Double-target chimeric antigen receptor co-expressing CD47 and IL-15, CAR-T cell and application of CAR-T cell

Abstract

The invention discloses a double-target chimeric antigen receptor for co-expression of CD47 and IL-15, a CAR-T cell and application of the CAR-T cell, and belongs to the technical field of genetic engineering, the double-target chimeric antigen receptor comprises a single-chain antibody scFv-EGFRv III, a single-chain antibody scFv-VEGF, CD47 and IL-15; the nucleotide artificial sequence of the single-chain antibody scFv-EGFRv III is as shown in SEQ ID NO. 6; the nucleotide artificial sequence of the single-chain antibody scFv-VEGF is as shown in SEQ ID NO. 9. According to the application disclosed by the invention, by performing sequence optimization on targets EGFRvIII and VEGF, the anti-tumor effect of CAR-T cells in glioblastoma can be improved, and by integrating immune checkpoint molecules CD47 and interleukin-15, the immunosuppression function of a CD47-SIRP alpha signal channel can be locally exerted.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to a dual-target chimeric antigen receptor co-expressing CD47 and IL-15, a CAR-T cell and its application. Background Art

[0002] The design of inserting immunosuppressive factors into the CAR (chimeric antigen receptor) structure has been one of the research hotspots in the field of tumor immunotherapy in recent years. This strategy aims to enhance the persistence and anti-tumor effect of CAR-T cells by locally regulating the immunosuppression of the tumor microenvironment (TME). Its core principle lies in reversing the inhibitory signals of the TME on immune cells, improving the functional state of CAR-T cells, and remodeling the immune microenvironment. CD47, as a key immunosuppressive molecule, its mechanism mainly focuses on the interaction with the macrophage surface receptor SIRPα. After binding to SIRPα, it triggers the immunoreceptor tyrosine inhibitory motif in the intracellular segment of SIRPα, recruits SHP-1 / 2 phosphatases, and inhibits the phagocytosis of macrophages.

[0003] Glioblastoma (GBM) is the most common malignant primary brain tumor, accounting for about 57% of all gliomas and 48% of all primary malignant central nervous system tumors. It has a poor prognosis, a high recurrence rate, and a low survival rate. The 5-year survival rate is 4%-5%. EGFRvⅢ (epidermal growth factor receptor variant Ⅲ) and vascular endothelial growth factor (VEGF) are both important molecular targets in glioblastoma (GBM).

[0004] Among them, EGFRvⅢ is a mutant of the EGFR gene, caused by the deletion of exons 2-7 (ΔEGFR), which produces a constitutively activated receptor tyrosine kinase. It is hardly expressed in normal tissues but is highly expressed in a variety of tumors (especially GBM), making it an ideal target. Currently, CAR-T cells targeting EGFRvⅢ show anti-tumor activity in preclinical models but face challenges of the blood-brain barrier and tumor microenvironment inhibition.

[0005] Regarding the application of immunosuppressive factors in the preparation of CAR-T cells, in a study published in "Nature" in 2023, the researchers designed a PD-1-CD28 immunostimulatory fusion protein. By fusing the extracellular domain of PD-1 with the intracellular domain of CD28, the function of the PD-1 / PD-L1 signaling pathway was altered. The original intention of this design was to transform the originally inhibitory PD-1 signal into an activating signal through the conformational change of the fusion protein, thereby enhancing the activity of T cells. However, in clinical trials, when using the CAR-T cell therapy with the PD-1-CD28 fusion protein to treat patients with B-cell non-Hodgkin lymphoma, the complete remission rate and objective remission rate were 41.2% and 58.8% respectively, and no significant advantage was shown compared with the second-generation CAR-T therapy. [Theo L, L B C, Hannah O, et al. Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy. [J]. British journal of cancer, 2023, 129(4): 696-705.].

