NK cell transduction method, NK cell, composition and application

Abstract

The invention provides an NK cell transduction method, an NK cell, a composition and application, and the method comprises the following steps: knocking out CREM in the NK cell, and then transduction CAR. The research finds that after the CREM is knocked out, the killing efficiency of the CAR NK cells can be effectively improved indeed, and surprisingly, besides the killing efficiency, the CAR transduction positive rate of the NK cells can be improved by knocking out the CREM, and for expression of IL15 in different forms, the increasing amplitude of CREM expression, the increasing amplitude of the CAR transduction positive rate and the increasing amplitude of CAR NK killing efficiency are different. Through experimental comparison, it is finally found that the function improvement of CAR NK expressing secretory IL15 is most obvious after CREM is knocked out.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cellular immunotherapy of tumors, in particular to a method for transducing NK cells, NK cells, compositions and applications thereof. BACKGROUND

[0002] NK cells (Natural Killer cells) are one of the core cells of anti-tumor immunotherapy, and with their unique biological characteristics, they have shown significant potential in the treatment of hematological tumors and solid tumors. However, the recognition of natural NK cells to tumors lacks absolute specificity, and some tumor cells can escape NK cell killing by down-regulating NKG2D ligands and up-regulating MHC-I molecules, which is called immune escape. CAR (Chimeric Antigen Receptor) modified NK cells can be used to improve their targeting ability, and double or triple specific CARs can be designed to recognize multiple tumor antigens and reduce the risk of escape. At the same time, natural NK cells also have the problem of poor persistence in vivo, and the therapeutic effect is difficult to maintain. After NK cell infusion, the survival time in vivo is only 3-7 days, which cannot continuously kill tumor cells, leading to the recurrence of some patients in the short term. In clinical practice, the in vivo persistence of NK cells is prolonged by overexpressing IL15 on NK cells.

[0003] CREM (cAMP responsive element modulator) is a transcription factor widely present in eukaryotes, and is a key downstream regulatory factor of CAR signal and IL15 signal. CAR can induce up-regulation of CREM through PKA-CREB pathway stimulated by ITAM (Immunoreceptor Tyrosine-based Activation Motif) signal or IL-15. CREM inhibits NK cell effector function through epigenetic reprogramming, while activating exhaustion-related genes, leading to the "activation-exhaustion" paradox phenotype of NK cells, which weakens the anti-tumor activity. To solve this problem, we can knock out CREM through CRISPR-Cas9 to improve the anti-tumor ability of CAR NK. SUMMARY

[0004] To solve the above problems, the present application provides a method for transducing NK cells, which comprises knocking out CREM in NK cells, and then transducing CAR, wherein the CAR structure sequentially comprises CD19 ScFv, CD28 transmembrane domain, CD28 co-stimulatory domain, CD3 zeta activation domain, BCMA ScFv, CD8 transmembrane domain, 41BB co-stimulatory domain, CD3 zeta activation domain and IL15, wherein the IL15 is any one of membrane-bound IL15, secreted IL15 or enhanced IL15.

[0005] In an embodiment, the IL15 is secreted IL15.

[0006] In an embodiment, the CD19 ScFv is SEQ ID NO. 1, and the BCMA ScFv is SEQ ID NO. 2.

[0007] In an embodiment, the membrane-bound IL15 is SEQ ID NO. 3, the secreted IL15 is SEQ ID NO. 4, and the enhanced IL15 is SEQ ID NO. 5.

[0008] In an embodiment, the CD8 is SEQ ID NO. 6, the CD28 is SEQ ID NO. 7, the 41BB co-stimulatory domain is SEQ ID NO. 8, and the CD3 zeta activation domain is SEQ ID NO. 9.

[0009] In an embodiment, the present application provides an NK cell, which is prepared by knocking out CREM in NK cells, and then transducing CAR, wherein the CAR structure sequentially comprises CD19 ScFv, CD28 transmembrane domain, CD28 co-stimulatory domain, CD3 zeta activation domain, BCMA ScFv, CD8 transmembrane domain, 41BB co-stimulatory domain, CD3 zeta activation domain and IL15, wherein the IL15 is any one of membrane-bound IL15, secreted IL15 or enhanced IL15.

