Function-enhanced CAR-T or CAR-NK cell as well as preparation method and application thereof

Abstract

The invention discloses a function-enhanced CAR-T or CAR-NK cell as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides an MAP3K8 gene overexpression vector. The MAP3K8 gene overexpression vector comprises an expression vector of a chimeric antigen receptor targeting EGFR or CD133, a promoter and an MAP3K8 gene integrated with the expression vector. The invention further provides a function enhancement type CAR-T or CAR-NK cell, and the function enhancement type CAR-T or CAR-NK cell is obtained by using the MAP3K8 gene overexpression vector virus transduction. The function-enhanced CAR-T or CAR-NK cell disclosed by the invention is forced to express under the action of an EF1 alpha promoter, so that the anti-tumor function of the CAR-T or CAR-NK can be remarkably enhanced, and the activating and killing functions of immune cells are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a function-enhanced CAR-T or CAR-NK cell, a preparation method and an application thereof. Background Art

[0002] CAR-T therapy, or chimeric antigen receptor T-cell immunotherapy, is a method of bioengineering a patient's T cells in vitro to give them the ability to recognize tumor surface antigens. After a large number of amplifications in vitro, they are infused back into the patient's body to achieve the purpose of specific recognition and killing of cancer cells. In the past decade, CAR-T cell therapy has been effective in the treatment of hematological malignancies and has developed rapidly. To date, a number of CAR-T therapies have been approved by the FDA for the treatment of malignant hematological tumors such as refractory / relapsed leukemia and lymphoma that have failed to respond to traditional drug treatments, with an overall remission rate of 70% to 94%, and even clinical cure for some patients. This breakthrough has completely changed the treatment landscape for hematological tumors and marks the arrival of the era of cell therapy.

[0003] Solid tumors account for more than 90% of all malignant tumors, with a heavy medical burden and a greater clinical demand for effective therapies. Therefore, CAR immune cell therapy targeting solid tumors has attracted much attention and has been widely studied in the past few years. Although CAR immune cell therapy has shown good application potential in the treatment of some solid tumors, the clinical experimental results of CAR immune cell therapy for solid tumors have been largely disappointing so far. Both the response rate and the objective remission rate are far lower than those of CAR-T therapy for hematological tumors, and a breakthrough is urgently needed.

[0004] Unlike hematological tumors, the uniqueness of solid tumors in terms of tissue composition and distribution location poses more challenges to CAR immune cell therapy, especially the limitation of tumor microenvironment (TME) immunosuppression on its function, which urgently needs to be broken through. Solid tumors have complex TME: low pH, hypoxia, severe lack of nutrient supply and a large number of immunosuppressive cells (such as TAM, Treg and MDSC). These factors severely limit the activation, survival and anti-tumor effect of CAR-T or CAR-NK cells. Therefore, the function of CAR-T or CAR-NK targeting solid tumors needs to be further strengthened.

[0005] MAP3K8, also known as Cot / tpl2, is essential for both innate and non-adaptive immune responses. In macrophages, MAP3K8 is involved in the regulation of TLR4 signaling, which activates MAP2K1 / 2-MAPK1 / 2 to regulate c-jun and p70-S6K activity. MAP3K8 is also involved in the intracellular signaling of TNF-α, IL-1, adiponectin, IL-17, antigen receptors, and G protein-coupled receptors, thereby regulating the production of inflammatory cytokines. In addition, MAP3K8 is also essential for the body's anti-infective immune response. MAP3K8 knockout mice are more susceptible to Toxoplasma gondii, Listeria monocytogenes, Group B Streptococcus, Mycobacterium tuberculosis, Schistosoma mansoni, etc. Despite this, the function of MAP3K8 on T cells and NK cells is still unclear, and there is no direct evidence whether MAP3K8 can be targeted for enhanced engineered immune cell therapy. Summary of the invention

[0006] In view of the defects existing in the above-mentioned prior art, in order to solve the problem that in the treatment of solid tumors, after CAR-T or CAR-NK cells are reinfused, they need to face a complex immunosuppressive microenvironment and cannot be effectively activated, and the killing function is limited, the purpose of the present invention is to design a method for enhancing the tumor cell killing function of CAR-T or CAR-NK cells by targeting MAP3K8. Specifically, the cDNA of MAP3K8 is cloned into the pWPXLD vector, packaged into a lentivirus, and then infected with CAR-T or CAR-NK cells. The MAP3K8 gene is forcibly expressed under the drive of EF1α, thereby enhancing the anti-tumor function of CAR-T or CAR-NK cells.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a function-enhanced CAR-T or CAR-NK cell, which can overexpress the MAP3K8 gene.

