CAR-T cell drug capable of simultaneously targeting two tumor antigens and application of CAR-T cell drug

Abstract

The invention relates to the field of cell drugs, in particular to a CAR-T cell drug capable of simultaneously targeting two tumor antigens and application of the CAR-T cell drug, tumors are solid glioma, the CAR-T cell drug can reduce the tumor recurrence risk, and when a traditional single-targeting CAR-T cell therapy is used for treating certain types of brain glioma, the CAR-T cell drug can be used for treating brain glioma. The CAR-T cell has the advantages that the treatment effect is not ideal due to tumor heterogeneity and an antigen escape mechanism, however, through the double-targeting strategy, the CAR-T cell can recognize and combine two different tumor-associated antigens at the same time, so that the possibility of tumor cell escape is reduced, and the treatment effect is improved; the antitumor activity of CAR-T cells is remarkably enhanced by simultaneously targeting an IL13 receptor alpha2 and an NKG2D ligand, a double-targeting CAR-T strategy provides a novel and effective means for treatment of brain glioma, and the double-targeting CAR-T cell has wide clinical application potential and possibly provides a new thought in the aspects of improving the treatment effect and reducing off-target toxicity.

Description

Technical Field

[0001] The present invention relates to the field of cell medicines, and specifically to a CAR-T cell medicine that simultaneously targets two tumor antigens and its application. Background Art

[0002] CAR-T therapy, or chimeric antigen receptor T cell immunotherapy, uses genetic engineering technology to reprogram the patient's immune T cells in vitro to recognize antigens on the surface of tumor cells, and then infuse these cells back into the patient for treatment. In this way, T cells can accurately identify and kill tumor cells in a non-major histocompatibility complex (MHC)-dependent manner. In the past decade, CAR-T therapy has made great progress in the field of tumor treatment and treated previously incurable blood tumors. Scientists have also been working hard to apply CAR-T therapy to other types of cancer besides blood cancers, but CAR-T cell therapy technology still faces some problems in the treatment of solid tumors. After CAR-T cells successfully enter solid tumors such as glioblastoma (GBM), they will encounter two major obstacles: ① Immunosuppressive cells and glioma immune microenvironment 1. The inhibitory molecules in GBM, such as TGF-β, IL-6, IL-10, IL-23, etc., inhibit and damage CAR-T cells. Many inhibitory leukocytes, such as regulatory T cells, myeloid suppressor cells, tumor-associated macrophages, etc., accumulate in GBM, thereby inhibiting CAR-T cells; 2. The interference of tumor antigen recognition is mainly caused by the heterogeneity of brain gliomas and the lack of specific antigens. These are the important reasons for the failure of CAR-T cell function. There are differences in the types of tumor antigens expressed by brain gliomas of different pathological types. Therefore, the selection of appropriate tumor antigens is crucial for the efficacy of CAR-T cells.

[0003] In brain glioma, the expression of IL-13Rα2 is positively correlated with the grade of brain glioma, and high expression of IL-13Rα2 is associated with poor prognosis. IL13Rα2 is one of the two subunits of the cytokine IL-13 receptor. IL-13 binds to IL13Rα1 with low affinity, and then IL13Rα1 forms a heterodimer with IL-4Rα, activates the STAT6 signaling pathway, and induces cell apoptosis. Based on the above findings, targeted therapy and immunotherapy for IL-13Rα2 have been launched. It has been reported that patients with recurrent glioblastoma achieved good results after treatment with CAR-T cells targeting IL-13Rα2. There were no toxic side effects after treatment, the tumor volume was reduced, and the content of immune factors and immune cells in the cerebrospinal fluid increased. In summary, IL-13Rα2 as a specific target of CAR-T cells can achieve a guiding role in killing brain glioma cells, and it plays an important role in the research on the treatment of glioma.

