A dual-target chimeric antigen receptor co-expressing CD47 and IL-15, CAR-T cell and its application

Abstract

The present invention discloses a dual-target chimeric antigen receptor, CAR-T cells and applications thereof that co-express CD47 and IL-15, belonging to the field of genetic engineering technology. The dual-target chimeric antigen receptor includes a single-chain antibody scFv-EGFRvⅢ, a single-chain antibody scFv-VEGF, CD47, and IL-15; the artificial nucleotide sequence of the single-chain antibody scFv-EGFRvⅢ is shown in SEQ ID NO.6; the artificial nucleotide sequence of the single-chain antibody scFv-VEGF is shown in SEQ ID NO.9. The present invention can enhance the anti-tumor effect of CAR-T cells in glioblastoma by sequence optimization of the targets EGFRvⅢ and VEGF, and can locally exert the immunosuppressive function of the CD47-SIRPα signaling pathway by integrating the immune checkpoint molecules CD47 and interleukin-15.

Description

Technical Field

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

[0002] The design of inserting immunosuppressive factors into CAR (chimeric antigen receptor) structures has been a research hotspot in the field of tumor immunotherapy in recent years. This strategy aims to enhance the persistence and anti-tumor efficacy of CAR-T cells by locally modulating the immunosuppressive properties of the tumor microenvironment (TME). Its core principle is to reverse the inhibitory signals of the TME on immune cells, improve the functional state of CAR-T cells, and reshape the immune microenvironment. CD47, as a key immunosuppressive molecule, mainly interacts with the macrophage surface receptor SIRPα. Upon binding to SIRPα, it triggers the immunoreceptor tyrosine inhibitory motif in the intracellular domain of SIRPα, recruiting SHP-1 / 2 phosphatases and inhibiting macrophage phagocytosis.

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

[0004] EGFRvIII is a mutant of the EGFR gene caused by the deletion of exons 2-7 (ΔEGFR), resulting in a constitutively activated receptor tyrosine kinase that is barely expressed in normal tissues but highly expressed in various tumors, particularly GBM, making it an ideal target. Currently, CAR-T cells targeting EGFRvIII have demonstrated anti-tumor activity in preclinical models but face challenges with the blood-brain barrier and tumor microenvironment.

[0005] In order to apply immunosuppressive factors to the preparation of CAR-T cells, in a study published in Nature in 2023, 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 changed. The original intention of this design was to transform the originally inhibitory PD-1 signal into an activating signal through conformational changes in the fusion protein, thereby enhancing the activity of T cells. However, in clinical trials, CAR-T cell therapy using PD-1-CD28 fusion protein had a complete remission rate and objective remission rate of 41.2% and 58.8%, respectively, in the treatment of patients with B-cell non-Hodgkin's lymphoma, which did not show a significant advantage over the second-generation CAR-T therapy. [Theo L, LBC, Hannah O, et al. Rational design of PD-1-CD28 immunostimulatoryfusionproteins 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 using CAR-T cells targeting EGFRvIII. This patent prepares a CAR-T cell that targets EGFRvIII and is genetically modified to knock out the PD-1 gene, but does not verify its anti-tumor effect or secretory factors. VEGF is only expressed at low levels in glial cells in normal brain tissue, but is significantly overexpressed in GBM, making it an ideal target for CAR-T cell therapy of glioblastoma. However, in actual use, the therapeutic effect falls short of expectations due to limitations such as VEGF expression. Therefore, it is necessary to further improve the chimeric antigen receptor to enhance its ability to target tumor antigens. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a dual-target chimeric antigen receptor, CAR-T cells and their applications that co-express CD47 and IL-15. 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 molecules CD47 and interleukin-15 (IL-15), the activity of the targeted tumor antigens VEGF and EGFRvⅢ is enhanced while the immunosuppressive function of the CD47-SIRPα signaling pathway is locally exerted.

[0008] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0009] A dual-target chimeric antigen receptor co-expressing CD47 and IL-15, the dual-target chimeric antigen receptor comprising a single-chain antibody scFv-EGFRvⅢ, a single-chain antibody scFv-VEGF, CD47, and IL-15;

[0010] The artificial nucleotide sequence of the single-chain antibody scFv-EGFRvIII is shown in SEQ ID NO.6; the artificial nucleotide sequence of the single-chain antibody scFv-VEGF is shown in SEQ ID NO.9; the artificial nucleotide sequence of CD47 is shown in SEQ ID NO.15; and the artificial nucleotide sequence of IL-15 is shown in SEQ ID NO.16.

