Bispecific chimeric antigen receptor targeting dll3 and cd155 and car-t cells and uses

Abstract

The application provides a bispecific chimeric antigen receptor targeting DLL3 and CD155, a CAR-T cell and application, and belongs to the technical field of biological medicine. The application provides a chimeric antigen receptor, which comprises, from N-terminal to C-terminal, a signal peptide, a first antigen binding domain, a first connecting peptide, a second antigen binding domain, a hinge region, a transmembrane domain, a costimulatory signal domain and a CD3 zeta signal domain; the costimulatory signal domain comprises an intracellular signal domain of a 4-1BB protein and an intracellular signal domain of an OX40 protein. The bispecific chimeric antigen receptor can significantly improve the killing efficiency on double-positive tumors, has stronger specificity and significantly enhanced tumor inhibition effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific chimeric antigen receptor targeting DLL3 and CD155, CAR-T cells, and their applications. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly aggressive and rapidly progressing neuroendocrine tumor. Although it is initially sensitive to chemotherapy and radiotherapy, patients are highly prone to recurrence, and the 5-year survival rate for extensive-stage SCLC is low. In recent years, immune checkpoint inhibitors have improved survival to some extent, but overall efficacy remains limited.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved great success in hematological malignancies, but its application in solid tumors, especially SCLC, faces many challenges. First, SCLC exhibits high tumor heterogeneity, and single-target CAR-T therapy easily leads to immune escape by antigen-negative tumor cells, causing relapse. Second, the solid tumor microenvironment can induce T-cell exhaustion, resulting in insufficient persistence of CAR-T cells in vivo. Furthermore, the potential for cytokine release syndrome (CRS) and neurotoxicity during treatment are also significant factors limiting its application.

[0004] DLL3 (Delta-like ligand 3) is highly specifically expressed on the surface of SCLC cells but expressed at extremely low levels in normal tissues, making it an ideal target for SCLC immunotherapy. CD155 (poliovirus receptor) is highly expressed in various solid tumors, including SCLC, and participates in tumor immune escape, also making it a potential therapeutic target. Therefore, developing a CAR-T cell that can simultaneously target DLL3 and CD155 holds promise for overcoming tumor heterogeneity.

[0005] In CAR structure optimization, the selection of co-stimulatory signaling domains is crucial. The 4-1BB signaling domain can promote T cell longevity and facilitate the formation of memory T cells; the OX40 signaling domain can enhance T cell proliferation and inhibit activation-induced cell death. Currently, there are no reports of using 4-1BB and OX40 in tandem as co-stimulatory signaling domains, simultaneously targeting DLL3 and CD155, for the treatment of SCLC. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a bispecific chimeric antigen receptor targeting DLL3 and CD155, CAR-T cells, and their applications. Through the synergistic effect of dual target recognition domains and dual co-stimulatory signaling domains, it achieves highly efficient and specific killing of SCLC cells, reduces the risk of immune escape, and enhances the persistence of CAR-T cells in vivo. The invention also provides its preparation method and applications.

[0007] On the one hand, the present invention provides a chimeric antigen receptor whose structure, from the N-terminus to the C-terminus, sequentially comprises: a signal peptide, a first antigen-binding domain, a first linker peptide, a second antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and a CD3ζ signaling domain. The co-stimulatory signal domain includes the intracellular signal domain of the 4-1BB protein and the intracellular signal domain of the OX40 protein; The amino acid sequence of the intracellular signaling domain of the 4-1BB protein is shown in SEQ ID NO: 7; the amino acid sequence of the intracellular signaling domain of the OX40 protein is shown in SEQ ID NO: 8.

[0008] SEQ ID NO:7: RRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPQEEAGGCEL; SEQ ID NO:8: ALYLLRRDQRLPPNAHKKPPGGGSFRAPIQEEQADARSTLAKI.

[0009] Specifically, the 4-1BB intracellular signaling domain and the OX40 intracellular signaling domain are tandemly linked by a second linker peptide; Furthermore, the aforementioned tandem structure can simultaneously activate two signaling pathways, synergistically enhancing the activation, proliferation, and anti-apoptotic capabilities of T cells.

[0010] Furthermore, the second linker peptide is a flexible peptide, and its amino acid sequence is shown in SEQ ID NO:10.

[0011] SEQ ID NO:10: GGGGS.

[0012] Specifically, the signal peptide is selected from human CD8α signal peptide, and its amino acid sequence is shown in SEQ ID NO: 1.

