Recombinant construct, CAR-NK cell and application

Abstract

The invention relates to the field of biotechnology and cell therapy, in particular to a recombinant construct, a CAR-NK cell and application. Experiments prove that the effect and mechanism of the NK cells for killing tumor cells are enhanced through cooperation of IL-10 and a CD19 antigen target; the treatment effect of immune cells is enhanced, and meanwhile, the treatment risk is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and cell therapy, in particular to a recombinant construct, CAR-NK cell and application. BACKGROUND

[0002] CAR-NK (chimeric antigen receptor natural killer) cell therapy has become a research hotspot in the field of tumor immunotherapy in recent years. Compared with CAR-T, CAR-NK not only retains the innate recognition and killing ability of NK cells to tumors, but also is endowed with precise targeting ability through genetic engineering, can eliminate tumor cells expressing and not expressing target antigens, and has lower side effects, such as significantly reduced incidence of cytokine release syndrome (CRS) and graft-versus-host disease (GVHD). At present, CAR-NK clinical research for hematological malignancies (such as CD19 target lymphoma, leukemia, etc.) shows considerable efficacy, with a complete remission rate of 25% to 83% and good safety.

[0003] Interleukin 10 (IL-10) is an important immunoregulatory cytokine and shows a "double-edged sword" effect in the process of tumor occurrence and development. On the one hand, IL-10 can weaken the body's immune response to tumor cells by inhibiting the antigen presentation ability of dendritic cells and macrophages, reducing the expression of pro-inflammatory cytokines (such as TNF-α, IL-6, etc.), and promoting the immune escape of tumor cells and the immunosuppressive state of tumor microenvironment. On the other hand, studies have shown that high-dose IL-10 can enhance the survival, expansion and cytotoxicity of CD8-positive T lymphocytes, promote their killing of tumor cells, and improve the efficacy of immunotherapy. Some new IL-10 fusion proteins and pegylated IL-10 drugs have shown anti-tumor potential in various solid tumor models and clinical studies. Therefore, IL-10 targeting strategies need to be finely regulated according to tumor types and microenvironments to exert both anti-inflammatory and immunosuppressive effects and activate their role in promoting anti-tumor immunity.

[0004] At present, whether it is a hematological tumor or a solid tumor, a number of clinical trials have been carried out in full swing, but challenges such as insufficient activation, increased treatment resistance, and immunosuppressive tumor microenvironment still exist. SUMMARY

[0005] Based on the dual function of IL-10 in promoting and inhibiting tumors, the effect of modifying CAR-NK cells with IL-10 cannot be predicted, and the present application creatively applies IL-10 to the modification of CAR-NK cells, which has unexpected technical effects.

[0006] The present application aims to provide a recombinant construct, CAR-NK cell and application to solve the problems raised in the background art.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] A recombinant construct that simultaneously encodes IL-10 and CAR, fragments or variants thereof, wherein the antigen of the CAR is CD19.

[0009] Preferably, the nucleic acid sequence of the recombinant construct is as shown in SEQ ID NO.1, or a fragment thereof or a variant thereof.

[0010] Preferably, the recombinant construct encodes an amino acid sequence as shown in SEQ ID NO.6, or a fragment thereof, or a variant thereof.

[0011] A CAR-NK cell, comprising a recombinant construct as described in any of the preceding claims.

[0012] Preferably, the CAR-NK cells are autologous, allogeneic, or xenogeneic.

[0013] A pharmaceutical composition comprising a recombinant construct as described in any of the preceding claims or a CAR-NK cell as described in any of the preceding claims.

[0014] Preferably, it comprises at least one pharmaceutically acceptable carrier.

[0015] Use of any of the recombinant constructs described in any of the foregoing, or any of the CAR-NK cells described in any of the foregoing, or any of the pharmaceutical compositions described in any of the foregoing, in the preparation of a kit for the diagnosis / treatment of malignant tumors or autoimmune diseases.

[0016] The use of any of the recombinant constructs described in any of the foregoing, or any of the CAR-NK cells described in any of the foregoing, or any of the pharmaceutical compositions described in any of the foregoing, in the preparation of a medicament for the diagnosis / treatment of malignant tumors or autoimmune diseases.

[0017] Preferably, the malignant tumor is any one of gastric adenocarcinoma, B-cell malignant tumor, or myeloma.

[0018] Compared with existing technologies, the beneficial effects achieved by this invention are: based on the antigen target CD19, targeting CD19 CAR-NK primarily eliminates B cells, making it applicable to the treatment of B-cell malignancies, such as B-cell leukemia and lymphoma, or autoantibody-related autoimmune diseases such as systemic lupus erythematosus. It secretes a cytokine, enhancing the long-term therapeutic effect of immune cells while reducing treatment risks. Attached Figure Description

[0019] The accompanying drawings are included to provide a further understanding of the application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the application, and together with the description serve to explain the principles of the application. In the drawings:

[0020] Figure 1 Schematic diagram of lentiviral vector structure of secretory CD19 CAR-NK;

[0021] Figure 2 Figure of flow cytometry detection of CAR expression of CD19 CAR-NK-IL10;

[0022] Figure 3 Figure of ELISA detection of IL-10 expression in supernatant of CD19 CAR-NK-IL10 culture medium;

[0023] Figure 4 Figure of flow cytometry detection of CD19 CAR-NK-IL10 cell surface receptor expression;

[0024] Figure 5 Figure of in vitro proliferation of CD19 CAR-NK-IL10 cells;

[0025] Figure 6 Figure of luciferase-labeled cell killing experiment results;