[0006] Chinese patent CN108913718A discloses a method for preparing and applying CAR-T cells targeting EGFRvⅢ. This patent prepared CAR-T cells targeting EGFRvⅢ and genetically modified to knockout the PD-1 gene, but no verification was carried out regarding the anti-tumor effect and secreted factors. VEGF is only expressed at a low level in glial cells in normal brain tissue, but is significantly overexpressed in GBM, which makes VEGF an ideal target for CAR-T cell therapy for glioblastoma. However, in actual applications, due to reasons such as the VEGF expression level, the treatment effect is different from the expectation. Therefore, it is necessary to further improve the chimeric antigen receptor to enhance the targeting and binding ability of the chimeric antigen receptor to tumor antigens. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the present invention provides a dual-target chimeric antigen receptor co-expressing CD47 and IL-15, CAR-T cells and their applications. By optimizing the sequences of the targets EGFRvⅢ and VEGF, the anti-tumor effect of CAR-T cells in glioblastoma can be enhanced. By integrating the immune checkpoint molecule CD47 and interleukin-15 (IL-15), while enhancing the activity of targeting tumor antigens VEGF and EGFRvⅢ, the immunosuppressive function of the CD47-SIRPα signaling pathway is locally exerted.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A dual-target chimeric antigen receptor co-expressing CD47 and IL-15, the dual-target chimeric antigen receptor comprising single-chain antibody scFv-EGFRvⅢ, single-chain antibody scFv-VEGF, CD47, IL-15; The nucleotide artificial sequence of the single-chain antibody scFv-EGFRvⅢ is shown in SEQ ID NO.6; the nucleotide artificial sequence of the single-chain antibody scFv-VEGF is shown in SEQ ID NO.9; the nucleotide artificial sequence of CD47 is shown in SEQ ID NO.15; the nucleotide artificial sequence of IL-15 is shown in SEQ ID NO.16; The dual-target chimeric antigen receptor is obtained by sequentially connecting the following modules: leader, single-chain antibody scFv-EGFRvⅢ, linker, single-chain antibody scFv-VEGF, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, self-cleaving region T2A, CD47, self-cleaving region T2A, IL-15; The nucleotide artificial sequence of the leader is shown in SEQ ID NO.4; the nucleotide artificial sequence of the linker is shown in SEQ ID NO.7; the nucleotide artificial sequence of the CD8 Hinge region is shown in SEQ ID NO.10; the nucleotide artificial sequence of the CD28 transmembrane region is shown in SEQ ID NO.11; the nucleotide artificial sequence of the CD28-4-1BB co-stimulatory region is shown in SEQ ID NO.12; the nucleotide artificial sequence of the CD3ζ intracellular region is shown in SEQ ID NO.13; the nucleotide artificial sequence of the self-cleaving region T2A is shown in SEQ ID NO.14; The nucleotide artificial sequence of the dual-target chimeric antigen receptor is shown in SEQ ID NO.3.

[0009] A CAR-T cell, the CAR-T cell is obtained by modifying a T cell with the dual-target chimeric antigen receptor.

[0010] Use of the dual-target chimeric antigen receptor in the preparation of a drug for treating glioblastoma multiforme.

[0011] Use of the CAR-T cell in the preparation of a drug for treating glioblastoma multiforme.

[0012] Compared with the prior art, the beneficial effects of the present invention are: (1) Early clinical trials of preparing CAR-T cells targeting EGFRvⅢ have shown partial efficacy, but limited by antigen escape. Therefore, in the present invention, the VEGF target is combined as a dual target to reduce antigen escape, and the immunosuppressive factor CD47 is also added to block the CD47-SIRPα signaling pathway on the surface of tumor cells, relieve immunosuppression, and enhance the phagocytosis of tumor cells by macrophages; the CD47 expressed by CAR-T cells binds to SIRPα to prevent being phagocytosed by macrophages. At the same time, by blocking the CD47-SIRPα signaling pathway of tumor cells, the phagocytosis of tumor cells by macrophages is enhanced, and these macrophages are activated. This synergistic effect not only enhances the phagocytic function of macrophages, but also further enhances the anti-tumor effect by promoting antigen presentation and activating T cell responses; (2) For the CAR-EGFRvⅢ-VEGF-3 cells prepared in the present invention, when the effector-to-target ratio is 10:1, the release amount of IFN-γ is 16939 pg / mL, and the release amount is significantly increased, which can significantly enhance the killing ability of cells. The in vitro killing rate of tumor cells is as high as 95.1%, and the cell safety is good after toxicity verification. Description of the Drawings