[0010] In an embodiment, the present application provides a composition for treating tumors, which comprises the above-mentioned NK cell, and a pharmaceutically acceptable carrier.

[0011] In an embodiment, the tumor is acute lymphoblastic leukemia.

[0012] In an embodiment, the present application provides use of the above-mentioned NK cell in the preparation of a medicament for treating acute lymphoblastic leukemia.

[0013] In one embodiment, the present application provides a method for inhibiting acute lymphoblastic leukemia cells in vitro, which comprises co-incubating the above-mentioned NK cells or the above-mentioned composition with tumor cells.

[0014] The present application first realizes specific recognition of tumor cells through the CAR structure targeting CD19 / BCMA, thereby significantly improving the accuracy of treatment. Secondly, the CAR-NK cells designed to express IL-15 provide a sustained survival and expansion signal in vivo, avoiding the risk of systemic toxicity caused by relying on exogenous cytokine support. Finally, because the presence of IL15 upregulates the expression of CREM, accelerating the exhaustion of NK cells, in order not to affect the killing efficiency and expansion of NK cells, the CREM is knocked out and then the CAR is retransduced, thereby effectively improving the anti-tumor effect of CAR NK cells. The present study found that knocking out CREM can indeed effectively improve the killing efficiency of CAR NK cells, and surprisingly, in addition to the killing efficiency, knocking out CREM can also improve the CAR transduction positive rate of NK cells, and the increase in the expression of CREM, the improvement in the CAR transduction positive rate, and the improvement in the CAR NK killing efficiency are different for different forms of IL15 expression. Through comparison experiments, we finally found that the CAR NK expressing secreted IL15 (sIL15) has the most obvious function improvement after knocking out CREM. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 is a schematic diagram of four CAR structures designed in the present application; Figure 2 is a colony PCR result graph of 256-parallel CD19 / BCMA CAR-mbIL15; Figure 3 is a colony PCR result graph of 336-parallel CD19 / BCMA CAR-sIL15; Figure 4 is a colony PCR result graph of 338-parallel CD19 / BCMA CAR-RLI; Figure 5 is a graph of the detection results of the biological titer of three retroviral vectors; Figure 6 is a graph of the detection results of the transduction positive rate of seven kinds of NK cells; Figure 7 Figure 8 is a graph showing the results of detection of expression of eight NK cell CREMs; Figure 8 Figure 9 is a graph showing the results of killing efficiency of eight NK cells on NALM6-BCMA cells; Figure 9 Figure 10 is a graph showing the results of persistent killing of six NK cells on NALM6-BCMA cells. DETAILED DESCRIPTION

[0017] In order to make the technical field of the art better understand the technical solutions in the present application, the following will be combined with examples to further illustrate the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative labor should belong to the scope of protection of the present application.

[0018] Cell source: HEK293T, Phoenix-Ampho cells are commercial cell lines, which are purchased from ATCC. The reagents and raw materials used in the present application are from commercial reagents if not specified.

[0019] Sequence information: CD19 scfv (SEQ ID No. 1): DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS BCMA scfv (SEQ ID No. 2): DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAA mbIL15 (SEQ ID No. 3): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL sIL15 (SEQ ID No. 4): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS RLI (SEQ ID No. 5): MAPRRARGCRTLGLPALLLLLLLRPPATRGDYKDDDDKIEGRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS CD8 (SEQ ID No. 6): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC CD28 (SEQ ID No. 7): KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 41BB costimulatory domain (SEQ ID No. 8): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3 zeta activation domain (SEQ ID No. 9): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0020] Example One Design and construction of parallel CD19 / BCMA CAR combined with IL15 plasmid.

[0021] 1. Plasmid design As shown in Figure 1 , the present application designs three kinds of parallel CD19 / BCMA CAR combined with IL15 plasmid, which are specifically described as follows.