[0026] Figure 7 Figure of CD107a degranulation and cytokine secretion experiment results;

[0027] Figure 8 Figure of real-time cell killing (RTCA) experiment results;

[0028] Figure 9 Figure of in vivo imaging results of CD19 CAR-NK-IL10 treatment of tumor-bearing mice;

[0029] Figure 10 Figure of dynamic change of tumor load fluorescence signal of CD19 CAR-NK-IL10 treatment of tumor-bearing mice;

[0030] Figure 11 Figure of survival curve comparison of CD19 CAR-NK-IL10 treatment of tumor-bearing mice;

[0031] Figure 12 Figure of sample gene expression principal component analysis (PCA);

[0032] Figure 13 Figure of differential gene up-regulation and down-regulation volcano plot;

[0033] Figure 14 Figure of KEGG signaling pathway bubble plot enriched by differential genes;

[0034] Figure 15 GSEA enrichment analysis result graph of mitochondrial translation gene set;

[0035] Figure 16 GSEA gene enrichment expression heat map of mitochondrial translation gene set;

[0036] Figure 17 Flow cytometry detection of mitochondrial membrane potential result graph. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only 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 creative labor fall within the scope of protection of the present application.

[0038] The main materials in the following examples are as follows: PBS (Gino, item number GNM20012-5); Ficoll (Tianjin Haoyang, item number LTS10770125); CD3 magnetic beads (Meitianyi, item number 130-097-043); erythrocyte lysis solution (Dawn Biotechnology, item number WB9510P-500mL); CryoStor CS5 (STEMCELL, item number 7933); Opti-MEM medium (Thermo, item number 31985070); pLP1, pLP2 and pBaEV (all purchased from Thermo fisher, item number K497500); expression plasmid pLenti (purchased from Addgene, item number 21474); transfection reagent Neofect (Lingkechuangzhi, item number TF201201); X-VIVO 15 serum-free medium (Lonza, item number 04-418Q); IL-2 (Shandong Quanqi); protamine (Sigma, item number P3369-10G); APC-anti-FMC63 (Bioswan, item number 300402); FITC-anti-CD56 (Biolegend, item number 304603), PE-anti-NKG2A (Biolegend, item number 375103), Percep-anti-LAG3 (Biolegend, item number 375103), PE-Cy7-anti-NKp44 (Biolegend, item number 375103), APC-anti-CD69 (Biolegend, item number 310909), FITC-anti-TIM3 (Biolegend, item number 345021), PE-anti-NKp46 (Biolegend, item number 250803), Percep-anti-NKG2D (Biolegend, item number 320817), PE-Cy7-anti-TIGIT (Biolegend, item number 372713), APC-anti-NKG2A (Biolegend, item number 375107); PE-cy7-anti-CD107a (BioLegend, item number 328618); PE-cy7-anti-CD56 (BioLegend, item number 304628); Pacific Blue-anti-CD45 (BioLegend, item number 304029); APC-anti CD3 (BioLegend, item number 300312); 7AAD-Solution (Biogems, item number 61410-00-200); ELISA detection kit of IL-10 (Yikexi, item number EH006-96); lymphoma cell Raji (ATCC);Mouse fibroblast 3T3 (China National Culture Collection Cell Bank / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); NK-92MI cell line (China National Culture Collection Cell Bank / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); trophoblast cells (ZY Biosciences, item number ZY-NKZ-0104); E-plate 16 plate (Agilent, item number 300600890).

[0039] Example 1. Preparation of CD19 CAR-NK-IL10 cells

[0040] Based on the FMC63 monoclonal antibody scFv sequence targeting CD19 (Genebank: HM852952.1), a CAR structure lentiviral vector using 4-1BB as a co-stimulatory factor and CD3 zeta activation domain was designed and constructed, and IL-10 was connected by Furin-T2A short peptide; the formed IL-10 secreting CD19 CAR-NK targeting CD19 is CD19 CAR-NK-IL10, and the schematic diagram of the lentiviral vector structure is shown in Figure 1 .

[0041] 1. The specific steps of preparing CD19 CAR-NK-IL10 are as follows:

[0042] Obtain umbilical cord blood mononuclear cells, remove CD3 positive T cells in umbilical cord blood, and then aliquot and freeze, the specific steps are as follows: 1) Measure the volume of umbilical cord blood: after the surface of fresh umbilical cord blood is disinfected with alcohol, use a 50 mL syringe to take it out from the blood collection bag, transfer it to a sealed T75 culture bottle, and at the same time measure the volume of the umbilical cord blood;

[0043] 2) Dilute the umbilical cord blood: dilute the umbilical cord blood sample with an equal volume of PBS (Geno, item number GNM20012-5) and mix well;

[0044] 3) Ficoll (Tianjin Haoyang, item number LTS10770125) separates mononuclear cells: take out a 50 mL centrifuge tube, add 20 mL of Ficoll separation solution to the lower layer, slowly add 25 mL of diluted umbilical cord blood to the upper layer, keep the interface clear, 1000g, 30 minutes, speed up 1, speed down 0;

[0045] 4) Obtain peripheral blood mononuclear cells: after centrifugation, a clear white membrane layer of cells can be seen, and the plasma is discarded as much as possible, and the white membrane layer of cells is transferred to a new centrifuge tube, and 5 times the volume of sterile flow cytometry washing solution (2% fetal bovine serum-containing PBS, i.e. 2% FBS+PBS, hereinafter referred to as FASC Buffer) is added;

[0046] 5) Wash cells with FASC Buffer: 300g, 10 minutes, wash twice, then resuspend the cell pellet in an appropriate amount of FACS Buffer and count a small number of cells.