[0009] In the functionally enhanced CAR-T or CAR-NK cell, the nucleic acid sequence of the MAP3K8 gene is as shown in SEQ ID NO.11 or its degenerate sequence.

[0010] In a second aspect, the present invention provides a method for preparing a function-enhanced CAR-T or CAR-NK cell, comprising the following steps:

[0011] (1) synthesizing a nucleic acid sequence encoding a chimeric antigen receptor targeting EGFR or CD133, and cloning the nucleic acid sequence into a lentiviral packaging backbone plasmid to construct a CAR expression vector;

[0012] (2) inserting the human MAP3K8 coding sequence into the lentiviral packaging backbone plasmid to construct a MAP3K8 overexpression plasmid;

[0013] (3) packaging and concentrating the CAR expression vector obtained in step (1) with lentivirus, and then infecting NK92 cells or T cells to obtain CAR-NK cells or CAR-T cells;

[0014] (4) After lentiviral packaging and concentration of the MAP3K8 overexpression plasmid obtained in step (2), the plasmid is infected with the CAR-NK cells or CAR-T cells obtained in the above step (3) to obtain MAP3K8 overexpressing CAR-NK or CAR-T cells.

[0015] In the preparation method, the chimeric antigen receptor targeting EGFR or CD133 in step (1) comprises a CD8α signal peptide, an anti-EGFR scFv, a tag, CD8αH / T, a 4-1BB or CD28 co-stimulatory domain, and CD3ζ connected in sequence;

[0016] Alternatively, the chimeric antigen receptor targeting EGFR or CD133 comprises a CD8α signal peptide, a tag, an anti-CD133 scFv, CD8αH / T, a 4-1BB or CD28 co-stimulatory domain, and CD3ζ which are sequentially linked.

[0017] The preparation method, the nucleic acid sequence of the CD8α signal peptide is shown as SEQ ID NO.3;

[0018] The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.4;

[0019] The nucleic acid sequence of the CD8αH / T is shown in SEQ ID NO.6;

[0020] The nucleic acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO.7;

[0021] The nucleic acid sequence of CD3ζ is shown in SEQ ID NO.8;

[0022] The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.10.

[0023] In the preparation method, the tag is one of a Myc tag and a Flag tag;

[0024] Wherein, the nucleic acid sequence of the Myc tag is shown as SEQ ID NO.5;

[0025] The nucleic acid sequence of the Flag tag is shown in SEQ ID NO.9.

[0026] In a third aspect, the present invention provides a MAP3K8 gene overexpression vector, comprising an expression vector of a chimeric antigen receptor targeting EGFR or CD133, a promoter, and a MAP3K8 gene integrated with the expression vector;

[0027] Preferably, the integration is carried out via lentiviral infection, IRES or 2A peptide.

[0028] In a fourth aspect, the present invention provides a method for preparing function-enhanced CAR-T or CAR-NK cells, using viral transduction of the MAP3K8 gene overexpression vector as described to obtain function-enhanced CAR-T or CAR-NK cells.

[0029] In a fifth aspect, the present invention provides the use of the CAR-T or CAR-NK cells or the MAP3K8 gene overexpression vector in the preparation of a drug for treating solid tumors;

[0030] Preferably, the solid tumor is caused by inflammation or infection.