[0013] SEQ ID NO: 1: MALPVTALLLPLALLLHAARP.

[0014] Furthermore, the human CD8α signal peptide is used to guide nascent peptide chains to the endoplasmic reticulum for post-translational modification and expression on the cell membrane surface.

[0015] Specifically, the first antigen-binding domain is a single-chain antibody (scFv) that specifically recognizes the DLL3 protein, and the second antigen-binding domain is a single-chain antibody that specifically recognizes the CD155 protein.

[0016] Specifically, the first antigen-binding domain is a single-chain antibody against human DLL3, which includes a heavy chain variable region (VH) and a light chain variable region (VL), which are linked by a (G4S)3 linker peptide.

[0017] Furthermore, the amino acid sequence of the first antigen-binding domain is shown in SEQ ID NO:2; SEQ ID NO:2: EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINPSGSTIYNPSLKSRVTISLDTSKNQFSLKLTSVTAADTAVYYCARNDYYYGMDVWGQGTTVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNNYPYTFGQGTKVDIK.

[0018] Specifically, the first linker peptide is a flexible linker peptide used to connect the first antigen-binding domain and the second antigen-binding domain; Furthermore, the amino acid sequence of the first linker peptide is shown in SEQ ID NO:3; SEQ ID NO:3: EAAAKEAAAKEAAAK; Furthermore, the sequence shown in SEQ ID NO:3 can form a stable α-helix structure, effectively separating the two single-chain antibodies, preventing mutual interference, and ensuring their respective antigen-binding activities.

[0019] Specifically, the second antigen-binding domain is a single-chain antibody against human CD155.

[0020] Furthermore, the amino acid sequence of the second antigen-binding domain is shown in SEQ ID NO:4; SEQ ID NO:4: EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS GGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL.

[0021] Specifically, the hinge area is selected from the CD8α hinge area or the CD28 hinge area; Furthermore, the hinge area is a CD28 hinge area, which provides sufficient flexibility.

[0022] Furthermore, the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 5.

[0023] SEQ ID NO:5: KILSCKGEKHTLNCTVLNARVGVEVERKQQKSVDFVLTGVNESSHGTYR.

[0024] Specifically, the transmembrane region is the human CD28 transmembrane region, which is beneficial for the stable expression and dimerization of CAR molecules; Furthermore, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 6.

[0025] SEQ ID NO:6:FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0026] Specifically, the activation signal domain is the CD3ζ intracellular signal domain, which is responsible for transmitting a signal for T cell activation.

[0027] Furthermore, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO:9.

[0028] SEQ ID NO:9: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0029] On the other hand, the present invention provides a nucleic acid molecule encoding the above-mentioned chimeric antigen receptor.

[0030] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecules.

[0031] Specifically, the expression vector is selected from any one of retroviral vectors, lentiviral vectors, and adeno-associated virus vectors.

[0032] Furthermore, the expression vector is a lentiviral vector.

[0033] Specifically, nucleic acid molecules are cloned into lentiviral transfer plasmids to construct recombinant vectors.

[0034] Furthermore, the nucleic acid molecule was cloned into a lentiviral transfer plasmid (pCDH-CMV-MCS-EF1-copGFP) to construct a recombinant lentiviral expression vector.

[0035] Furthermore, the vector contains an EF1α promoter to drive CAR gene expression and a WPRE enhancing element to improve expression stability.

[0036] In another aspect, the present invention provides an immune cell comprising: expressing the chimeric antigen receptor described above, carrying the nucleic acid molecule described above, or the expression vector described above.

[0037] Specifically, the immune cells are selected from any one of T cells, NK cells, macrophages, and iPSCs.

[0038] Furthermore, the immune cells mentioned are T cells.

[0039] In another aspect, the present invention provides a method for preparing immune cells, comprising the following steps: (1) Isolate human peripheral blood mononuclear cells and sort them to obtain CD3+ T cells; (2) Activate the T cells obtained in step (1) using CD3 / CD28 antibody-conjugated magnetic beads; (3) Transducing activated T cells using the recombinant lentiviral vector described above; (4) CAR-T cells were obtained by expanding and culturing transduced T cells in serum-free medium containing IL-7 and IL-15 cytokines.

[0040] Specifically, in step (1), density gradient centrifugation is used to obtain human peripheral blood mononuclear cells.

[0041] Specifically, in step (1), CD3+ T cells are obtained by sorting using CD3 / CD4 / CD8 magnetic beads.