[0013] Figure 1 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 in Example 1; Figure 2 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 in Example 1; Figure 3 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 in Example 1; Figure 4 Flow cytometry diagram of detecting the expression rate of the T cell activation index CD69 in Example 2; Figure 5 Fluorescence diagram of lentivirus transfection of 293T cells in Example 2; Figure 6 Infection rate diagram of recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 on activated T cells in Example 2; Figure 7 Infection rate diagram of recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 on activated T cells in Example 2; Figure 8Infection rate graph of recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 for activated T cells in Example 2; Figure 9 IFN-γ release results in the in vitro killing activity study of three types of cells in Example 3; Figure 10 In vitro killing rate results of four types of cells against tumor cells (effector-target ratio 10:1) in Example 4; Figure 11 Graph showing the change in mouse body weight in the in vivo toxicity experiment of CAR-T cells in Example 5. Detailed implementation manners

[0014] To have a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed implementation manners of the present invention are described below.

[0015] Example 1 Construction of recombinant expression vector The sequences of each module of CAR-EGFRvⅢ-VEGF are as follows: (1) Leader (SEQ ID NO.4); (2) Single-chain antibody scFv-EGFRvⅢ, the artificial nucleic acid sequence before optimization is shown as SEQ ID NO.5 in the sequence listing; the optimized artificial nucleic acid sequence is shown as SEQ ID NO.6 in the sequence listing; (3) Linker (SEQ ID NO.7); (4) Single-chain antibody scFv-VEGF, the artificial nucleic acid sequence before optimization is shown as SEQ ID NO.8 in the sequence listing; the optimized artificial nucleic acid sequence is shown as SEQ ID NO.9 in the sequence listing; (5) CD8 Hinge region (SEQ ID NO.10); (6) CD28 transmembrane region (SEQ ID NO.11); (7) CD28-4-1BB co-stimulatory region (SEQ ID NO.12); (8) CD3ζ intracellular region (SEQ ID NO.13); (9) Self-cleaving region T2A (SEQ ID NO.14); (10) CD47 (SEQ ID NO.15); (11) IL-15 (SEQ ID NO.16); The artificial nucleic acid sequences of SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 were ligated in sequence. The ligated sequence is shown as SEQ ID NO.1 in the sequence listing. Entrust Shandong Hongnuo Biotechnology Co., Ltd. to synthesize its entire expression cassette, insert it into the pLent-EF1α vector (purchased from Vigene), transform it into E. coli (Top10), and after correct sequencing, extract the plasmid using the plasmid extraction kit from OMEGA to obtain the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-1. The schematic diagram of its CAR structure is as Figure 1 shown. Extract the recombinant plasmid pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 from the positive clone, dilute it to 2 μg / μL, and store it at -80 °C for later use.

[0016] Using the same method, the artificial nucleic acid sequences of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 were ligated in sequence. The ligated sequence is shown as SEQ ID NO.2 in the sequence listing. The pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 expression vector was successfully constructed according to the above method. The schematic diagram of its CAR structure is as Figure 2 shown. Extract the recombinant plasmid pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 from the positive clone, dilute it to 2 μg / μL, and store it at -80 °C for later use.

[0017] Using the same method, the artificial nucleic acid sequences of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.14, and SEQ ID NO.16 were ligated in sequence. The ligated sequence is shown as SEQ ID NO.3 in the sequence listing. The pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 expression vector was successfully constructed according to the above method. The schematic diagram of its CAR structure is as Figure 3 shown. Extract the recombinant plasmid pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 from the positive clone, dilute it to 2 μg / μL, and store it at -80 °C for later use.