[0031] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an effective therapeutic amount of the functionally enhanced CAR-T or CAR-NK cells, and / or the functionally enhanced CAR-T or CAR-NK cells prepared by the preparation method.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention provides a method for enhancing the function of CAR-T or CAR-NK cells, and the application of CAR immune cells constructed based on the method in the treatment of solid tumors. After the MAP3K8 gene is transduced into CAR-T or CAR-NK cells by virus, it is forced to express under the action of EF1α promoter, which can significantly enhance the anti-tumor function of CAR-T or CAR-NK and improve the activation and killing function of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the CAR structure;

[0035] Figure 2 Schematic diagram of MAP3K8 overexpression plasmid;

[0036] Figure 3 is the expression of CAR on NK92MI cells;

[0037] Figure 4 ZsGreen detection for CAR-NK92MI cells overexpressing MAP3K8;

[0038] Figure 5 qPCR was used to detect MAP3K8 overexpression in CAR-NK92MI cells;

[0039] Figure 6 Western blot was used to detect the overexpression of MAP3K8 in CAR-NK92MI cells;

[0040] Figure 7 The killing of target cells by CAR-NK92MI overexpressing MAP3K8;

[0041] Figure 8 Cytokine secretion after co-culture of CAR-NK92MI overexpressing MAP3K8 with target cells. DETAILED DESCRIPTION

[0042] The present invention will be further described below by means of the accompanying drawings and examples. The present invention will be described in detail below by means of specific examples, which are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples, unless otherwise specified, are conventional methods, and the experimental materials used are all commercially available products.

[0043] Embodiment 1:

[0044] 1. Plasmid structure design and construction

[0045] according to Figure 1 The structure shown inserts the corresponding nucleic acid sequence into the lentiviral packaging backbone plasmid pWPXLd to construct a CAR expression plasmid named EGFR-BBz (nucleic acid sequence as shown in SEQ ID NO.1) and CD133-BBz (nucleic acid sequence as shown in SEQ ID NO.2). Among them, the nucleic acid sequence of CD8α signal peptide (i.e., CD8αSP) is shown in SEQ ID NO.3; the nucleic acid sequence of anti-EGFR scFv is shown in SEQ ID NO.4; the nucleic acid sequence of Myc tag is shown in SEQ ID NO.5; the nucleic acid sequence of CD8αH / T (or CD8α hinge / transmembrane domain) is shown in SEQ ID NO.6; the nucleic acid sequence of 4-1BB co-st (or 4-1BB costimulatory domain) is shown in SEQ ID NO.7; the nucleic acid sequence of CD3ζ is shown in SEQ ID NO.8; the nucleic acid sequence of Flag tag is shown in SEQ ID NO.9; the nucleic acid sequence of anti-CD133 scFv is shown in SEQ ID NO.10.

[0046] according to Figure 2The structure shown inserts the human MAP3K8 coding sequence (the nucleotide sequence of MAP3K8 is shown in SEQ ID NO.11) into the lentiviral packaging backbone plasmid pWPXLd to construct a MAP3K8 overexpression plasmid named pWPXLd-MAP3K8.

[0047] 2. Lentivirus packaging and concentration

[0048] 1) HEK293T cells were seeded in 10 cm 2 Incubate the cells in a CO2 incubator overnight in a cell culture dish to allow the cell confluence to reach 70%-90% on the next day;

[0049] 2) Liposome transfection: Add the target plasmid (10 μg), lentiviral packaging plasmid psPAX2 (7.5 μg), and envelope plasmid pMD2.G (2.5 μg) to 1 mL of opti-MEM medium at a ratio of 4:3:1, then add 60 μL of PEI transfection reagent, mix well, let stand at room temperature for 15 min, slowly add to HEK293T cells, gently shake the culture dish to mix, place in a 37°C incubator, and replace with fresh complete medium after 6 hours of culture;

[0050] 3) After 48 hours, the culture medium in the culture dish was collected and filtered through a 0.45 μm filter to obtain the lentiviral supernatant;

[0051] 4) Add 2 mL of 5×PEG8000 to every 8 mL of viral supernatant and shake at 4°C overnight;

[0052] 5) Centrifuge at 4000 g for 30 min at 4°C, remove the supernatant completely, and resuspend the virus precipitate in 100 μL PBS to obtain the concentrated lentivirus.

[0053] 3. Preparation of CAR-NK cells

[0054] 2 × 10 5 For NK92MI cells, add concentrated EGFR-BBz and CD133-BBz lentiviruses directly into the wells, add polybrene at a final concentration of 8 μg / mL to enhance infection, replace with fresh culture medium the next day, and detect the CAR positivity rate after another two days.