[0004] NKG2D is an activating receptor expressed on the surface of NK cells and CD8+T cells. It plays an important role in natural immunity and is involved in the recognition of virus-infected cells and the killing of tumor cells by NK. Many studies have shown that NKG2D ligands (NKG2DL) such as MICA / B are highly expressed in brain glioma cells, especially in high-grade gliomas (such as glioblastoma). Studies have found that high expression of NKG2DL is associated with poor prognosis in patients with brain glioma. This may be because tumor cells with high expression of NKG2DL are more invasive or more likely to escape the surveillance of the immune system. Previous studies have shown that targeted killing of cancer stem cells (CSC) can effectively inhibit the occurrence of glioblastoma and prolong the survival of glioma mice. It is reported that NKG2D ligands (NKG2DL) are found to be expressed in large quantities in glioblastoma stem cells (SC), so NKG2D-specific CAR-T cells can be used for treatment. At the same time, NKG2DL is also an ideal target for the treatment of brain glioma.

[0005] Single-target CAR-T cells may not be able to recognize and kill all tumor cells. As the treatment progresses, tumor cells may survive through antigen escape mechanisms, leading to recurrence of residual tumors. Multi-target CAR-T cells can reduce the possibility of antigen escape by targeting multiple antigens, maintain long-term therapeutic effects, and reduce nonspecific attacks on normal cells and the occurrence of side effects. The efficacy of dual-target CAR-T cell therapy has been verified in some clinical trials. The safety of dual-target CAR-T cells is also better than that of single-target CAR-T cells. The incidence of severe cytokine release syndrome (CRS) and neurotoxicity (NT) is low. The main CAR structures used for dual-target CAR-T cell therapy include: single-target CAR hybrid, bivalent tandem CAR, bivalent circular CAR and bicistronic CAR. These innovative designs provide new hope and possibilities for the application of CAR-T cell therapy in complex tumors such as brain glioma.

[0006] This invention is the first to simultaneously target IL13Rα2 and NKG2D ligands, aiming to more effectively identify and attack glioma cells, improve the specificity and efficiency of treatment, overcome tumor heterogeneity and antigen escape, and enhance tumor killing ability. This innovative design is expected to bring new hope and better treatment effects to glioma patients. Summary of the invention

[0007] The purpose of the present invention is to provide a CAR-T cell drug that simultaneously targets two tumor antigens and its application, in order to more effectively identify and attack glioma cells, improve the specificity and efficiency of treatment, overcome tumor heterogeneity and antigen escape, and enhance tumor killing ability, so as to solve the problems raised in the above-mentioned background technology.

[0008] According to a first aspect of the present invention, a CAR-T cell drug that simultaneously targets two tumor antigens and its application is provided, wherein the CAR-T cell drug simultaneously targets IL13 receptor α2 and NKG2D ligand;

[0009] The dual-target chimeric antigen receptor of the CAR-T cell drug includes:

[0010] The signal peptides connected in sequence are: human CD8α signal peptide, CD8αleader;

[0011] IL13(E13Y) mutant targeting IL13Rα2;

[0012] Linker fragment linker;

[0013] NKG2D extracellular domain targeting NKG2D ligand: NKG2DECD;

[0014] CD8α hinge region: human CD8α hinge region, CD8αHinge;

[0015] Transmembrane region: human CD8α transmembrane region, CD8αtransmembrane;

[0016] Co-stimulatory factors and intracellular signal peptides: human CD3ζ intracellular signal, CD3ζsignal.

[0017] According to a second aspect of the present invention, a method for constructing a dual-target chimeric antigen receptor is provided, comprising the following steps:

[0018] S1. Vector linearization: The plasmid pLL3.7-NKG2D-CAR stored in the laboratory was linearized by double digestion with NheⅠ and EcoRⅠ. The enzyme digestion system included: 1μL 10× buffer; 1μg plasmid pLL3.7-NKG2D-CAR; 1μL Nhe I enzyme; 1μL EcoR I enzyme, and ddH2O was added to make up to 10μL;

[0019] S2. PCR amplification of linker fragment sequences: The ((4S)3 linker sequence was designed on the primers used to amplify the NKG2D extracellular region fragment and the IL13 (E13Y) fragment, and two large fragments containing the CD8α signal peptide-IL13 (E13Y)-linker sequence and the NKG2D extracellular region-CD8α hinge region-transmembrane region-co-stimulatory factor and intracellular signal peptide were obtained by PCR amplification;

[0020] S3, gel recovery and plasmid recombination: using homologous recombination method, the recovered vector pLL3.7-NKG2D-CAR large fragment and the two small fragments amplified by PCR in step S2 were seamlessly cloned using a one-step cloning kit;

[0021] S4. Transformation: The ligation product was transferred into DH5α competent cells, placed in an ice bath for 30 minutes, and then heat-shocked at 42°C, and then placed in an ice bath for another 5 minutes to obtain the transformation product. The transformation product was added to 1 mL of liquid LB medium without antibiotics, and cultured on a shaking platform at 37°C for 1 hour. After centrifugation to obtain a precipitate, the precipitate was resuspended in 100 μL of LB medium and smeared on an Amp-resistant LB plate, and cultured overnight.