[0022] (1) Parallel CD19 / BCMA-mbIL15 CAR structure, CD19 ScFv is FMC63, connected with CD28 transmembrane domain, CD28 co-stimulatory domain, CD3 zeta activation domain, BCMA ScFv is C11D5.3, connected with CD8 transmembrane domain, 41BB co-stimulatory domain, CD3 zeta activation domain and membrane-bound IL15 (mbIL15), named 256.

[0023] (2) Parallel CD19 / BCMA-sIL15 CAR structure: replace mbIL15 in parallel CD19 / BCMA-mbIL15 CAR structure with secreted IL15 (sIL15), named 336.

[0024] (3) Parallel CD19 / BCMA-RLI CAR structure: replace mbIL15 in parallel CD19 / BCMA-mbIL15 CAR structure with enhanced IL15 (RLI), named 338.

[0025] 2. Plasmid construction and identification PCR primers for recombinant antibody fragments were designed using SnapGene software, and the recombinant antibody fragments were amplified using the original CAR plasmid as a template. The PCR products were subjected to agarose gel electrophoresis and recovered and purified. The MFG retrovirus vector was double-cut with XhoI / NotI restriction endonuclease and recovered and purified. The recombinant antibody fragment and linearized vector were subjected to seamless cloning, and the product was transformed into DH5a E. coli. After shaking culture, the plate was incubated overnight, and a single colony was picked and subjected to shaking culture and sequencing verification.

[0026] (1) Amplification of target fragments The base sequences of parallel CD19 / BCMA CAR-mbIL15 (256), CD19 / BCMA CAR-sIL15 (336), and CD19 / BCMACAR-RLI (338) were synthesized by Genescript Biotech Co., Ltd. The target fragments were amplified by PCR using designed primers to obtain double-stranded DNA fragments.

[0027] (2) Target fragment insertion into Z203 vector The commercial retrovirus packaging vector plasmid Z203 was cut with XhoI and BamH I restriction endonuclease, and the vector fragment with XhoI and BamH I restriction sites was recovered by agarose gel electrophoresis, with a fragment size of about 7.6 kb. The target fragment and the vector fragment were connected by seamless cloning reagent, and the molar ratio of the target fragment to the vector fragment was 3:1-5:1.

[0028] (3) Plasmid transformation and screening The recombinant plasmid was transformed into DH5α competent cells and cultured overnight at 37°C with 5% CO2. The next day, single colonies were picked and colony PCR was performed. Gel electrophoresis was used to verify the size of the target fragment. Subsequently, the correct bacterial culture was sent to Qingke Biotechnology for sequencing. Table 1 shows the primers for colony PCR.

[0029] Table 1 .

[0030] (4) Extraction of endotoxin-free plasmids Take the correctly sequenced bacterial strains, add 60 μL to 15 mL of LB liquid medium (final Amp concentration 100 μg / mL), and incubate at 37 ℃ and 200 rpm for 12-16 h on a shaker. Harvest the bacterial pellet by centrifugation, extract the plasmid using an endotoxin-free plasmid extraction kit, measure the DNA concentration, and store the plasmid at -20 ℃ for later use.

[0031] 3. Experimental Results (1) Colony PCR identification results PCR identification of 256-mesh bacterial culture revealed a fragment size of 3.1 kb (e.g., ...). Figure 2 (As shown); PCR identification of 336-mesh bacterial culture showed a fragment size of 1.2kb (as shown). Figure 3 (As shown); PCR identification of 338-target bacterial culture showed a fragment size of 1.4 kb (e.g. Figure 4 (As shown in the image); the above gel electrophoresis results show that the band sizes are all correct and can be sent for further sequencing verification.

[0032] (2) Successful construction of plasmids 256, 336, and 338: The bacterial culture with correct sequencing results ( Figures 2-4 (Labeling was performed) Plasmids were extracted and sent for sequencing. Sequencing results showed that plasmid sequences 256, 336, and 338 were completely correct.