[0055] 4. Detection of CAR expression by flow cytometry

[0056] 1) Collect CAR-NK92MI cells with good growth status in a 1.5 mL centrifuge tube, and collect wild-type NK92MI cells as a negative control, centrifuge at 4°C, 200g for 3 minutes, and remove the culture medium;

[0057] 2) Resuspend the cell pellet in 1 mL of pre-cooled PBS, centrifuge at 200 g for 3 min at 4°C, and remove the supernatant;

[0058] 3) Add 100 μL of diluted anti-Myc or anti-Flag antibody working solution to each sample (the specific dilution ratio is determined according to the antibody instructions), protect from light, and incubate at 4°C for 20 minutes;

[0059] 4) Add 1 mL of pre-cooled PBS, centrifuge at 4°C, 200 g for 3 min, remove the supernatant, and repeat once;

[0060] 5) After resuspending in 200 μL PBS, flow cytometry was performed to analyze the expression of CAR on NK92MI cells.

[0061] 5. Flow cytometry sorting of positive CAR-NK cells

[0062] 1) Collect approximately 5 × 10 6 CAR-NK92MI cells with good growth status were collected, and wild-type NK92MI cells were collected as negative control, centrifuged at 4°C, 200g for 3 minutes, and the culture medium was removed;

[0063] 2) Resuspend the cell pellet in 5 mL of pre-cooled PBS, centrifuge at 200 g for 3 min, and remove the supernatant;

[0064] 3) Add 500 μL of diluted anti-Myc or anti-Flag antibody working solution (the specific dilution ratio is determined according to the antibody instructions), protect from light, and incubate at 4°C for 20 minutes;

[0065] 4) Add 5 mL of pre-cooled PBS to resuspend, centrifuge at 200 g for 3 min, remove the supernatant, and repeat once;

[0066] 5) Resuspend in 500 μL complete medium and transfer to a 15 mL centrifuge tube and place on ice away from light;

[0067] 6) In the flow cytometer, determine the gate of positive cells based on the negative control and sort out the CAR-NK92MI cells that are CAR-positive;

[0068] 7) The sorted cells were centrifuged at 300 g for 5 min, resuspended in complete culture medium containing 3% cyan / chain dual antibody, and cultured in a CO2 incubator to obtain completely positive CAR-NK92MI cells. Figure 3 As shown, it can be seen that the sorted CAR-NK92MI cells are all CAR-positive cells.

[0069] 6. Preparation of MAP3K8 overexpressing CAR-NK cells

[0070] 2 × 10 5 For CAR-NK92MI cells, add the concentrated pWPXLD and pWPXLD-MAP3K8 lentivirus directly into the wells, add polybrene with a final concentration of 8 μg / mL to enhance infection, replace the culture medium with fresh one the next day, and detect the ZsGreen positivity rate two days later. After the infected CAR-NK92MI cells are expanded, the ZsGreen-positive cells are sorted by flow cytometry to obtain fully positive MAP3K8 overexpressing CAR-NK92MI cells. The ZsGreen expression of the sorted CAR-NK92MI is shown in Figure 2. Figure 4 As shown, it can be seen that the sorted CAR-NK92MI cells are all cells that overexpress MAP3K8.

[0071] Example 2: Overexpression of MAP3K8 in CAR-NK cells

[0072] 1. qPCR detection of MAP3K8 overexpression in CAR-NK cells

[0073] 1) Collect 1×10 6 EGFR-BBz and CD133-BBz CAR-NK92MI infected with pWPXLD and pWPXLD-MAP3K8 were centrifuged at 200 g for 3 min, the supernatant was removed, 1 mL of TRIzol was added to lyse the cells, and RNA was extracted using the TransZol UpPlus RNAKit;

[0074] 2) Use Tiangen brand FastKing one-step genomic cDNA first-strand synthesis premix reagent to reverse transcribe RNA;

[0075] 3) After the reverse transcription product was diluted 10 times with DEPC water, qPCR SYBR Green Master Mix (No Rox) was used to detect MAP3K8 expression by qPCR, with GAPDH as an internal reference. The upstream primer sequence of MAP3K8 is shown in SEQ ID NO.12, the downstream primer sequence of MAP3K8 is shown in SEQ ID NO.13, the upstream primer sequence of GAPDH is shown in SEQ ID NO.14, and the downstream primer sequence of GAPDH is shown in SEQ ID NO.15.