[0018] Example 2 Preparation of T cells modified with three plasmids 1. Preparation of activated T cells Take 75 ml of the patient's autologous peripheral blood, and isolate peripheral blood mononuclear cells with Ficoll-Paque lymphocyte separation solution. The separated cells are sorted into CD8+ T cells using the CD8+ sorting reagent provided by BD, and cell counting is performed. Inoculate at 1×10 6 cells / mL, and add KBM551 cell culture medium (purchased from Corning, product number: 88-551-CM) containing IL-2 at a final concentration of 1500 IU / mL. Then add CD3CD28 magnetic beads in the same number as the cells to activate T cells. After 24 hours of activation, remove the magnetic beads to obtain activated T cells. Use a flow cytometer to detect the expression rate of CD69, and the expression rate of CD69 is 65.6%, as shown in Figure 4 .

[0019] 2. Lentivirus packaging Resuscitate 293T cells and culture for 3 days. Passage according to cell density. When the cell confluence reaches 80% after 1 passage, perform transfection. Inoculate 6×10 5 cells / well in a six-well plate, and the total volume per well is 2 mL of DMEM medium (purchased from Gibco, product number 11960-044). Incubate overnight in a 37°C, 5% CO2 incubator. The next day, perform transfection. Before transfection, replace the medium in the six-well plate with fresh DMEM medium at 2 mL / well and incubate in a 37°C, 5% CO2 incubator for 1 hour.

[0020] Preparation of transfection reagent: In a 5 mL centrifuge tube, prepare reagent tubes A and B respectively; The composition of reagent tubes A and B is shown in Table 1.

[0021] Table 1 Composition of reagent tubes A and B

[0022] After preparation, let it stand for 5 minutes, then slowly add tube A to tube B, mix well, and let it stand at room temperature for 20 minutes to form a liposome-DNA mixture. Add the mixture to the culture flask, mix gently, and incubate in a 37°C, 5% CO2 incubator.

[0023] After 48 hours, observe the morphological changes of 293T cells after transfection under a microscope, as shown in Figure 5As shown. After 72 h, the cell culture supernatant containing the virus was collected into a centrifuge tube, centrifuged at 3500 rpm for 10 min to remove cell debris, filtered through a 4.5 μm filter, and then centrifuged at 4 °C at a centrifugal force of 70000 g for 2 h. The precipitate was resuspended in 100 μL of PBS, aliquoted and stored at -80 °C, and the virus titer was measured simultaneously. The virus titer of the virus solution containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 was 1.96×10 8 TU / mL, the virus titer of the virus solution containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 was 2.03×10 8 TU / mL, and the virus titer of the virus solution containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 was 2.01×10 8 TU / mL.

[0024] 3. Lentivirus infection of activated T cells Take out the above three virus solutions from -80 °C, thaw and add them to KBM551 serum-free medium containing IL-2 at a final concentration of 1500 IU / mL, and dilute the virus titer to 3×10 7 TU / mL to obtain the diluted virus solution. Resuspend 1×10 6 activated T cells obtained in the first step with 100 μL of the diluted virus solution to make the ratio of the number of virus particles to the number of activated T cells 3:1, and obtain the virus and cell suspension. Add the virus and cell suspension to a 6-well plate, 2 mL per well, and culture in a 37 °C, 5% CO2 incubator for 48 hours. Collect the cells, centrifuge at 400 g for 5 min, discard the supernatant, count the cells, and inoculate at a density of 1×10 6 cells / mL, add KBM551 serum-free medium containing IL-2 at a final concentration of 1500 IU / mL, and add liquid at a ratio of 1:1 on the basis of the original solution volume every 3 days. Culture in a 37 °C, 5% CO2 incubator for 13 days to expand the cells to sufficient amounts, and obtain T cells infected with recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-1, simply referred to as CAR-EGFRvⅢ-VEGF-1 cells, T cells infected with recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-2, simply referred to as CAR-EGFRvⅢ-VEGF-2 cells, and T cells infected with recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3, simply referred to as CAR-EGFRvⅢ-VEGF-3 cells.

[0025] Detect the expression of chimeric antigen receptor by flow cytometry, as Figures 6 - 8As shown, the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 in activated T cells in the present invention is 55.3%, the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 in activated T cells is 63.4%, and the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 in activated T cells is 73.2%.

[0026] Example 3 In vitro IFN-γ Release Experiment Using U87 MG cells (glioblastoma cell line) as target cells, and the effector cells are CAR-EGFRvⅢ-VEGF-1 cells, CAR-EGFRvⅢ-VEGF-2 cells, and CAR-EGFRvⅢ-VEGF-3 cells prepared in Example 2.