[0022] S5. Identification: Select positive clones for sequencing and double enzyme digestion for identification. Double enzyme digestion with NheI and EcoR I yields 6841 bp and 1548 bp fragments. After sequencing is correct, an endotoxin-free plasmid extraction kit is used to extract the plasmid of the bacteria.

[0023] Furthermore, the enzyme digestion reaction conditions in step S1 are as follows: the enzyme digestion temperature is 37° C., the enzyme digestion time is 30 min, and the total volume of the enzyme digestion system is 10 μL.

[0024] Further: in step S4, the thermal shock time is 45s.

[0025] According to a second aspect of the present invention, a CAR-T cell drug that simultaneously targets two tumor antigens is provided for use in anti-tumor activity, wherein the tumor is a solid glioma.

[0026] Furthermore: the CAR-T cell drug can reduce the risk of tumor recurrence.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. Traditional single-target CAR-T cell therapy may not be effective in treating certain types of brain gliomas due to tumor heterogeneity and antigen escape mechanisms. However, through the dual-targeting strategy of the present invention, CAR-T cells can simultaneously recognize and bind to two different tumor-associated antigens, thereby reducing the possibility of tumor cell escape and improving the treatment effect;

[0029] 2. By simultaneously targeting IL13 receptor α2 (IL13Rα2) and NKG2D ligand, the anti-tumor activity of CAR-T cells was significantly enhanced. The dual-targeting CAR-T strategy provides a novel and effective means for the treatment of brain glioma and has broad clinical application potential. The dual-targeting strategy may provide new ideas for improving therapeutic effects and reducing off-target toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the dual-target chimeric antigen receptor CAR plasmid structure of the present invention;

[0031] Figure 2 This is the enzyme digestion identification diagram of the recombinant plasmid of the present invention;

[0032] Figure 3 This is a schematic diagram of the lentivirus titer determination of the present invention;

[0033] Figure 4 This is a CAR positive rate detection diagram of the present invention;

[0034] Figure 5 This is a schematic diagram of U251-luciferase killing (highly expressing IL13 ligand) of the present invention;

[0035] Figure 6 This is a schematic diagram of PANC-1-luciferase killing (highly expressed NKG2D ligand) of the present invention;

[0036] Figure 7 It is a schematic diagram of U87-luciferase killing (highly expressing NKG2D ligand and IL13 ligand) of the present invention;

[0037] Figure 8 This is a schematic diagram of GzmB secretion by U251 cells (highly expressing IL13 ligand) as target cells of the present invention;

[0038] Fig. 9 This is a schematic diagram of the secretion of GzmB by PANC-1 cells (highly expressing NKG2D ligands) as target cells of the present invention;

[0039] Fig.10 Schematic diagram of GzmB secretion by U87 as target cell of the present invention (high expression of NKG2D ligand and IL13 ligand). DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with the various embodiments shown in the accompanying drawings, but it should be noted that these embodiments are not limitations of the present invention, and any equivalent transformations or substitutions in functions, methods, or structures made by ordinary technicians in the field based on these embodiments are all within the scope of protection of the present invention.

[0041] See also Figure 1-10 The specific implementation process of the present invention is described below through the preparation of a CAR-T cell drug that simultaneously targets two tumor antigens and multiple experiments.