[0047] 6) Removal of CD3-positive T lymphocytes: CD3 magnetic beads (Mitteni, catalog number 130-097-043) were used to remove CD3-positive T lymphocytes from peripheral blood mononuclear cells.

[0048] 7) Red blood cell lysis: Add red blood cell lysis buffer (DaVen Biotech, catalog number WB9510P-500mL) according to the total number of cells after T lymphocyte removal and the degree of red blood cell residue, at a rate of approximately 2-3 × 10⁻⁶ cells / mL. 7 Add 30-40 mL of erythrocyte lysis buffer to each cell and incubate at 4 degrees Celsius for 10-20 minutes.

[0049] 8) Cell identification: Wash cells as in step 5), take a small number of cells for counting, and simultaneously detect the expression of CD56 / CD3 / CD45 by flow cytometry using PE-cy7-anti-CD56 (BioLegend, catalog number 304628), Pacific Blue-anti-CD45 (BioLegend, catalog number 304029) and APC-antiCD3 (BioLegend, catalog number 300312).

[0050] 9) Cell cryopreservation: The obtained cells were cryopreserved using CryoStor CS5 (STEMCELL, catalog number 7933) cryopreservation solution at a ratio of 1×10⁻⁶ cells / mL. 7 Cells were resuspended at a density of 1 × 10⁶ cells / mL. 7 One vial of cells was cryopreserved, and the obtained cells were stored at -80°C in a liquid nitrogen tank.

[0051] 2. Preparation of lentiviral vectors

[0052] The specific steps for preparing a lentiviral vector for infecting NK cells expressing CAR molecules are as follows:

[0053] 1) Transfect when the confluence of 293T cells reaches 70-80%. Replace the Opti-MEM medium (Thermo, catalog number 31985070) specifically for packaging the virus 2 hours before transfection.

[0054] 2) Four-plasmid packaging system for lentivirus preparation: including three helper plasmids, including pLP1, pLP2 and pBaEV (all purchased from Thermo fisher, item number K497500); transfer plasmid pLenti is a lentivirus expression plasmid, the backbone is based on pLenti-CMV-V5-LUC Blast (purchased from Addgene, item number 21474), the original CMV promoter is replaced by EF1ɑ promoter, and CAR is inserted into the expression framework to obtain CD19 CAR-IL10. After mixing the four plasmids in proportion, add transfection reagent Neofect (Zero guest wisdom, item number TF201201) to prepare the transfection reagent mixture;

[0055] 3) Mix well and incubate at room temperature for 15-3 minutes, and add 293T cells along the side wall;

[0056] 4) Collect the virus supernatant: collect the virus supernatant at 48 and 72 hours respectively;

[0057] 5) After filtering the cell debris with a 0.45 μm pore size filter membrane, use the ultracentrifugation method to concentrate the lentivirus; 6) Use the NK-92MI cell line (Chinese Academy of Sciences Typical Culture Collection Cell Bank / Cell Resource Center of Shanghai Institutes for Biological Sciences) to detect the lentivirus titer, and the functional titer result is in the range of 0.5-1×10 8 TU / mL, obtain lentivirus that can effectively transfect cells, and can perform subsequent experiments.

[0058] 3、Preparation of CD19 CAR-NK-IL10 cells

[0059] 1) On day 0, resuscitate the umbilical cord blood mononuclear cells and count them;

[0060] 2) Add feeder cells (Zhongying biological, item number ZY-NKZ-0104) at a ratio of 2:1 of mononuclear cells to feeder cells, and the culture medium is X-VIVO 15 serum-free medium (Lonza, item number 04-418Q))+10%FBS+100IU / mL IL-2 (referred to as X-VIVO);

[0061] 3) On day 5, calculate the amount of lentivirus added according to the following formula: (cell number x MOI) / virus titer, where MOI is 5, and add the transfection aid protamine (Sigma, item number P3369-10G) at a final concentration of 10 μg / mL;

[0062] 4) Add lentivirus and transfection aid to the NK cells according to the above step 3) and mix well, 1-3×10 5 / 200μL of the system into a 96-well flat-bottom plate, centrifuge the flat-bottom plate at 1200g, 37°C for 90 minutes;

[0063] 5) After centrifugation, place in the incubator for 4 hours, then change the cell liquid, discard the virus liquid, and replace it with fresh NK cell culture medium;

[0064] 6) On the 7th day, analyze the proportion of CD56 positive NK cells and the expression of CAR by flow cytometry; add feeder cells at a ratio of NK cells: feeder cells 1:1 and continue to culture;

[0065] 7) On the 12th day, obtain proliferated CAR-NK cells for subsequent functional research experiments.

[0066] Example 2. Expression identification of CD19 CAR-NK-IL10

[0067] Prepare CD19 CAR-NK-IL10 cells according to Example 1, and flow cytometry is used to identify the expression of CAR on the 10th day of cell culture. Collect the cell culture supernatant and use ELISA to detect the secretion of IL-10.

[0068] 1) Prepare CD19 CAR-NK-IL10 cells according to the above scheme, and count the number of CD19 CAR-NK-IL10 and NK cells (NK cells without CAR transduction) on the 6th / 8th / 10th / 13th / 15th day during the preparation of CD19 CAR-NK-IL10.