[0033] PCR identification of 256-mesh bacterial culture revealed a fragment size of 3.1 kb (e.g., ...). Figure 2 (As shown); PCR identification of 336-mesh bacterial culture showed a fragment size of 1.2kb (as shown). Figure 3 (As shown); PCR identification of 338-target bacterial culture showed a fragment size of 1.4 kb (e.g. Figure 4 (As shown in the image); the above gel electrophoresis results show that the band sizes are all correct, and sequencing can be sent for further verification. (2) Successful construction of plasmids 256, 336, and 338: The bacterial culture with correctly sequenced bacterial culture ( Figures 2-4 (Labeling was performed) Plasmids were extracted and sent for sequencing. Sequencing results showed that plasmid sequences 256, 336, and 338 were completely correct.

[0034] Example 2: Preparation of Retroviral Vectors and Detection of Viral Biological Titer The three retroviral vectors mentioned above, 256, 336, 338, are prepared by a retroviral vector packaging cell line. The retroviral vector packaging cell line (Ampho and BAEV-WT) is selected, the plasmid is transfected into the Ampho cell by Lipomaster 2000 transfection reagent, and virus production is carried out. The supernatant is collected 48 h and 72 h after transfection, and the BAEV-WT cell is transduced by horizontal centrifugation to construct a BAEV-WT retroviral vector stable cell line. The culture supernatant of the BAEV-WT retroviral vector stable cell line is collected, and the 293T cell is transduced to detect the supernatant virus vector biological titer. The specific experimental process is as follows.

[0035] 1. Plasmid transfection The constructed plasmids 256, 336, 338 are transfected into Ampho cells by Lipomaster 2000, and are cultured in a 37℃, 5% CO2 incubator for 16-24 h. When the cell density reaches 80%-90%, the cells are placed in a 32℃, 5% CO2 incubator after changing the liquid, and then the virus vectors collected 24 h, 48 h and 72 h after changing the liquid are collected for subsequent cell transduction experiments.

[0036] 2. Construction of stable cell line The collected virus vectors are transfected into BAEV-WT cells by polybrene, and three stable cells BAEV-WT-256, BAEV-WT-336 and BAEV-WT-338 are constructed and expanded. When the cell density reaches 80%-90%, the cells are changed and placed in a 32℃, 5% CO2 incubator. The retroviral vectors collected 24 h, 48 h, 72 h, 96 h and 120 h are named H1, H2, H3, H4 and H5. The vectors collected for 5 days are mixed and divided, and the titer is detected.

[0037] 3. Titer detection The virus vector is added with polybrene to promote the infection of the virus vector to the 293T cells. After the infected 293T cells are cultured in a 37°C, 5% CO2 incubator for 48 hours, the cells are digested with trypsin, and the transduction efficiency of the 293T is detected by APC-G4S antibody staining and flow cytometry. The formula is: 293T transduction positive rate x 293T cell number on the day of transduction / virus volume (ml) for transduction, and the virus titers of the three stable cell production of BAEV-WT-256, BAEV-WT-336 and BAEV-WT-338 are obtained, and the results of the detection of the biological titer of the retrovirus vector are shown in FIG. 5, and the numerical values of the detection results of the biological titer of the retrovirus vector are shown in Table 2, and the unit is TU / ml. The titer is above 3E5 TU / ml, which indicates that the vector preparation is successful and can be used for NK cell transduction.

[0038] Table 2 .

[0039] Example Three Preparation of CAR-NK Cells with CREM Knockout 1. NK cell transduction

[0040] Experimental process: NK cells are sorted from peripheral blood mononuclear cells (PBMC) of healthy volunteers by NK sorting kit, and the NK cells are knocked out of CREM before being transduced with CAR. NK-256-CREM (KO), NK-336-CREM (KO), and NK-338-CREM (KO) cells are used as experimental groups. The three CAR NK cells without CREM knockout, NK-256, NK-336, and NK-338, are used as control groups. The untreated NK cells are used as negative controls of the control group. The NK cells with CREM knockout, NK-CREM (KO), are used as negative controls of the experimental group. There are a total of 8 groups of NK cells.