[0076] qPCR results Figure 5 As shown, it can be seen that the expression level of MAP3K8 in CAR-NK92MI after sorting was significantly upregulated.

[0077] 2. Western blot detection of MAP3K8 overexpression in CAR-NK cells

[0078] 1) Collect 1×10 6 EGFR-BBz and CD133-BBz CAR-NK92MI infected with pWPXLD and pWPXLD-MAP3K8 were centrifuged at 200 g for 3 min, the supernatant was removed, 1 mL of pre-cooled PBS was added to resuspend the cells, and then centrifuged at 200 g for 3 min, the supernatant was removed, and 50 μL of RIPA lysis buffer (strong) was added to the cell pellet;

[0079] 2) The sample was ultrasonically disrupted for 30 seconds at 40% power and then centrifuged at 14000g for 15 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube;

[0080] 3) Add 10 μL of 6× Loading Buffer, boil at 100°C for 10 min, add 10 μL of sample to SDS-PAGE gel for Western blot experiment to detect MAP3K8 expression, using GAPDH as internal reference.

[0081] Western blot results Figure 6 As shown, it can be seen that the MAP3K8 protein expression level of CAR-NK92MI after sorting was significantly upregulated.

[0082] Example 3: Luciferase bioluminescence assay to determine the tumor killing effect of CAR-NK cells

[0083] 1) Huh7, a liver cancer cell line with good growth status and positive for EGFR and CD133, was digested and resuspended and counted at 1×10 per well. 4 The cells were seeded in a black, light-proof 96-well ELISA plate and cultured overnight;

[0084] 2) Remove the target cell culture medium, add 100 μL of different CAR-NK92MI cells according to different effector-target ratios, and culture them in a CO2 incubator for 4-6 hours. Set up 3 replicates for each effector-target ratio in each group, and set up a target cell culture group (negative control group) and a culture medium group (blank group);

[0085] 3) Prepare D-luciferin potassium salt working solution: Dilute 30 mg / mL D-luciferin potassium salt stock solution to 0.15 mg / mL with PBS in a dark place. All subsequent operations must be kept away from light.

[0086] 4) Remove the cell culture medium, add 100 μL of D-luciferin potassium salt working solution to each well using an 8-channel pipette, and incubate at 37°C for 5 min;

[0087] 5) The fluorescence intensity was collected in a microplate reader, the blank group signal was subtracted from the signal in all wells, and then the killing rate of CAR-NK on target cells was calculated according to the following formula: (1-co-culture group reading / negative control group reading)×100%.

[0088] Figure 7 The results show that under different effector-target ratios, the killing effect of EGFR-BBz and CD133-BBz CAR-NK92MI cells in the present invention after overexpressing MAP3K8 on Huh7 cells is significantly enhanced after forced overexpression of MAP3K8.

[0089] Example 4: CBA method to measure cytokine secretion of CAR-NK cells

[0090] 1) Huh7 cells with good growth status were digested and resuspended and counted at 1×10 per well. 4 The cells were seeded in a black, light-proof 96-well ELISA plate and cultured overnight;

[0091] 2) Collect and adjust the density of CAR-NK92MI to 2×10 5 cells / mL;

[0092] 3) Remove the target cell culture medium from the ELISA plate, add 100 μL of different CAR-NK92MI cells (target ratio 2:1) to each well, and culture in a CO2 incubator, with 3 replicate wells per group;

[0093] 4) After 24 hours, the culture medium of each well was collected and centrifuged at 1000 g for 5 min to remove cells and debris. TM Detect cytokine concentrations in the culture medium.

[0094] Figure 8 The cytokine secretion levels of EGFR-BBz and CD133-BBz CAR-NK92MI cells overexpressing MAP3K8 and co-cultured with Huh7 in the present invention are shown. It can be seen that after forced overexpression of MAP3K8, CAR-NK92MI cells can secrete more anti-tumor cytokines (IFN-γ, TNF-α, Granzyme B, Granulysin) when co-cultured with target cells.