[0027] The effector-to-target ratios are 1:1, 5:1, and 10:1 respectively, and the number of target cells is 1×10 5 / well, corresponding to the effector cells according to different effector-to-target ratios. Each group has 3 replicate wells, and the average value of the 3 replicate wells is taken. 200 μL of DMEM medium containing 10 vol% FBS is added to each well. After the effector cells and target cells are co-cultured in a 37°C, 5% CO2 incubator for 24 h, the cell supernatant is collected, and the content of IFN-γ is detected using an ELISA kit. The results are shown in Table 2 Figure 9 As shown, the release amount of IFN-γ in the CAR-EGFRvⅢ-VEGF-2 cells and CAR-EGFRvⅢ-VEGF-3 cells of the present invention is increased compared with that in the CAR-EGFRvⅢ-VEGF-1 cells. The release amount of IFN-γ in the CAR-EGFRvⅢ-VEGF-2 cells is higher than that in the CAR-EGFRvⅢ-VEGF-1 cells, indicating that after codon optimization of the single-chain antibody, the killing ability of CAR-T cells can be improved. The release amount of IFN-γ in the CAR-EGFRvⅢ-VEGF-3 cells is higher than that in the CAR-EGFRvⅢ-VEGF-2 cells, indicating that CAR-T cells co-expressing CD47 and IL-15 have stronger killing ability.

[0028] Table 2 IFN-γ Release Amount (pg / mL) in the Study of the In vitro Killing Activity of CAR-EGFRvⅢ-VEGF Cells

[0029] Example 4 In vitro T Cell Killing Experiment Using U87 MG cells (glioblastoma cell line) as target cells, and CAR-EGFRvⅢ-VEGF-1 cells, CAR-EGFRvⅢ-VEGF-2 cells and CAR-EGFRvⅢ-VEGF-3 cells as effector cells, the killing activity was measured. According to the ratio of effector cells (1×10 5 / well) to target cells (1×10 4 / well) of 10:1, add them into a 48-well culture plate. The co-culture medium is DMEM medium containing 10 vol% FBS, 200 μL per well, and place it in a 5% CO2, 37 °C incubator for co-culture. After 24 h, add 10 μL of CCK-8 (Yeasen Biotech Co., Ltd., product number 40203ES60) to each well. After continuing to incubate for 2 h, use an enzyme-linked immunosorbent assay (ELISA) reader to detect at a wavelength of 450 nm and read the OD value.

[0030] The specific grouping is as follows: Experimental group A: Co-culture of CAR-EGFRvⅢ-VEGF-1 cells and target cells; Experimental group B: Co-culture of CAR-EGFRvⅢ-VEGF-2 cells and target cells; Experimental group C: Co-culture of CAR-EGFRvⅢ-VEGF-3 cells and target cells; Control group D: Co-culture of the activated T cells obtained in the first step of Example 2 and target cells.

[0031] Blank group: Target cells Calculate the cell killing rate according to the following formula: Killing rate (%) = [1 - (OD value of the blank group - OD value of the experimental group or control group) / OD value of the blank group] × 100%.

[0032] The results showed (see Figure 10 ), the killing rates of experimental groups A-C and control group D were 47.6%, 66.2%, 95.1%, and 14.3% in turn. The killing efficiencies of CAR-EGFRvⅢ-VEGF-2 cells and CAR-EGFRvⅢ-VEGF-3 cells were significantly higher than that of CAR-EGFRvⅢ-VEGF-1 cells, and all three were higher than the control group. Therefore, after the single-chain antibody sequence of this application is optimized, the killing rate of CAR-T cells can be improved. The CAR-T cells co-expressing CD47 and IL-15 can further enhance the killing ability of the codon-optimized CAR-T cells.