[0041] Experimental results: Figure 6 The CAR transduction positive rate is shown in Table 3. As can be seen from the specific numerical values of the CAR NK positive rate, under the same transduction conditions, the CAR transduction positive rate of the NK cells with CREM knockout is generally higher than that of the NK cells without CREM knockout, indicating that the knockout of CREM can improve the CAR transduction positive rate of the NK cells.

[0042] Table 3 .

[0043] 2. Detection of NK cell CREM knockout efficiency Experimental process: after knocking out CREM in NK cells, take 2E5 cells to extract genomic RNA, reverse transcription to obtain cDNA, and then detect the expression of CREM in the genome of NK cells by qPCR method to verify the knocking out effect of NK cell CREM.

[0044] Experimental results: Figure 7 For NK cell CREM expression detection results, from Figure 7 A can see that after knocking out CREM, the expression of NK and three CAR NK cell CREM is significantly reduced. Figure 7 B can see that NK cells without knocking out CREM, after expressing IL15, CREM is significantly increased, among which membrane-bound IL15 (256) is the most obvious, followed by enhanced IL15 (338-RLI), and finally secreted IL15 (336-sIL15). It is proved that overexpression of IL15 can indeed increase the expression of NK cell CREM, and there is difference among the three forms of IL15. From the results, secreted IL15 has the lowest effect on the expression of NK cell CREM. P <0.05, P <0.01, P <0.001), Table 4 is Figure 7 The specific numerical values of the expression level of CAR-NK-CREM are shown in Table 4.

[0045] Table 4 .

[0046] 3、Conclusion After knocking out CREM in NK cells and then transducing CAR, the positive rate of CAR transduction can be generally improved, and overexpression of IL15 in NK cells can increase the expression level of CREM, but the expression level of different forms of IL15 is different, among which secreted IL15 has the lowest effect on the expression of NK cell CREM.

[0047] Example four in vitro study of NK-CREM (KO) cell anti-tumor function 1、Luciferase detection of NK-CREM (KO) cell anti-tumor activity on NALM6-BCMA cells

[0048] Experimental Procedure: NK-256-CREM(KO), NK-336-CREM(KO), and NK-338-CREM(KO) cells were used as experimental groups. The three types of CAR NK cells without CREM knockout (NK-256, NK-336, and NK-338) served as control groups. Untreated NK cells served as negative controls for the control groups. NK cells with CREM knockout (NK-CREM(KO)) served as negative controls for the experimental groups, for a total of eight groups of NK cells. NALM6-BCMA (CD19+BCMA+) was used as the target cells. Different ratios of effector cells to target cells were co-cultured, and the killing efficiency of effector cells against target cells in each group was detected using luciferase assay.

[0049] Experimental Results: Based on the results, there was no significant difference in the killing efficiency of the three CAR NK cells without CREM knockout. Figure 8 A; Knockout of CREM significantly improved the killing efficiency of the three CAR NK cells, and NK-336-CREM(KO) cells showed superior antitumor activity compared to NK-256-CREM(KO) and NK-338-CREM(KO) cells, with statistically significant differences (*). P <0.05) Figure 8 B. Individual comparisons of the three CREM knockout cells with non-knockout CAR NK cells showed that NK-336-CREM (KO) exhibited the most significant improvement in killing efficiency (*). P <0.05,** P <0.01, *** P <0.001) such as Figure 8 The specific experimental data on the killing effect from C to 8E are shown in Table 5. Table 5 shows the specific data results of CAR-NK cell killing efficiency.

[0050] Table 5

[0051] 2. Detection of persistent killing effect of NK-CREM (KO) cells on NALM6-BCMA cells Experimental Procedure: Six groups of NK cells—NK-256-CREM (KO), NK-336-CREM (KO), NK-338-CREM (KO), NK-256, NK-336, and NK-338—were co-cultured with NALM6-BCMA (CD19+BCMA+). The effector-to-target ratio was 1:2. The same number of target cells were added every 24 hours for multiple rounds of stimulation of the effector cells, for a total of three additions. The killing efficiency over these three days was detected by luciferase assay, thereby observing the multi-round killing effect of NK cells on NALM6-BCMA.