[0011] The dual-target chimeric antigen receptor is obtained by sequentially connecting the following modules: a leader, a single-chain antibody scFv-EGFRvⅢ, a linker, a single-chain antibody scFv-VEGF, a CD8 Hinge region, a CD28 transmembrane region, a CD28-4-1BB costimulatory region, a CD3ζ intracellular region, a self-cleavage region T2A, CD47, a self-cleavage region T2A, and IL-15;

[0012] The artificial nucleotide sequence of the leader is shown in SEQ ID NO.4; the artificial nucleotide sequence of the linker is shown in SEQ ID NO.7; the artificial nucleotide sequence of the CD8 Hinge region is shown in SEQ ID NO.10; the artificial nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO.11; the artificial nucleotide sequence of the CD28-4-1BB co-stimulatory region is shown in SEQ ID NO.12; the artificial nucleotide sequence of the CD3ζ intracellular region is shown in SEQ ID NO.13; and the artificial nucleotide sequence of the self-cleavage region T2A is shown in SEQ ID NO.14.

[0013] The artificial nucleotide sequence of the dual-target chimeric antigen receptor is shown in SEQ ID NO.3.

[0014] A CAR-T cell, wherein the CAR-T cell is obtained from the dual-target chimeric antigen receptor modified T cell.

[0015] The dual-target chimeric antigen receptor is used in the preparation of a drug for treating glioblastoma.

[0016] The use of the CAR-T cells in the preparation of a drug for treating glioblastoma.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Currently, early clinical trials targeting EGFRvⅢ to prepare CAR-T cells have shown partial efficacy, but are limited by antigen escape. Therefore, the present invention combines VEGF as a dual target to reduce antigen escape, and also adds the immunosuppressive factor CD47 to block the CD47-SIRPα signaling pathway on the surface of tumor cells, relieve immunosuppression, and enhance the phagocytosis of tumor cells by macrophages; CD47 expressed by CAR-T cells binds to SIRPα to prevent phagocytosis by macrophages, and 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;

[0019] (2) The CAR-EGFRvⅢ-VEGF-3 cells prepared by the present invention release 16939 pg / mL of IFN-γ at an effector-target ratio of 10:1, which is a significant increase in the release amount. This can significantly enhance the cell killing ability, with an in vitro killing rate of up to 95.1% for tumor cells. Furthermore, the cell safety is good after toxicity verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvIII-VEGF-1 in Example 1;

[0021] Figure 2 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvIII-VEGF-2 in Example 1;

[0022] Figure 3 Schematic diagram of the CAR structure of the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 in Example 1;

[0023] Figure 4 This is a flow cytometric graph of the expression rate of CD69, an indicator of T cell activation, detected by flow cytometry in Example 2;

[0024] Figure 5 This is a fluorescence image of 293T cells transfected with lentivirus in Example 2;

[0025] Figure 6 This is a graph showing the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-1 on activated T cells in Example 2;

[0026] Figure 7This is a graph showing the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-2 in activated T cells in Example 2;

[0027] Figure 8 This is a graph showing the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-3 on activated T cells in Example 2;

[0028] Figure 9 The results of IFN-γ release in the in vitro cytotoxicity study of the three cells in Example 3 are as follows;

[0029] Figure 10 The results of the in vitro killing rate of tumor cells by the four cell types in Example 4 (effect-target ratio 10:1);

[0030] Figure 11 This is a graph showing changes in mouse body weight during the in vivo toxicity experiment to verify CAR-T cells in Example 5. DETAILED DESCRIPTION

[0031] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0032] Example 1 Construction of recombinant expression vector

[0033] The sequences of each module of CAR-EGFRvⅢ-VEGF are as follows:

[0034] (1) Leader (SEQ ID NO. 4);

[0035] (2) Single-chain antibody scFv-EGFRvⅢ, the artificial nucleic acid sequence before optimization is shown in SEQ ID NO.5 in the sequence listing; the artificial nucleic acid sequence after optimization is shown in SEQ ID NO.6 in the sequence listing;