[0095] In the in vitro killing experiment, forced overexpression of MAP3K8 in CAR-NK92MI cells in the present invention significantly enhanced its killing of tumor cells and the secretion of anti-tumor cytokines, verifying that the overexpression of MAP3K8 disclosed in the present invention can significantly enhance the anti-tumor function of CAR-NK.

[0096] In summary, overexpression of the MAP3K8 gene can significantly enhance the killing function of CAR-T or CAR-NK, and the new CAR immune cells constructed based on this method have better anti-tumor potential.

Claims

1. A functionally enhanced CAR-T or CAR-NK cell, characterized in that: The CAR-T or CAR-NK cells may overexpress the MAP3K8 gene.

2. A function-enhanced CAR-T or CAR-NK cell according to claim 1, characterized in that: The nucleic acid sequence of the MAP3K8 gene is shown in SEQ ID NO.11 or its degenerate sequence.

3. The method for preparing a functionally enhanced CAR-T or CAR-NK cell according to claim 1 or 2, characterized in that: The following steps are involved: (1) synthesizing a nucleic acid sequence encoding a chimeric antigen receptor targeting EGFR or CD133, and cloning the nucleic acid sequence into a lentiviral packaging backbone plasmid to construct a CAR expression vector; (2) inserting the human MAP3K8 coding sequence into the lentiviral packaging backbone plasmid to construct a MAP3K8 overexpression plasmid; (3) packaging and concentrating the CAR expression vector obtained in step (1) with lentivirus, and then infecting NK92 cells or T cells to obtain CAR-NK cells or CAR-T cells; (4) After lentiviral packaging and concentration of the MAP3K8 overexpression plasmid obtained in step (2), the CAR-NK cells or CAR-T cells obtained in the above step (3) are infected to obtain MAP3K8 overexpressing CAR-NK or CAR-T cells.

4. The preparation method according to claim 3, characterized in that: The chimeric antigen receptor targeting EGFR or CD133 in step (1) comprises a CD8α signal peptide, an anti-EGFR scFv, a tag, a CD8αH / T, a 4-1BB or CD28 co-stimulatory domain, and CD3ζ connected in sequence; Alternatively, the chimeric antigen receptor targeting EGFR or CD133 comprises a CD8α signal peptide, a tag, an anti-CD133 scFv, CD8αH / T, a 4-1BB or CD28 co-stimulatory domain, and CD3ζ which are sequentially linked.

5. The preparation method according to claim 4, characterized in that: The nucleic acid sequence of the CD8α signal peptide is shown in SEQ ID NO.3; The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.4; The nucleic acid sequence of the CD8αH / T is shown in SEQ ID NO.6; The nucleic acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO.7; The nucleic acid sequence of CD3ζ is shown in SEQ ID NO.8; The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.

10.

6. The preparation method according to claim 4, characterized in that: The tag is one of a Myc tag and a Flag tag; Wherein, the nucleic acid sequence of the Myc tag is shown as SEQ ID NO.5; The nucleic acid sequence of the Flag tag is shown in SEQ ID NO.

9.

7. A MAP3K8 gene overexpression vector, characterized in that: An expression vector comprising a chimeric antigen receptor targeting EGFR or CD133, a promoter, and a MAP3K8 gene integrated with the expression vector; Preferably, the integration is carried out via lentiviral infection, IRES or 2A peptide.

8. A method for preparing functionally enhanced CAR-T or CAR-NK cells, characterized in that: Functionally enhanced CAR-T or CAR-NK cells are obtained by viral transduction using the MAP3K8 gene overexpression vector as described in claim 7.

9. Use of the CAR-T or CAR-NK cell according to claim 1 or the MAP3K8 gene overexpression vector according to claim 7 in the preparation of a drug for treating solid tumors; Preferably, the solid tumor is caused by inflammation or infection.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective therapeutic amount of the function-enhanced CAR-T or CAR-NK cells according to claim 1, and / or the function-enhanced CAR-T or CAR-NK cells prepared by the preparation method according to claim 2 or 8.

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