[0052] Experimental results: According to the results, under the condition of continuous addition of tumor cells, the killing efficiency of NK-336-CREM (KO) is still higher than that of other NK cells, such as Figure 9 The specific experimental data of the killing results are shown in Table 6, which is the killing efficiency results of the multi-round killing results of CAR-NK cells.

[0053] Table 6

[0054] 3. Conclusion Knocking out CREM can improve the killing efficiency of CAR NK cells on NALM6-BCMA, and after multi-round stimulation, it still has obvious killing effect on NALM6, and the best structure is the CAR NK cell overexpressing secreted IL15, NK-336-CREM (KO).

[0055] It should be understood that the disclosed application is not limited only to the particular methods, schemes and materials described herein, as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing only particular embodiments and is not intended to be limiting, as the scope of the application will be limited only by the appended claims.

Claims

1. A method for NK cell transduction, characterized in that, The method involves first knocking out CREM in NK cells, and then transducing CAR; the CAR structure sequentially includes: CD19 ScFv, connecting to the CD28 transmembrane domain, CD28 co-stimulatory domain, CD3ζ activation domain, BCMA ScFv, connecting to the CD8 transmembrane domain, 41BB co-stimulatory domain, CD3ζ activation domain, and IL15, wherein the IL15 is any one of membrane-bound IL15, secreted IL15, or enhanced IL15.

2. The method according to claim 1, characterized in that, The IL15 mentioned is secreted IL15.

3. The method according to claim 1, characterized in that, The CD19 ScFv is SEQ ID NO.1, and the BCMA ScFv is SEQ ID NO.

2.

4. The method according to claim 1, characterized in that, The membrane-bound IL15 is SEQ ID NO.3, the secreted IL15 is SEQ ID NO.4, and the enhanced IL15 is SEQ ID NO.

5.

5. The method according to claim 1, characterized in that, The CD8 is SEQ ID NO.6, the CD28 is SEQ ID NO.7, the 41BB co-stimulatory domain is SEQ ID NO.8, and the CD3ζ activation domain is SEQ ID NO.

9.

6. An NK cell, characterized in that, First, CREM is knocked out of NK cells, and then CAR is prepared by transduction. The CAR structure sequentially includes: CD19 ScFv, CD28 transmembrane domain, CD28 co-stimulatory domain, CD3ζ activation domain, BCMA ScFv, CD8 transmembrane domain, 41BB co-stimulatory domain, CD3ζ activation domain, and IL15. The IL15 is any one of membrane-bound IL15, secreted IL15, or enhanced IL15.

7. The NK cell according to claim 6, characterized in that, The IL15 mentioned is secreted IL15.

8. The NK cell according to claim 6, characterized in that, The CD19 ScFv is SEQ ID NO.1, and the BCMA ScFv is SEQ ID NO.

2.

9. The NK cell according to claim 6, characterized in that, The membrane-bound IL15 is SEQ ID NO.3, the secreted IL15 is SEQ ID NO.4, and the enhanced IL15 is SEQ ID NO.

5.

10. The NK cell according to claim 6, characterized in that, The CD8 is SEQ ID NO.6, the CD28 is SEQ ID NO.7, the 41BB co-stimulatory domain is SEQ ID NO.8, and the CD3ζ activation domain is SEQ ID NO.

9.

11. A composition for treating tumors, characterized in that, The composition comprises NK cells as described in any one of claims 6-10, and a pharmaceutically acceptable carrier.

12. The composition according to claim 11, characterized in that, The tumor is acute lymphoblastic leukemia.

13. The use of NK cells as described in any one of claims 6-10 in the preparation of drugs for treating acute lymphoblastic leukemia.

14. A method for inhibiting acute lymphoblastic leukemia cells in vitro, the method comprising co-incubating tumor cells with NK cells of any one of claims 6-10 or the composition of claim 11.

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