[0036] (3) Linker (SEQ ID NO. 7);

[0037] (4) Single-chain antibody scFv-VEGF, the artificial nucleic acid sequence before optimization is shown in SEQ ID NO.8 in the sequence listing; the artificial nucleic acid sequence after optimization is shown in SEQ ID NO.9 in the sequence listing;

[0038] (5) CD8 Hinge region (SEQ ID NO. 10);

[0039] (6) CD28 transmembrane region (SEQ ID NO. 11);

[0040] (7) CD28-4-1BB co-stimulatory region (SEQ ID NO. 12);

[0041] (8) CD3ζ intracellular region (SEQ ID NO. 13);

[0042] (9) Self-cleavage region T2A (SEQ ID NO. 14);

[0043] (10) CD47 (SEQ ID NO. 15);

[0044] (11) IL-15 (SEQ ID NO. 16);

[0045] 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 connected in sequence, and the connected sequence is shown in SEQ ID NO.1 in the sequence list. Shandong Hongnuo Biotechnology Co., Ltd. was commissioned to synthesize the entire expression cassette, inserted into the pLent-EF1α vector (purchased from Vigene), and transformed into E. coli (Top10). After correct sequencing, the plasmid was extracted using the plasmid extraction kit of OMEGA to obtain the recombinant expression vector pLent-EF1α-CAR-EGFRvⅢ-VEGF-1. The schematic diagram of its CAR structure is shown below. Figure 1 As shown, the pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 recombinant plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80°C until use.

[0046] 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 connected in sequence. The connected sequence is shown in 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 shown in FIG. Figure 2 As shown, the pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 recombinant plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80°C until use.

[0047] 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 connected in sequence. The connected sequence is shown in 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 shown in FIG. Figure 3 As shown, the pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 recombinant plasmid was extracted from the positive clones, diluted to 2 μg / μL, and stored at -80°C until use.

[0048] Example 2 Preparation of T cells modified with three plasmids

[0049] 1. Preparation of activated T cells

[0050] 75 ml of the patient's autologous peripheral blood was collected and peripheral blood mononuclear cells were separated using Ficoll-Paque lymphocyte separation medium. The separated cells were sorted out using CD8+ sorting reagent provided by BD to separate CD8+ T cells. The cells were counted and the number of cells was calculated as 1×10 6 The cells were inoculated at 100 cells / mL and KBM551 cell culture medium (purchased from Corning, catalog number: 88-551-CM) containing a final concentration of 1500 IU / mL IL-2 was added. The same number of CD3CD28 magnetic beads as the cells were then added to activate the T cells. After 24 hours of activation, the magnetic beads were removed to obtain activated T cells. The expression rate of CD69 was detected by flow cytometry, and the expression rate of CD69 was 65.6%. Figure 4 .

[0051] 2. Lentiviral Packaging

[0052] Resuscitate 293T cells, culture for 3 days, and passage according to cell density. Transfection is performed when the cell confluence reaches 80% after passage 1. 5 Cells were seeded per well in a total volume of 2 mL of DMEM medium (Gibco, Cat. No. 11960-044) per well and incubated overnight at 37°C in a 5% CO2 incubator. Transfection was performed the next day. Before transfection, the six-well plate was replaced with fresh DMEM medium at a rate of 2 mL per well and incubated in a 37°C, 5% CO2 incubator for 1 hour.

[0053] Preparation of transfection reagents: Prepare Tube A and Tube B reagents in 5 mL centrifuge tubes.

[0054] The composition of tube A and tube B is shown in Table 1.

[0055] Table 1 Composition of tube A and tube B

[0056]

[0057] After preparation, let stand for 5 minutes, then slowly add tube A to tube B, mix thoroughly, and let stand at room temperature for 20 minutes to form a liposome-DNA mixture. Add the mixture to a culture flask, mix gently, and incubate in a 37°C, 5% CO2 incubator.