[0042] Specifically, in step (2), the T cells are resuspended in AIM-V medium containing 10% FBS; Furthermore, in step (2), the ratio of cells to CD3 / CD28 antibody-conjugated magnetic beads is 1:3.

[0043] Furthermore, in step (2), 80-120 IU / mL IL-2 and 5-15 ng / mL IL-15 are added and activated in a 37°C, 5% CO2 incubator for 20-25 hours.

[0044] Furthermore, in step (2), 100 IU / mL IL-2 and 10 ng / mL IL-15 were added and activated for 24 hours in a 37°C, 5% CO2 incubator.

[0045] Specifically, lentivirus transduction in step (3) includes the following steps: 1) Coat culture plates or culture flasks with recombinant human fibronectin fragments; 2) The activated T cells were seeded at a density of 1×10^6 cells / mL, and lentivirus concentrate was added at an MOI of 5-10. The cells were centrifuged at 37°C (1000g, 30 minutes) for auxiliary infection, and then cultured for 8-12 hours.

[0046] Specifically, the concentration of IL-7 mentioned in step (4) is 5-20 ng / mL; Furthermore, the concentration of IL-7 mentioned in step (4) is 10 ng / mL.

[0047] Specifically, the concentration of IL-15 in step (4) is 2-10 ng / mL; Furthermore, the concentration of IL-15 in step (4) is 5 ng / mL.

[0048] Specifically, the amplification culture time in step (4) is 10-14 days.

[0049] In another aspect, the present invention provides the application of the above-mentioned immune cells in the preparation of drugs for treating lung cancer.

[0050] Specifically, the lung cancers mentioned include small cell lung cancer and non-small cell lung cancer.

[0051] Furthermore, the lung cancer mentioned is small cell lung cancer.

[0052] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0053] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0054] Specifically, the dosage form of the drug is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, lyophilized powder for injection, gel, suppository or aerosol.

[0055] In another aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor, the nucleic acid molecule, the expression vector, or the immune cell described above.

[0056] Specifically, the pharmaceutical composition is used to treat lung cancer.

[0057] Furthermore, the lung cancers mentioned include small cell lung cancer and non-small cell lung cancer.

[0058] Furthermore, the lung cancer mentioned is small cell lung cancer.

[0059] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0060] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0061] Specifically, the dosage form of the drug is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, lyophilized powder for injection, gel, suppository or aerosol.

[0062] The beneficial effects of this invention are as follows: (1) Significantly improves the killing efficiency against DLL3 / CD155 double-positive tumors. In vitro experiments showed that the CAR-T of this invention had a killing efficiency of up to 85.6% against human small cell lung cancer H446 cells, effectively solving the problem of insufficient killing effect of single-target CAR-T.

[0063] (2) Antigen recognition is more precise, more specific, and less off-target toxicity. This invention employs a dual-antigen recognition mode, which has no significant killing effect on double-negative cells, significantly improving treatment safety and reducing non-specific damage to normal tissues.

[0064] (3) Higher cytokine release levels and stronger anti-tumor immune response Under antigen stimulation, the CAR-T cells of this invention can efficiently secrete IFN-γ and IL-2, indicating that their T cell activation is more complete and their immune killing effect is stronger.

[0065] (4) The tumor-suppressing effect in vivo is significantly enhanced, and complete tumor regression without recurrence can be achieved. In vivo anti-tumor experiments showed that the CAR-T of this invention could completely regress tumors in tumor-bearing mice within 20 days, with no recurrence and a 100% survival rate after 60 days. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0067] Example 1: Construction of CAR structure and lentivirus packaging 1. Gene Synthesis: Based on the designed CAR amino acid sequence (SEQ ID NO: 1-10), the nucleotide sequence encoding the full-length CAR was obtained through whole-gene synthesis. Appropriate restriction enzyme sites (NheI and NotI) were added to both ends of the sequence.

[0068] 2. Vector Construction: The synthesized gene fragment was cloned into the lentiviral vector pCDH-EF1-MCS-IRES-Puro, which had also been digested with the same enzymes, by enzyme digestion and ligation. The original MCS region was replaced to construct the recombinant plasmid pCDH-EF1-DLL3-CD155-CAR. The sequence correctness was verified by sequencing.