[0033] Example 5 In vivo toxicity experiment of CAR-T cells C57BL6 mice at 6-8 weeks old (purchased from Nanjing Junke Bioengineering Co., Ltd.) were divided into 5 groups, with 10 mice in each group, to verify the in vivo toxicity experiment of CAR-T cells. The experimental groups were respectively: a. Control group: Inject the same volume of normal saline into the tail vein; b. Experimental group 1: Inject 2×10 7 activated T cells obtained in Step 1 of Example 2 at a dose of 2×10 cells per mouse into the tail vein; c. Experimental group 2: Inject 2×10 7 CAR-EGFRvⅢ-VEGF-1 cells at a dose of 2×10 cells per mouse into the tail vein; d. Experimental group 3: Inject 2×10 7 CAR-EGFRvⅢ-VEGF-2 cells at a dose of 2×10 cells per mouse into the tail vein; e. Experimental group 4: Inject 2×10 7 CAR-EGFRvⅢ-VEGF-3 cells at a dose of 2×10 cells per mouse into the tail vein.

[0034] Observe the behavioral performance of the mice daily after injection, weigh the mice once a week, and use an in vivo imaging system to monitor the presence of CAR-T cells in the mice. After 45 days, dissect the mice and perform pathological observations on the main tissues of the mice, such as the brain, heart, lung, liver, colon, and kidney.

[0035] During the experiment, no abnormal behavioral manifestations were observed in the mice, such as Figure 11 As shown in Table 3, there was no significant difference in the weight gain of the mice, and CAR-T cells were continuously detected in the blood for 35 days. After dissecting the mice, no tissue lesions were found in the CAR-T mice during the pathological observation of the main tissues.

[0036] Table 3 Changes in body weight gain of mice in each experimental group after 45 days of culture

Claims

1. A dual-target chimeric antigen receptor co-expressing CD47 and IL-15, characterized in that, The dual-target chimeric antigen receptor comprises single-chain antibody scFv-EGFRvⅢ, single-chain antibody scFv-VEGF, CD47, and IL-15; The nucleotide artificial sequence of the single-chain antibody scFv-EGFRvⅢ is as shown in SEQ ID NO.6; the nucleotide artificial sequence of the single-chain antibody scFv-VEGF is as shown in SEQ ID NO.9; the nucleotide artificial sequence of CD47 is as shown in SEQ ID NO.15; the nucleotide artificial sequence of IL-15 is as shown in SEQ ID NO.

16.

2. The dual-target chimeric antigen receptor co-expressing CD47 and IL-15 according to claim 1, characterized in that, The dual-target chimeric antigen receptor is obtained by sequentially connecting the following modules: Leader, single-chain antibody scFv-EGFRvⅢ, linker, single-chain antibody scFv-VEGF, CD8 Hinge region, CD28 transmembrane region, CD28-4-1BB co-stimulatory region, CD3ζ intracellular region, self-cleaving region T2A, CD47, self-cleaving region T2A, IL-15.

3. The dual-target chimeric antigen receptor co-expressing CD47 and IL-15 according to claim 2, characterized in that, The nucleotide artificial sequence of the Leader is as shown in SEQ ID NO.4; the nucleotide artificial sequence of the linker is as shown in SEQ ID NO.7; the nucleotide artificial sequence of the CD8 Hinge region is as shown in SEQ ID NO.10; the nucleotide artificial sequence of the CD28 transmembrane region is as shown in SEQ ID NO.11; the nucleotide artificial sequence of the CD28-4-1BB co-stimulatory region is as shown in SEQ ID NO.12; the nucleotide artificial sequence of the CD3ζ intracellular region is as shown in SEQ ID NO.13; the nucleotide artificial sequence of the self-cleaving region T2A is as shown in SEQ ID NO.

14.

4. The dual-target chimeric antigen receptor co-expressing CD47 and IL-15 according to claim 1, characterized in that, The nucleotide artificial sequence of the dual-target chimeric antigen receptor is as shown in SEQ ID NO.

3.

5. A CAR-T cell, characterized in that, The CAR-T cell is obtained by modifying a T cell with the dual-target chimeric antigen receptor described in claim 1.

6. Use of the dual-target chimeric antigen receptor according to any one of claims 1-4 in the preparation of a drug for treating glioblastoma multiforme.

7. Use of the CAR-T cell according to claim 5 in the preparation of a drug for treating glioblastoma multiforme.

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