[0069] 2) Flow cytometry is used to identify the expression of CAR and cell surface receptors:

[0070] (a) On the 10th day, take 1x10 5 CD19 CAR-NK-IL10 cells and NK cells, respectively, and centrifuge and wash the cells twice with FACS Buffer, and resuspend the cells with 50μL of FACS Buffer;

[0071] (b) Surface Marker staining: APC-anti-FMC63 (Bioswan, Cat# 300402) was added at 1 :500 in 50 pL system, while FITC-anti-CD56, PE-anti-NKG2A, Percep-anti-LAG3, PE-Cy7-anti-NKp44, APC-anti-CD69, FITC-anti-TIM3, PE-anti-NKp46, Percep-anti-NKG2D, PE-Cy7-anti-TIGIT, APC-anti-NKG2A were added at 1 :200 in 50 pL system, avoid light, 4°C, staining for 20 minutes;

[0072] (c) After washing twice with FACS buffer, resuspend the cells with 200 pL FACS buffer, add 1 pL dead-live dye 7AAD-Solution (Biogems, Cat# 61410-00-200) per sample, and detect on flow cytometry.

[0073] 3) Collect cell culture supernatant

[0074] (a) Adjust cell density: take 2 x 10 6 cells from CD19 CAR-NK-IL10 and NK cells, adjust the cell density to 1 x 10 6 cells / mL, and inoculate into 12-well plates for culture for three days;

[0075] (b) Collect cell culture supernatant: take the CD19 CAR-NK-IL10 cells and NK cells cultured for three days and transfer them to 15 mL centrifuge tubes, centrifuge at 400 g for 5 minutes to obtain cell supernatant, place it in a 1.5 mL EP tube, label each supernatant with name, CAR%, cell density, culture time, collection date, and store it at -80°C for standby;

[0076] 4) ELISA detection of IL-10 secretion (a) Take the IL-10 ELISA detection kit (Ekoase, Cat# EH006-96) out of the refrigerator and equilibrate it to room temperature; (b) Take the required strips from the sealed bag that has been equilibrated to room temperature, and place the unused strips and desiccant back into the aluminum foil bag and store it in the 2-8°C refrigerator;

[0077] (c) Preparation of standard and sample: after the cell culture supernatant stored at -80°C was dissolved, the cell culture supernatant stock solution, 5-fold dilution and 10-fold dilution were prepared respectively, and three duplicate wells were prepared for each sample; the standard protein IL-10 in the ELISA kit was dissolved into 1000 pg / mL sample with sample dissolution solution Assay Diluent, and was left for 15 minutes, and was diluted into 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL sample by two-fold gradient, and three duplicate wells were prepared for each standard;

[0078] (d) Different concentrations of standard or sample were added into the corresponding wells respectively, 100 μL / well, and the blank wells were left as control, and the reaction wells were sealed with sealing tape, and were incubated in a 37°C constant temperature incubator for 90 minutes;

[0079] (e) Preparation of biotinylated antibody working solution: according to the required amount for the experiment, the Human 100XBiotin-Antibody was diluted 100 times with Assay Diluent to prepare the biotinylated antibody working solution, which was prepared 30 minutes before use and was used only on the same day; (f) 20X Wash Buffer Concentrate was diluted into Wash Buffer with deionized water, the liquid in the well plate was discarded, the well plate was washed with Wash Buffer five times, the biotinylated antibody working solution was added, 100 μL / well, the reaction wells were sealed with sealing tape, and were incubated in a 37°C constant temperature incubator for 60 minutes;

[0080] (g) Preparation of enzyme binding working solution: according to the required amount for the experiment, the 100X HRP-Streptavidin was diluted 100 times with Assay Diluent to prepare the enzyme binding working solution, which was prepared 30 minutes before use and was used only on the same day;

[0081] (h) The liquid in the well plate was discarded, the well plate was washed with Wash Buffer five times, the enzyme binding working solution was added, 100 μL / well, and was incubated in a 37°C constant temperature incubator for 60 minutes;

[0082] (i) The liquid in the well plate was discarded, the well plate was washed with Wash Buffer five times, the Substrate Solution was added, 100 μL / well, and was incubated in a 37°C constant temperature incubator for 15 minutes;

[0083] (j) The Stop Solution was added, 100 μL / well, and after mixing, the CD 450 value was detected immediately with an enzyme label instrument;

[0084] The results are shown in Figure 2 and Figure 3As shown, CD19 CAR-NK-IL10 cells CAR was expressed on the surface of NK cells; ELISA results showed that CD19 CAR-NK-IL10 had a large amount of IL-10 secretion, while NK had almost no IL-10 cytokine secretion, indicating that the CD19 CAR-NK-IL10 structure we constructed could normally express IL-10 and did not affect the expression of CAR; Figure 4 The results showed that the expression of the activating receptors NKG2D and NKp44 of CD19 CAR-NK-IL10 was inhibited, but the expression of the inhibitory receptor TIGIT was also inhibited. Figure 5 The results showed that after transduction of CD19 CAR-NK-IL10 lentivirus, the umbilical cord blood NK could still proliferate in large quantities.

[0085] Example 3. In vitro functional verification of CD19 CAR-NK-IL10 cells

[0086] A cell line Raji-CBR-Luc-GFP (CBR is click beetle red luciferase, a kind of beetle luciferase that can catalyze the luciferin substrate to excite red spectrum; CBR and GFP are introduced into Raji cells at the same time) was constructed using lymphoma cells Raji (ATCC) to express luciferase CBR and green fluorescent protein GFP, and this tumor cell line was used as a target cell for short-term in vitro killing and CD107a degranulation experiment of CD19 CAR-NK-IL10 cells; At the same time, a cell line 3T3-CD19-Luc-GFP was constructed using mouse fibroblasts 3T3 (Chinese Academy of Sciences Typical Culture Collection Committee Cell Bank / Cell Resource Center of Shanghai Institutes for Biological Sciences) to overexpress human CD19 antigen, and this cell line was used as a target cell for long-term in vitro killing function verification of CD19 CAR-NK-IL10 cells.

