptides to stimulate and activate human T cells and mouse T cells respectively, flow cytometry detection found that after P1 stimulation, the CD25, CD69, and CD137 of human CD8+T cells increased by 3 times compared with the control group, indicating that P1 can significantly activate human T cells; flow cytometry detection found that after P2 and P3 stimulation, the CD25, CD69, and CD137 of mouse CD8+T cells increased by 3 times compared with the control group, indicating that P2 and P3 can significantly activate mouse T cells.

[0138] 4. The effector cell killing experiment found that P2 and P3 specific cells can significantly increase the tumor killing ability of mouse T cells.

[0139] 5. In the 4T1 and CT26 subcutaneous tumor models, it was found that P2 and P3 peptides could significantly inhibit tumor growth in the presence of CpG adjuvant.

[0140] Therefore, the short peptide P1 (GTFDLVQLK) targeting human CD73 and the short peptides P2 (PFLSNPGTNL) and P3 (HYQGSFPLV) targeting mouse CD73 have good immunogenicity, and the induced specific T cells have strong targeted killing ability and the ability to inhibit tumor growth. They can be used for antigen-specific T cell immunotherapy of patients with CD73-positive tumor tissue, and can also be used in the preparation of vaccines and engineered T cells in the future.

[0141] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A tumor antigen peptide targeting CD73, characterized in that: Based on the amino acid sequence of CD73 protein and combined with MHC typing, an antigen peptide is synthesized, and its amino acid sequence is at least one of the following sequences: SEQ ID NO 1: GTFDLVQLK; SEQ ID NO 3: HYQGSFPLV.

2. The tumor antigen peptide targeting CD73 according to claim 1, characterized in that: The CD73 protein is human CD73 protein or mouse CD73 protein; when the CD73 protein is human CD73 protein, the sequence of the antigenic peptide is SEQ ID NO 1; when the CD73 protein is mouse CD73 protein, the sequence of the antigenic peptide is SEQ ID NO 3.

3. The method for preparing a tumor antigen peptide targeting CD73 according to claim 1 or 2, characterized in that: The following steps are involved: Analyze the amino acid sequence of CD73 protein, combine with MHC typing, use T cell epitope prediction tools to predict CD8 T cell epitopes for the full-length polypeptide sequence, and synthesize polypeptide fragments based on the predicted amino acid polypeptide sequence; Evaluate the antigen-specific killing ability of reactive T cells induced by the synthesized polypeptide fragments, screen out immunogenic polypeptide fragments, and obtain tumor antigen peptides targeting CD73.

4. The method for preparing a tumor immune antigen peptide targeting CD73 according to claim 3, characterized in that: The T cell epitope prediction tool is at least one of SYFPEITHI, RANKPEP, and NetMHCpan4.

1.

5. A method for preparing a tumor peptide vaccine targeting CD73, characterized in that: An adjuvant is added to the CD73-targeting tumor antigen peptide described in claim 1 or 2 to prepare a CD73-targeting tumor peptide vaccine.

6. The method for preparing a tumor peptide vaccine targeting CD73 according to claim 5, characterized in that: The adjuvant is CpG ODN.

7. A tumor peptide vaccine targeting CD73 prepared by the method of claim 5 or 6.

8. Use of the CD73-targeting tumor antigen peptide according to claim 1 or 2 in the preparation of targeted therapeutic or immunotherapeutic drugs for breast cancer or colorectal cancer.

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