[0042] 1. Plasmid construction:

[0043] The dual-target chimeric antigen receptor includes: a signal peptide (human CD8α signal peptide, CD8αleader), an IL13(E13Y) mutant targeting IL13Rα2, a linker, an NKG2D extracellular region (NKG2DECD) targeting an NKG2D ligand, a CD8α hinge region (human CD8α hinge region, CD8αHinge), a transmembrane region (human CD8α transmembrane region, CD8αtransmembrane), a costimulatory factor and an intracellular signal peptide (human CD3ζ intracellular signal, CD3ζsignal) connected in sequence, such as Figure 1 As shown, the specific construction process:

[0044] (1) Vector linearization: The plasmid pLL3.7-NKG2D-CAR stored in the laboratory was linearized by double restriction enzyme digestion with NheⅠ and EcoRⅠ. The restriction enzyme digestion reaction conditions were as follows: the restriction enzyme digestion temperature was 37°C, the restriction enzyme digestion time was 30 min, and the restriction enzyme digestion system (total volume 10 μL) included: 1 μL of 10× buffer; 1 μg of plasmid pLL3.7-NKG2D-CAR; 1 μL of Nhe I enzyme; 1 μL of EcoR I enzyme, and ddH2O was added to make up to 10 μL.

[0045] (2) PCR amplification of the linker fragment sequence: The (G4S)3 linker sequence was designed on the primers used to amplify the NKG2D extracellular region fragment and the IL13 (E13Y) fragment. PCR amplification was used to obtain two large fragments containing the CD8α signal peptide-IL13 (E13Y)-linker sequence and the NKG2D extracellular region-CD8α hinge region-transmembrane region-co-stimulatory factor and intracellular signal peptide, respectively.

[0046] (3) Gel recovery and plasmid recombination: Using the homologous recombination method, the recovered vector pLL3.7-NKG2D-CAR large fragment and the two small fragments obtained by PCR amplification in the previous step were seamlessly cloned using a one-step cloning kit;

[0047] (4) Transformation: The ligation product was transformed into DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then incubated on ice for another 5 min to obtain the transformation product. The transformation product was added to 1 mL of liquid LB medium without antibiotics, incubated on a shaking platform at 37°C for 1 h, and centrifuged to obtain a precipitate. The precipitate was resuspended in 100 μL of LB medium and smeared on an Amp-resistant LB plate for overnight incubation.

[0048] (5) Positive clones were selected for sequencing and double enzyme digestion for identification. Double enzyme digestion with NheI and EcoR I yielded 6841 bp and 1548 bp fragments. The identification results were as follows: Figure 2 As shown, after the sequencing was correct, an endotoxin-free plasmid extraction kit was used to extract the plasmid of the bacteria according to the instructions.

[0049] 2. Lentiviral vector plasmid packaging:

[0050] 293T cells with a density of about 80% were selected, and the packaging system was as follows: 8 μg of the target plasmid, 6.5 μg of psPAX2, 3.5 μg of pMD2.G, 50 μL of PEI, and DMEM basal medium were added to 1 mL per dish. After the mixed system was evenly added to 293T, the medium was replaced with 1% DMEM medium for 6-8 hours. After 48 hours and 72 hours of replacement, the supernatant containing the lentiviral vector plasmid was collected, and after centrifugation at 4000g for 10 minutes, the supernatant was filtered with a 0.22 μm filter membrane to obtain the filtrate containing the lentiviral vector plasmid. The filtrate containing the lentiviral vector plasmid was transferred to an ultracentrifuge tube and centrifuged at 15000 rpm for 2 hours at 4°C. After the centrifugation, the supernatant was discarded, and the precipitate was dissolved with 400 μL of X-VIVO complete medium at 4°C overnight.

[0051] 3. Determine the lentiviral titer:

[0052] Prepare 293T cells in good growth state, plate them in a 24-well plate at a density of 2×105 cells / well, add 1μL, 2μL, and 4μL of each virus to different wells, place the cell culture plate in a 37°C, 5% CO2 incubator for 48 hours, collect the infected cells by centrifugation, discard the supernatant, suspend the precipitate with 100μL PBS buffer, and add 0.3μL APC anti-human NKG2D antibody and PE anti-human IL-13 antibody to different viruses respectively, stain in a 4°C refrigerator for 30 minutes, add 1mL PBS buffer and centrifuge, finally suspend the precipitate with 250μL PBS buffer and transfer it to a flow tube for flow detection, and analyze it with FlowJo V10 software: HEK-293T cells not infected with the virus are used as negative controls for gating, and the proportion of CAR-positive expressing cells after different volumes of virus concentrate are infected with HEK-293T cells is analyzed. The titer test results are shown in the figure. Figure 3 Select samples with a positive rate of about 20%-30% to calculate the virus titer (Titer).