[0087] 1. Luciferase-labeled cell killing experiment

[0088] 1) Target cell plating: Collect tumor cells Raji, add PBS buffer for washing, count, adjust the cell density to 1×10 5 6 / mL, and inoculate into a 96-well U-bottom plate at 100 μL / well, i.e. 1×10 4 6 target cells per well;

[0089] 2) Effect cell plating: Collect CD19 CAR-NK-IL-10 / NK cells, calculate the number of cells needed for plating, resuspend the cells at the highest required CAR-NK cell concentration for target ratio (calculate the cell density according to 100 μL per well), and dilute and plate the remaining target ratios, while setting up positive controls (K max, adding 100 μL of 1% Triton X-100) and negative control (K min , adding 100 μL of medium);

[0090] 4) Mix the cell mixture in each well gently, and incubate in the incubator for 4-6 hours. Preheat the Luciferin substrate in advance. After the cells are washed twice with FACS Buffer, add 10 dg / ml of fluorescent substrate, and incubate at 37°C in the dark for 10 minutes. Select the chemiluminescence mode on the microplate reader to read the data K;

[0091] 5) Calculate the killing efficiency % = (K min -K) / (K min -K max ) x 100%.

[0092] The results are shown in Figure 6 , compared with NK, CD19 CAR-NK-IL-10 showed significant killing ability to Raji cells expressing CD19 antigen under different effector-target ratio conditions.

[0093] 2. CD107a degranulation experiment

[0094] 1) Target cell plating: Collect the Raji lymphoma cell line, wash with PBS buffer, count, and inoculate 1 x 10 5 cells per well into a 96-well U-bottom plate, 100 μL per well;

[0095] 2) Effector cell plating: Adjust the cell density of each CD19 CAR-NK-IL10 / NK cell according to the effector-target ratio of 1:1 and inoculate into a 96-well U-bottom plate. Add Protein transport inhibitor (BD, item number 554724) at 1:1500 and PE-cy7 anti-CD107a (BioLegend, item number 328618) antibody at 1:200 to the total volume, mix with the target cells, and co-culture for 4-6 hours. Leave the NK cells without tumor as a control for each group;

[0096] 3) Wash twice with FACS buffer according to the flow staining method, stain the surface marker antibody, and add 100 μL of the system 1:500 antibody of APC anti-FMC63, stain at 4°C in the dark for 20 minutes;

[0097] 4) After washing twice with FACS buffer, resuspend the cells with 200 μL of FACS buffer, and add 1 μL of dead and live dye 7AAD-Solution to each sample. Detect on the flow cytometer.

[0098] The results are shown inFigure 7 As shown, CD19 CAR-NK-IL10 cells had stronger CD 107a degranulation reaction than NK cells after contacting target cells.

[0099] 3. Long-term real-time in vitro killing experiment (RTCA experiment)

[0100] 1) Take out the E-plate 16 plate (Agilent, item number 300600890), preheat the medium at 37°C in advance, add 50 μL of medium to each well, measure the baseline value, and ensure that the optical density (OD) value of each well is the same;

[0101] 2) Target cell plating: collect 3T3-CD19-Luc-GFP cells, adjust the cell density to 10000 cells per well with a volume of 150 μL;

[0102] 3) Add 150 μL of cell suspension to each well of the E-plate 16 plate, and after standing at room temperature for 30 minutes, place it in the xCELLigence RTCA instrument to detect the OD value of the cells in real time;

[0103] 4) When the OD value is about 1.0, centrifuge and wash the CAR-NK / NK cells, resuspend them with preheated medium, count and adjust the CAR-NK / NK density to 1.25 x 10 4

[0104] 5) Effector cell plating: interrupt the RTCA experiment, take out the E-plate 16 plate, discard the original medium, and add 200 μL of CAR-NK cells with adjusted density to each well according to the grouping, and keep the negative control (only target cells);

[0105] 6) Put the E-plate 16 plate back into the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-NK cells on target cells.

[0106] The results are shown in Figure 8 As shown, CD19 CAR-NK-IL10 can significantly kill 3T3 cells expressing CD19 antigen at a 1:4 effector-target ratio compared to NK cells.

[0107] Example 4. Mouse xenograft model to verify the in vivo anti-tumor function of CD19 CAR-NK-IL10 cells

[0108] Raji-CBR-Luc-GFP cells with luciferase were injected into the tail vein of severely immunodeficient mice (NSG), CD19 CAR-NK-IL10 cells were prepared, and intravenous infusion therapy was performed on tumor-bearing mice (NSG). ​

[0109] 1) Whole body tumor formation of lymphoma cells: Collect log phase Raji-CBR-Luc-GFP cells, 400g, 5min centrifugation, discard supernatant, wash twice with PBS, ensure no serum residue;

[0110] 2) Count, resuspend cells with pre-cooled PBS, 8x10 4 tumor cells per NSG mouse, volume 200μL / mouse, adjust tumor cell density to 4x10 5 μL / mouse, tail vein injection;

[0111] 3) Small animal live imaging 48h later, evaluate tumor formation according to imaging results and randomly group, specific grouping as follows:

[0112] (a) NK group: injection of normal umbilical cord blood NK cells without transduction of CAR (4 animals);

[0113] (b) CD19 CAR-NK-IL10 group: injection of umbilical cord blood NK cells transduced with CD19 CAR-IL10 (4 animals);

[0114] 4) NK cell tail vein treatment 72h later (a) collect log phase CAR-NK / NK cells, take the same total number of CD19 CAR-NK-IL10 and NK cells; (b) wash twice with pre-cooled PBS without serum, ensure no serum residue;

[0115] (c) count, adjust cell density to 1.2x10 7 / mL with pre-cooled PBS without serum, prepare for tail vein treatment;

[0116] (d) according to the grouping in step 4), inject corresponding CD19 CAR-NK-IL10 and NK cells into each mouse with a volume of 200μL tail vein;

[0117] (e) live animal imaging twice a week in the first week of intravenous treatment, and then live animal imaging once a week, evaluate tumor progression;

[0118] 6) According to tumor load and mouse survival, statistics fluorescence curve and survival curve.

[0119] Results are shown in Figure 9 , Figure 10 and Figure 11As shown, with the increase of tumor load time, the tumor load of NK group increased sharply, the tumor load of CD19 CAR-NK-IL10 group was obviously inhibited, and increased slowly after one week; the CD19 CAR-NK-IL-10 group began to die after 25 days of treatment ("X" represents death); it can be seen from the fluorescence curve of NSG mice that the CD19 CAR-NK-IL10 cells have a significant effect on treating lymphoma in vivo; from the survival graph of NSG mice, CD19 CAR-NK-IL10 cells can significantly prolong the survival time of mice.

[0120] Example 5. Transcriptome sequencing analysis of CD19 CAR-NK cells expressing IL-10

[0121] After co-incubation of CD19 CAR-NK-IL10 / NK cells with Raji-CBR-Luc-GFP cells carrying luciferase for 24 hours at an effector to target ratio of 2:1, transcriptome sequencing analysis was performed.

[0122] 1. Antigen stimulation of CD19 CAR-NK-IL10 / NK cells

[0123] 1) Target cell plating: Collect lymphoma cells Raji-CBR-Luc-GFP in logarithmic growth phase, wash twice with PBS, count, take 2x10 5 tumor cells, resuspend in 100 μL volume with X-VIVO medium and add to a 12-well plate;

[0124] 2) Effector cell plating: Collect 1x10 6 CD19 CAR-NK-IL10 / NK cells, resuspend in 1 ml with X-VIVO medium and add to a 12-well plate;

[0125] 3) After gently mixing the cells in each well, incubate in a 37°C incubator for 24 hours, then wash the cell mixture twice with serum-free PBS;

[0126] 4) As much as possible to absorb the residual PBS, then use liquid nitrogen to quickly freeze the cell mixture into a lump and store at -80°C.

[0127] 2. RNA extraction

[0128] 1) Take the cell lump and extract total RNA using the Trizol method, measure the A260 / A280 ratio using a spectrophotometer to evaluate purity;

[0129] 3. Detect RNA integrity (RIN value) using Agilent Bioanalyzer 2100 and measure RNA concentration using Qubit to screen qualified samples for library construction and sequencing.

[0130] 1) With total RNA as input, specific molecular tags (Unique Molecular Identifier, UMI) are used to label cDNA molecules to correct biases and errors in PCR amplification and sequencing;

[0131] 2) Enrichment of 200-500 bp fragment PCR products, after quantification, paired-end 150bp (PE150) sequencing was performed on the DNBSEQ-T7 (Huada) platform.

[0132] 4. Flow cytometry detection of mitochondrial membrane potential

[0133] 1) Plating: Collect lymphoma cells Raji and CD19 CAR-NK-IL10 / NK cells in the logarithmic growth phase, count after centrifugation, adjust the cell density according to the effector target ratio of 2:1 and inoculate into 96-well U-bottom plates, mix with target cells and co-culture for 24 hours, and leave NK cells without tumors as controls for each group;

[0134] 2) According to the method of flow cytometry staining, wash twice with FACS buffer, stain surface marker antibodies, add APC anti-FMC63 at 1:500 in 50μL system and FITC-anti-CD56 (Biolegend, Cat. No. 304603) at 1:200 in 50μL system, add antibodies, avoid light, stain at 4°C for 20 minutes;

[0135] 4) After washing twice with FACS buffer, resuspend the cells with 200μL FACS buffer, add 1μL dead and live dye 7AAD-Solution per sample, and detect on the flow cytometer.

[0136] The results are shown in the figure, Figure 12 The principal component analysis chart shows that there are significant differences in the overall gene expression profile after incubation of CD19 CAR-NK-IL10 / NK cells with Raji; Figure 13 The volcano plot of differential genes shows that the expression of immune-related differential genes (IL21R, CXCL8, CCR7, etc.) of CD19 CAR-NK-IL10 cells is up-regulated; Figure 14 The bubble chart of KEGG functional enrichment analysis of differential genes shows that the differential genes are significantly enriched in immune-related functions such as "immune response", "T cell receptor signaling pathway", "cytokine-mediated signaling pathway", etc., suggesting that IL-10 expression affects multiple immune pathways regulated by CD19 CAR-NK-IL10 cells in the tumor microenvironment; Figure 15GSEA enrichment analysis curve showed that the mitochondria translation related function was significantly enhanced after CD19CAR-NK-IL10 cells expressing IL-10 were co-incubated with Raji, suggesting that energy metabolism and mitochondrial function might play an important role in regulating immune effect. Figure 16 The heat map results of differentially expressed mitochondria related genes suggested that IL-10 expression significantly affected the expression of mitochondrial function related genes of CD19CAR-NK-IL10 cells, which might be related to its enhanced anti-tumor effect. Figure 17 The results of flow cytometry detection showed that the mitochondrial membrane potential of CD19 CAR-NK-IL10 group cells was higher, indicating that the energy metabolism of CD19 CAR-NK-IL10 cells was in a more active state.