[0069] 3. Lentiviral Packaging: The recombinant plasmid and helper plasmids (psPAX2 and pMD2.G) were co-transfected into HEK293T cells. The specific steps were as follows: 293T cells in logarithmic growth phase were seeded in 10cm culture dishes. When confluence reached 80%-90%, 15μg pCDH-EF1-DLL3-CD155-CAR, 10μg psPAX2, and 5μg pMD2.G were mixed and transfected into the cells using the liposome transfection reagent Lipofectamine 3000. The culture medium was replaced with fresh medium 6-8 hours after transfection. The cell supernatant was collected at 48 and 72 hours post-transfection, and the virus was concentrated by ultracentrifugation (25,000 rpm, 4°C for 2 hours), aliquoted, and stored at -80°C for later use.

[0070] Example 2 Preparation of CAR-T cells 1. T cell isolation and activation: 50 mL of peripheral blood from healthy volunteers was collected and PBMCs were obtained by density gradient centrifugation using Ficoll-Paque PLUS. Purified CD3+ T cells were obtained using the EasySep Human T Cell Sorting Kit, following the manufacturer's instructions. T cells were suspended at a density of 1 × 10^6 cells / mL in AIM-V medium containing 10% FBS, 100 IU / mL IL-2, and 10 ng / mL IL-15. CD3 / CD28 magnetic beads were added at a cell:bead ratio of 1:3, and the mixture was incubated at 37°C for 24 hours for activation.

[0071] 2. Lentiviral infection: Coat 24-well plates with 20 μg / mL RetroNectin and incubate overnight at 4°C. After washing with PBS containing 2% FBS, add 1 × 10^6 activated T cells and lentivirus (MOI = 10) to each well, and add Polybrene to a final concentration of 8 μg / mL. Centrifuge the plates at 1000g for 90 minutes at 32°C. After centrifugation, incubate at 37°C for 12 hours. Discard the supernatant and replace with fresh culture medium.

[0072] 3. Cell Expansion: Transfer cells to T75 or G-Rex flasks. Use TexMACS medium supplemented with IL-7 (10 ng / mL) and IL-15 (5 ng / mL). Count cells every 2 days and maintain a cell density of 0.5–1 × 10^6 cells / mL. On day 10, collect cells for analysis.

[0073] Comparative Example 1 1. CAR Structure Construction The only difference from Example 1 is that it contains only the first antigen-binding domain (anti-DLL3 scFv), and the single-target CAR structure is: signal peptide-anti-DLL3 scFv-CD28 hinge-CD28 transmembrane-4-1BB+OX40 co-stimulatory-CD3ζ.

[0074] 2. Lentiviral Packaging Same as Example 1.

[0075] 3. Preparation of CAR-T cells Same as Example 2.

[0076] Example 3: In vitro functional verification 1. Target cells: The human SCLC cell line H446 (highly expressing DLL3 and CD155) was selected as the target cells. K562 cells, which are DLL3-negative and CD155-negative, were used as negative controls.

[0077] 2. Killing Assay: CAR-T cells and target cells were co-cultured in 96-well plates at effector-to-target ratios of 10:1, 5:1, and 1:1, with three replicates per group. The cells were incubated at 37℃ and 5% CO2 for 18 hours. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2-4 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the killing efficiency was calculated according to the formula.

[0078] The in vitro cytotoxic activity of CAR-T cells against target cells was detected using the CCK-8 assay. Specific cytotoxicity was calculated using the following formula: Kill efficiency (%) = (1 - experimental group OD) 450 / Target cell control group OD 450 ) × 100%.

[0079] The experiment included a target cell control group (target cells only), an experimental group (target cells + CAR-T cells), a negative control group, and a non-target cell (NC) group. Each group had three replicates. Results are expressed as mean ± SD.

[0080] The results are shown in Table 1. At an effector-to-target ratio of 5:1, the killing efficiency of the DLL3-CD155 CAR-T cell in this invention against H446 cells reached 85.6% ± 4.2%, significantly higher than that of the single-target DLL3-CAR-T group (62.3% ± 5.1%) and the NC group. No significant killing effect was observed on K562 cells.

[0081] Table 1

[0082] 3. Cytokine Release: The supernatant from the co-cultured culture for 24 hours was collected, and the levels of IFN-γ and IL-2 were detected using an ELISA kit. The results are shown in Table 2 below. The IFN-γ release level of the DLL3-CD155 CAR-T group of this invention was 3500 ± 420 pg / mL, and the IL-2 content was 286 ± 35 pg / mL, which was significantly higher than that of the control group (DLL3 CAR-T).