[0053] Calculation formula: Titer (TU / mL) = (number of positive cells × dilution factor) / inoculated virus volume (mL).

[0054] The results are as follows: the titer of LV-NKG2D-CAR was 1.2×108TU / mL, the titer of LV-IL13-CAR was 0.5×108TU / mL, and the titer of LV-IL13-NKG2D-CAR was 0.5×108TU / mL.

[0055] 4.CAR-T cell preparation:

[0056] Mononuclear cells (PBMCs) were isolated from the donor's peripheral blood, and T cells were activated using anti-CD3 and anti-CD28 antibodies. The activated T cells were cultured in a medium containing IL-2. The virus suspension was prepared at a ratio of MOI = 10 for each virus. The required virus volume was calculated based on the virus titer. The calculated virus suspension was added to 5 × 105 T cells in a 24-well plate, mixed, and the T cells were cultured in a medium containing IL-2, usually at 37 ° C, 5% CO2 incubator for 24-48 hours. The expression of CAR molecules was detected by flow cytometry to determine the infection efficiency. The flow cytometry steps were the same as above. The positive rate is as follows: Figure 4 shown.

[0057] 5. Comparison of anti-tumor effects of CAR-T cells in vitro:

[0058] This experiment first detected the expression of NKG2DL and IL13Rα2 on the existing cell lines in the laboratory. For example, U251-LUC cells were finally selected as the IL13Rα2+NKG2DL- cell line. Due to resource limitations, PANC-1-LUC cells were used as an alternative to the IL13Rα2-NKG2DL+ glioma cell line, and U87-LUC was used as the IL13Rα2+NKG2DL+ cell line. Then, killing experiments were performed on these three cell lines respectively. This experiment set up MOCK T, NKG2D-CAR-T, IL13-CAR-T, and TanCAR-T (IL13-NKG2D-CAR-T), with three effector-target ratios of 1:1, 2:1, and 4:1 in each group. Three replicates were made for each gradient to eliminate errors caused by accidental factors. 39 wells were selected from the 96-well low adsorption plate for the experiment, 7.8×105 U251-LUC cells were taken and resuspended in 3.9mL X-VIVO complete medium, 100μL of target cell suspension was added to each well, 2×104 effector T cells were added to the effector-target ratio group of 1:1, 4×104 effector T cells were added to the effector-target ratio group of 2:1, and 8×104 effector T cells were added to the effector-target ratio group of 4:1, and the total volume in the well was filled to 200μL. The above plating process was repeated with PANC-1-LUC cells and U87-LUC cells. The 96-well low-adsorption plate was placed in a 37°C incubator for 16 h. After 16 h, the liquid in each well was removed and marked. After centrifugation, the well was resuspended with 100 μL of firefly luciferase reporter gene cell lysis buffer. The sample was transferred to an all-white detection plate, 20 μL of firefly luciferase detection reagent was added, and the absorbance was detected on a luminometer. Cell killing efficiency = [target cell control well only - experimental well) / target cell control well only] × 100%. The killing results are shown in Table 1. Figure 5 , 6 and 7. The results showed that the dual-target chimeric antigen receptor could not only specifically recognize tumor cells expressing the two single targets NKG2DL or IL13Rα, but also recognize tumor cells co-expressing NKG2DL or IL13Rα. The dual-target CAR-T had stronger anti-tumor activity and could avoid immune escape of positive tumor cells expressing low-abundance antigens, thereby reducing the risk of recurrence.

[0059] 6. Detection of GzmB, an activation marker of CAR-T cells:

[0060] 1×105 tumor cells were added to each well of a 48-well plate, and 3×105 CAR-T cells were added at an effector-target ratio of 3:1. The volume was supplemented to 400 μL and placed in an incubator. After culturing at 37°C for 12 h, a Golgi blocker was added. After 4 h, the cells were transferred to a 1.5 mL centrifuge tube, and the supernatant was removed after centrifugation. 100 μL of a fixative permeabilizer was added for 1 h, and 1 mL of PBS buffer was added to wash once, and then the cells were resuspended in 100 μL of PBS buffer, and 0.3 μL of Anti-GzmB antibody was added for staining for 30 min. After washing with 1 mL of buffer, 250 μL of the cell pellet was resuspended for detection on the machine. The results are shown in the table. Figure 8 , 9 As can be seen from the figure, for target cells that highly express IL13 ligands or NKG2D ligands, the expression levels of GzmB in dual-targeted CAR-T cells are respectively different from those in IL13-CAR-T cells ( Figure 8 ) or NKG2D-CAR-T cells ( Fig. 9 ), but when co-cultured with target cells that highly express both NKG2D ligands and IL13 ligands, the expression level of GzmB in dual-targeted CAR-T cells was significantly higher than that in single-targeted CAR-T cells, indicating that dual-targeted CAR-T cells are more activated by simultaneously recognizing two different tumor-associated antigens, which may be the reason for their stronger tumor-killing effect.