[0137] The DNA sequence of CD19 CAR-NK-IL10 CAR molecule (SEQ ID NO. 1):

[0138] ATGGCACTGCCAGTGACCGCCTTACTCCTCCCCTTGGCCCTGCTACTGCACGCAGCTAG

[0139] GCCGGACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGATAGG

[0140] GTGACCATCTCCTGCCGCGCCTCTCAGGACATCAGCAAGTATCTGAACTGGTACCAGCA

[0141] GAAGCCTGACGGCACAGTGAAGCTGCTGATCTATCACACCTCCAGGCTGCACTCTGGC

[0142] GTGCCAAGCCGCTTTTCCGGCTCTGGCAGCGGCACAGATTACTCCCTGACCATCTCTAA

[0143] CCTGGAGCAGGAGGACATCGCCACCTATTTTTGCCAGCAGGGCAATACACTGCCATACA

[0144] CCTTCGGCGGCGGCACAAAGCTGGAGATCACCGGAGGAGGAGGATCCGGCGGAGGAG

[0145] GCTCTGGCGGCGGCGGCAGCGAGGTGAAGCTGCAGGAGTCCGGACCAGGACTGGTGG

[0146] CACCTTCCCAGTCTCTGAGCGTGACATGTACCGTGTCTGGCGTGAGCCTGCCCGACTAC

[0147] GGCGTGTCTTGGATCCGGCAGCCCCCTAGAAAGGGACTGGAGTGGCTGGGCGTGATCT

[0148] GGGGCAGCGAGACCACATACTATAATAGCGCCCTGAAGTCCCGGCTGACAATCATCAAG

[0149] GATAACTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACAGACGATACCGC

[0150] CATCTACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGACTACTGGGG

[0151] CCAGGGCACATCTGTGACCGTGTCTAGCACTACAACCCCAGCACCAAGGCCACCAACA

[0152] CCTGCACCAACCATCGCCTCTCAGCCACTGAGCCTGAGGCCAGAGGCATGTAGGCCTG

[0153] CAGCAGGAGGCGCCGTGCACACCAGAGGCCTGGATTTCGCCTGCGACATCTATATCTGG

[0154] GCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGTCCCTGGTCATCACCCTGTATTG

[0155] CAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGGCCAGTG

[0156] CAGACAACCCAGGAGGAGGATGGCTGCAGCTGTAGATTTCCAGAAGAGGAGGAGGGA

[0157] GGATGTGAGCTGCGGGTGAAGTTCAGCAGATCCGCCGACGCACCTGCATATCAGCAGG

[0158] GACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGC

[0159] TGGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCACAACGGAGAAAG

[0160] AACCCCCAGGAGGGCCTGTATAATGAGCTGCAGAAGGATAAGATGGCCGAGGCCTACT

[0161] CCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGACGGCCTGTATC

[0162] AGGGCCTGTCTACAGCCACCAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCT

[0163] CCAAGGGGCAGCGGAGCTACAAACTTCAGCCTGCTGAAGCAGGCCGGAGATGTGGAA

[0164] GAGAATCCTGGACCTATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTCCTGCTCACCGG

[0165] CGTGCGGGCCAGCCCCGGCCAGGGCACCCAATCTGAGAACAGCTGCACCCACTTCCCC

[0166] GGCAACCTGCCTAACATGCTGCGGGACCTGCGCGACGCCTTTTCTAGAGTGAAGACCTT

[0167] CTTCCAGATGAAAGACCAGCTGGACAATCTGCTGCTGAAGGAAAGCCTGCTGGAAGAT

[0168] TTTAAGGGCTACCTGGGCTGTCAGGCTCTGTCTGAGATGATCCAGTTCTACCTGGAGGA

[0169] AGTGATGCCTCAAGCCGAGAACCAGGACCCTGACATCAAGGCTCATGTGAACAGCCTC

[0170] GGCGAGAACCTGAAAACCCTGAGACTGAGACTGCGGAGATGTCACAGATTCCTGCCAT

[0171] GCGAAAACAAGTCCAAGGCCGTGGAACAGGTGAAAAACGCCTTCAACAAGCTGCAGG

[0172] AGAAGGGCATCTATAAGGCCATGAGCGAGTTCGACATCTTCATTAACTACATCGAGGCC

[0173] TACATGACAATGAAGATCAGAAACCGTGCACGGAGATCTGGGTCTGGAGAAGGCAGAG

[0174] GGTCTTTGTTGACATGCGGTGACGTGGAGGAGAACCCCGGCCCCATGAGAATCTCAAA

[0175] GCCACATCTTAGATCAATCTCAATCCAATGCTACCTTTGCCTTCTTCTTAACTCGCATTTC

[0176] TTGACGGAGGCAGGAATCCACGTGTTTATCCTTGGATGCTTCTCCGCCGGACTTCCTAA

[0177] GACTGAGGCAAACTGGGTGAACGTGATCTCAGATCTTAAGAAGATAGAGGATCTTATCC

[0178] AATCAATGCACATCGATGCAACACTTTACACAGAATCAGATGTGCACCCTTCATGCAAA

[0179] GTGACAGCAATGAAATGCTTTCTTCTTGAACTTCAAGTGATCTCACTTGAATCAGGAGA

[0180] TGCATCAATCCACGATACAGTGGAGAATCTGATCATCCTTGCAAACAATAGTCTATCTTC

[0181] CAATGGAAACGTGACAGAATCAGGATGCAAAGAATGCGAAGAACTTGAAGAGAAGAA

[0182] TATAAAGGAGTTCCTTCAATCATTTGTGCACATCGTGCAAATGTTTATCAACACATCATG

[0183] A

[0184] wherein

[0185] Signal peptide sequence (SEQ ID NO. 2):