[0058] After 48 hours, the morphological changes of 293T cells after transfection were observed under a microscope, such as Figure 5 As shown. After 72 hours, the cell culture supernatant containing the virus was collected into a centrifuge tube, centrifuged at 3500 rpm for 10 minutes to remove cell debris, filtered through a 4.5 μm filter, and centrifuged at 70,000 g for 2 hours at 4°C. The precipitate was resuspended in 100 μL of PBS, aliquoted, and stored at -80°C. The virus titer was determined at the same time. 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, the virus titer of the virus solution containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 was 2.01×10 8 TU / mL.

[0059] 3. Lentiviral infection of activated T cells

[0060] Take out the three virus solutions mentioned above from -80℃, thaw them, add 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 The activated T cells obtained in step 1 were added to obtain a virus and cell suspension at a ratio of 3:1 between the number of virus particles and the number of activated T cells. The virus and cell suspension was added to a 6-well plate, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator for 48 hours. The cells were collected and centrifuged at 400g for 5 minutes. The supernatant was discarded and the cells were counted according to the 1×10 6Cells were seeded at a density of 10 cells / mL and KBM551 serum-free medium containing IL-2 at a final concentration of 1500 IU / mL was added. The volume of the solution was increased every 3 days at a 1:1 ratio based on the original volume. The cells were cultured at 37°C in a 5% CO2 incubator for 13 days to expand to a sufficient volume. T cells infected with the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-1 (referred to as CAR-EGFRvIII-VEGF-1 cells), T cells infected with the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-2 (referred to as CAR-EGFRvIII-VEGF-2 cells), and T cells infected with the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvIII-VEGF-3 (referred to as CAR-EGFRvIII-VEGF-3 cells).

[0061] Chimeric antigen receptor expression was detected by flow cytometry. Figure 6-8 As shown, the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-1 in the present invention on activated T cells is 55.3%, the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-2 in the present invention on activated T cells is 63.4%, and the infection rate of the recombinant lentivirus containing pLent-EF1α-CAR-EGFRvⅢ-VEGF-3 on activated T cells is 73.2%.

[0062] Example 3 In vitro IFN-γ release experiment

[0063] U87 MG cells (glioblastoma cell line) were used as target cells, and the effector cells were the CAR-EGFRvIII-VEGF-1 cells, CAR-EGFRvIII-VEGF-2 cells, and CAR-EGFRvIII-VEGF-3 cells prepared in Example 2.

[0064] The effector-target ratios were 1:1, 5:1, and 10:1, respectively, and the number of target cells was 1×10 5 Each well was divided into 2 wells, each containing 200 μL of DMEM medium containing 10 vol% FBS. The effector and target cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The supernatant was collected and the IFN-γ content was detected using an ELISA kit. The results are shown in Table 2. Figure 9As shown, the CAR-EGFRvⅢ-VEGF-2 cells and CAR-EGFRvⅢ-VEGF-3 cells of the present invention release more IFN-γ than CAR-EGFRvⅢ-VEGF-1 cells. The IFN-γ release of CAR-EGFRvⅢ-VEGF-2 cells is higher than that of CAR-EGFRvⅢ-VEGF-1 cells, indicating that the cytotoxicity of CAR-T cells can be improved after codon optimization of single-chain antibodies. The IFN-γ release of CAR-EGFRvⅢ-VEGF-3 cells is higher than that of CAR-EGFRvⅢ-VEGF-2 cells, indicating that CAR-T cells co-expressing CD47 and IL-15 have stronger cytotoxicity.

[0065] Table 2 IFN-γ release in vitro in the study of CAR-EGFRvⅢ-VEGF cell killing activity (pg / mL)

[0066]

[0067] Example 4 T cell killing experiment in vitro

[0068] U87 MG cells (brain glioblastoma cell line) were used as target cells, and CAR-EGFRvⅢ-VEGF-1 cells, CAR-EGFRvⅢ-VEGF-2 cells, and CAR-EGFRvⅢ-VEGF-3 cells were used as effector cells to determine the cytotoxicity. 5 / well) and target cells (1×10 4 / well) were added into a 48-well culture plate at a ratio of 10:1. The co-culture medium was DMEM medium containing 10 vol% FBS, 200 μL per well, and the cells were placed in a 5% CO2, 37°C incubator for co-culture. After 24 hours, 10 μL CCK-8 (Yisheng Biotechnology Co., Ltd., Catalog No. 40203ES60) was added to each well. After incubation for another 2 hours, the OD value was read using a microplate reader at a wavelength of 450 nm.