[0083] Table 2

[0084] Example 4: In vivo antitumor experiment 1. Animal model: 6-8 week old female NSG mice were subcutaneously injected with 5×10^6 H446 cells (mixed with Matrigel 1:1) into the right abdomen. When the tumor volume reached 100-150 mm³, the mice were randomly divided into 3 groups (6 mice per group): PBS control group, DLL3-CAR-T control group, and DLL3-CD155-CAR-T experimental group.

[0085] 2. Cell reinfusion: Mice in each group were administered the drug via tail vein injection. PBS control group: 200 μL of PBS was injected via the tail vein; DLL3-CAR-T control group: 1×10⁻⁶ mmol / L via tail vein injection 7 One DLL3-CAR-T cell; DLL3-CD155-CAR-T experimental group: 1×10⁻⁶ mmol / L injected via tail vein 7 One DLL3×CD155-CAR-T cell.

[0086] 3. Results Observation: The long diameter (L) and short diameter (W) of the tumor were measured every 3 days, and the tumor volume was calculated according to the formula: Tumor volume (mm) 3 = 0.5 × L × W 2 Observe and record the mouse's body weight, tumor growth, and survival status over a period of 60 days.

[0087] The experimental results are shown in Table 3.

[0088] Table 3. Tumor growth and survival in mice of each group

[0089] The results showed that the tumors in the experimental group mice began to shrink significantly on day 10 after infusion and completely disappeared on day 20. By the end of the observation period on day 60, all 6 mice in the experimental group were alive and there was no tumor recurrence; while 3 mice in the DLL3-CAR-T control group had tumor recurrence; and all mice in the PBS control group died within 35 days.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that, The structure of the chimeric antigen receptor, from the N-terminus to the C-terminus, includes, in sequence: a signal peptide, a first antigen-binding domain, a first linker peptide, a second antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling domain, and a CD3ζ signaling domain. The co-stimulatory signal domain includes the intracellular signal domain of the 4-1BB protein and the intracellular signal domain of the OX40 protein; The amino acid sequence of the intracellular signaling domain of the 4-1BB protein is shown in SEQ ID NO: 7; the amino acid sequence of the intracellular signaling domain of the OX40 protein is shown in SEQ ID NO:

8.

2. The chimeric antigen receptor according to claim 1, characterized in that, The first antigen-binding domain is a single-chain antibody that specifically recognizes the DLL3 protein, and the second antigen-binding domain is a single-chain antibody that specifically recognizes the CD155 protein.

3. The chimeric antigen receptor according to claim 2, characterized in that, The amino acid sequence of the first antigen-binding domain is shown in SEQ ID NO:2; the amino acid sequence of the second antigen-binding domain is shown in SEQ ID NO:

4.

4. The chimeric antigen receptor according to claim 1, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 5, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 6, and the amino acid sequence of the CD3ζ intracellular signal domain is shown in SEQ ID NO:

9.

5. A nucleic acid molecule encoding the chimeric antigen receptor as described in any one of claims 1-4.

6. An expression carrier, characterized in that, The expression vector includes the nucleic acid molecule as described in claim 5.

7. An immune cell, characterized in that, The immune cells include those expressing the chimeric antigen receptor of any one of claims 1-4, carrying the nucleic acid molecule of claim 5, or the expression vector of claim 6.

8. The immune cells according to claim 7, characterized in that, The immune cells mentioned are selected from any one of T cells, NK cells, macrophages, and iPSCs.

9. The immune cells according to claim 7, characterized in that, The immune cells mentioned are T cells.

10. The method for preparing immune cells according to any one of claims 7-9, characterized in that, Includes the following steps: (1) Isolate human peripheral blood mononuclear cells and sort them to obtain CD3+ T cells; (2) Activate the T cells obtained in step (1) using CD3 / CD28 antibody-conjugated magnetic beads; (3) Transducing activated T cells using a recombinant lentiviral vector; (4) CAR-T cells were obtained by expanding and culturing transduced T cells in serum-free medium containing IL-7 and IL-15 cytokines.

11. The preparation method according to claim 10, characterized in that, The concentration of IL-7 in step (4) is 5-20 ng / mL, and the concentration of IL-15 is 2-10 ng / mL.

12. The preparation method according to claim 10, characterized in that, The amplification culture time in step (4) is 10-14 days.

13. The use of the immune cells according to any one of claims 7-9 in the preparation of a medicament for treating lung cancer.

14. The application according to claim 13, characterized in that, The lung cancer mentioned is non-small cell lung cancer.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the immune cell of any one of claims 7-9.

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