[0061] Finally, it should be noted that in the description of the present invention, it should be noted that the terms "vertical", "up", "down", "horizontal", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0062] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly stipulated and limited, the terms "set", "installed", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal connection of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances.

[0063] The above contents are further detailed descriptions of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope determined by the claims submitted by the present invention.

[0064]

Claims

1. A CAR-T cell drug that simultaneously targets two tumor antigens, characterized in that: The CAR-T cell drug simultaneously targets IL13 receptor α2 and NKG2D ligand; The dual-target chimeric antigen receptor of the CAR-T cell drug includes: The signal peptides connected in sequence are: human CD8α signal peptide, CD8αleader; IL13(E13Y) mutant targeting IL13Rα2; Linker fragment linker; NKG2D extracellular domain targeting NKG2D ligand: NKG2DECD; CD8α hinge region: human CD8α hinge region, CD8αHinge; Transmembrane region: human CD8α transmembrane region, CD8αtransmembrane; Co-stimulatory factors and intracellular signal peptides: human CD3ζ intracellular signal, CD3ζsignal.

2. A method for constructing a dual-target chimeric antigen receptor, characterized in that: The steps include: S1. Vector linearization: The plasmid pLL3.7-NKG2D-CAR stored in the laboratory was linearized by double digestion with NheⅠ and EcoRⅠ. The enzyme digestion system included: 1μL 10× buffer; 1μg plasmid pLL3.7-NKG2D-CAR; 1μL Nhe I enzyme; 1μL EcoR I enzyme, and ddH2O was added to make up to 10μL; S2. PCR amplification of linker fragment sequences: The (G4S)3 linker sequence was designed on the primers used to amplify the NKG2D extracellular region fragment and the IL13 (E13Y) fragment, and two large fragments containing the CD8α signal peptide-IL13 (E13Y)-linker sequence and the NKG2D extracellular region-CD8α hinge region-transmembrane region-co-stimulatory factor and intracellular signal peptide were obtained by PCR amplification; S3, gel recovery and plasmid recombination: using homologous recombination method, the recovered vector pLL3.7-NKG2D-CAR large fragment and the two small fragments amplified by PCR in step S2 were seamlessly cloned using a one-step cloning kit; S4. Transformation: The ligation product was transferred into DH5α competent cells, placed in an ice bath for 30 minutes, and then heat-shocked at 42°C, and then placed in an ice bath for another 5 minutes to obtain the transformation product. The transformation product was added to 1 mL of liquid LB medium without antibiotics, and cultured on a shaking platform at 37°C for 1 hour. After centrifugation to obtain a precipitate, the precipitate was resuspended in 100 μL of LB medium and smeared on an Amp-resistant LB plate, and cultured overnight. S5. Identification: Select positive clones for sequencing and double enzyme digestion for identification. Double enzyme digestion with NheI and EcoR I yields 6841 bp and 1548 bp fragments. After sequencing is correct, an endotoxin-free plasmid extraction kit is used to extract the plasmid of the bacteria.

3. The method according to claim 2, characterized in that: The enzyme digestion reaction conditions in step S1 are: the enzyme digestion temperature is 37° C., the enzyme digestion time is 30 min, and the total volume of the enzyme digestion system is 10 μL.

4. The method according to claim 2, characterized in that: In step S4, the thermal shock time is 45 seconds.

5. Application of a CAR-T cell drug that simultaneously targets two tumor antigens in anti-tumor activity, characterized in that: The tumor is a solid glioma.

6. The use according to claim 5, characterized in that: The CAR-T cell drug can reduce the risk of tumor recurrence.

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