[0069] The specific groups are as follows:

[0070] Experimental group A: co-culture of CAR-EGFRvⅢ-VEGF-1 cells and target cells;

[0071] Experimental group B: co-culture of CAR-EGFRvⅢ-VEGF-2 cells and target cells;

[0072] Experimental group C: co-culture of CAR-EGFRvⅢ-VEGF-3 cells and target cells;

[0073] Control group D: The activated T cells obtained in step 1 of Example 2 were co-cultured with target cells.

[0074] Blank group: target cells

[0075] The cell killing rate was calculated according to the following formula: killing rate (%) = [1-(OD value of blank group-OD value of experimental group or control group) / OD value of blank group] × 100%.

[0076] The results showed that (see Figure 10 ), the killing rates of experimental groups AC and control group D were 47.6%, 66.2%, 95.1%, and 14.3%, respectively. The killing efficiency of CAR-EGFRvⅢ-VEGF-2 cells and CAR-EGFRvⅢ-VEGF-3 cells was significantly higher than that of CAR-EGFRvⅢ-VEGF-1 cells, and all three were higher than the control group. Therefore, the optimization of the single-chain antibody sequence in this application can improve the killing rate of CAR-T cells. CAR-T cells co-expressing CD47 and IL-15 can further enhance the killing ability of CAR-T cells after codon optimization.

[0077] Example 5 In vivo toxicity experiment of CAR-T cells

[0078] 6-8 week old C57BL6 mice (purchased from Nanjing Junke Bioengineering Co., Ltd.) were divided into 5 groups, 10 mice in each group, to verify the in vivo toxicity experiment of CAR-T cells. The experimental groups were:

[0079] a. Control group, the same volume of normal saline was injected into the tail vein;

[0080] b. Experimental group 1, 2×10 7 cells / activated T cells obtained in step 1 of Example 2;

[0081] c. Experimental group 2, tail vein injection of 2×10 7 cells / CAR-EGFRvⅢ-VEGF-1 cells;

[0082] d. Experimental group 3, 2×10 7 cells / CAR-EGFRvⅢ-VEGF-2 cells;

[0083] e. Experimental group 4, tail vein injection of 2×10 7 cells / CAR-EGFRvⅢ-VEGF-3 cells.

[0084] After injection, the mice's behavior was observed daily, their weight was weighed weekly, and the presence of CAR-T cells in their bodies was monitored using an in vivo animal imaging system. After 45 days, the mice were dissected and pathological examinations were performed on major tissues, including the brain, heart, lungs, liver, colon, and kidneys.

[0085] During the experiment, no abnormal behavior of mice was observed, 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 detected in the blood for 35 days. Pathological observation of major tissues after autopsy showed no tissue lesions in the CAR-T mice.

[0086] Table 3 Changes in weight gain of mice in each experimental group after 45 days of culture

[0087]

Claims

1. A dual-target chimeric antigen receptor co-expressing CD47 and IL-15, characterized in that: The dual-target chimeric antigen receptor is obtained by sequentially connecting the following modules: a leader as shown in SEQ ID NO.4, a single-chain antibody scFv-EGFRvⅢ as shown in SEQ ID NO.6, a linker as shown in SEQ ID NO.7, a single-chain antibody scFv-VEGF as shown in SEQ ID NO.9, a CD8 Hinge region as shown in SEQ ID NO.10, a CD28 transmembrane region as shown in SEQ ID NO.11, a CD28-4-1BB co-stimulatory region as shown in SEQ ID NO.12, a CD3ζ intracellular region as shown in SEQ ID NO.13, a self-cleavage region T2A as shown in SEQ ID NO.14, CD47 as shown in SEQ ID NO.15, a self-cleavage region T2A as shown in SEQ ID NO.14, and IL-15 as shown in SEQ ID NO.

16.

2. A CAR-T cell, characterized in that: The CAR-T cells are obtained from the dual-target chimeric antigen receptor modified T cells according to claim 1.

3. Use of the dual-target chimeric antigen receptor according to claim 1 in the preparation of a drug for treating glioblastoma.

4. Use of the CAR-T cells according to claim 2 in the preparation of a medicament for treating glioblastoma.

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