[0186] ATGGCACTGCCAGTGACCGCCTTACTCCTCCCCTTGGCCCTGCTACTGCACGCAGCTAG

[0187] GCCG Anti-CD19 scFv sequence (SEQ ID NO. 3):

[0188] GACATCCAGATGACACAGACCACAAGCTCCCTGTCTGCCAGCCTGGGCGATAGGGTGA

[0189] CCATCTCCTGCCGCGCCTCTCAGGACATCAGCAAGTATCTGAACTGGTACCAGCAGAAG

[0190] CCTGACGGCACAGTGAAGCTGCTGATCTATCACACCTCCAGGCTGCACTCTGGCGTGCC

[0191] AAGCCGCTTTTCCGGCTCTGGCAGCGGCACAGATTACTCCCTGACCATCTCTAACCTGG

[0192] AGCAGGAGGACATCGCCACCTATTTTTGCCAGCAGGGCAATACACTGCCATACACCTTC

[0193] GGCGGCGGCACAAAGCTGGAGATCACCGGAGGAGGAGGATCCGGCGGAGGAGGCTCT

[0194] GGCGGCGGCGGCAGCGAGGTGAAGCTGCAGGAGTCCGGACCAGGACTGGTGGCACCT

[0195] TCCCAGTCTCTGAGCGTGACATGTACCGTGTCTGGCGTGAGCCTGCCCGACTACGGCGT

[0196] GTCTTGGATCCGGCAGCCCCCTAGAAAGGGACTGGAGTGGCTGGGCGTGATCTGGGGC

[0197] AGCGAGACCACATACTATAATAGCGCCCTGAAGTCCCGGCTGACAATCATCAAGGATAA

[0198] CTCCAAGTCTCAGGTGTTTCTGAAGATGAATAGCCTGCAGACAGACGATACCGCCATCT

[0199] ACTATTGCGCCAAGCACTACTATTACGGCGGCTCCTATGCCATGGACTACTGGGGCCAG

[0200] GGCACATCTGTGACCGTGTCTAGC

[0201] 4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO. 4):

[0202] AAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGGCCAGTGC

[0203] AGACAACCCAGGAGGAGGATGGCTGCAGCTGTAGATTTCCAGAAGAGGAGGAGGGAG

[0204] GATGTGAGCTGCGGGTGAAGTTCAGCAGATCCGCCGACGCACCTGCATATCAGCAGGG

[0205] ACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCT

[0206] GGATAAGAGGAGGGGAAGGGACCCAGAGATGGGAGGCAAGCCACAACGGAGAAAGA

[0207] ACCCCCAGGAGGGCCTGTATAATGAGCTGCAGAAGGATAAGATGGCCGAGGCCTACTC

[0208] CGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGACGGCCTGTATCA

[0209] GGGCCTGTCTACAGCCACCAAGGACACATACGATGCCCTGCACATGCAGGCCCTGCCTC

[0210] CAAGG

[0211] IL-10 sequence (SEQ ID NO. 5):

[0212] ATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTCCTGCTCACCGGCGTGCGGGCCAGCC

[0213] CCGGCCAGGGCACCCAATCTGAGAACAGCTGCACCCACTTCCCCGGCAACCTGCCTAA

[0214] CATGCTGCGGGACCTGCGCGACGCCTTTTCTAGAGTGAAGACCTTCTTCCAGATGAAAG

[0215] ACCAGCTGGACAATCTGCTGCTGAAGGAAAGCCTGCTGGAAGATTTTAAGGGCTACCT

[0216] GGGCTGTCAGGCTCTGTCTGAGATGATCCAGTTCTACCTGGAGGAAGTGATGCCTCAAG

[0217] CCGAGAACCAGGACCCTGACATCAAGGCTCATGTGAACAGCCTCGGCGAGAACCTGAA

[0218] AACCCTGAGACTGAGACTGCGGAGATGTCACAGATTCCTGCCATGCGAAAACAAGTCC

[0219] AAGGCCGTGGAACAGGTGAAAAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTAT

[0220] AAGGCCATGAGCGAGTTCGACATCTTCATTAACTACATCGAGGCCTACATGACAATGAA

[0221] GATCAGAAACCD19 CAR-NK-IL10 CAR molecule amino acid sequence: (SEQ ID NO. 6)

[0222] MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP

[0223] DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT

[0224] KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP

[0225] RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY

[0226] GGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA

[0227] CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE

[0228] EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRK

[0229] NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

[0230] RGSGATNFSLLKQAGDVEENPGPMHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLP

[0231] NMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA

[0232] ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAM

[0233] SEFDIFINYIEAYMTMKIRNRARRSGSGEGRGSLLTCGDVEENPGPMRISKPHLRSISIQCYL

[0234] CLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVH

[0235] PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*

[0236] wherein

[0237] Signal peptide sequence (SEQ ID NO. 7): MALPVTALLLPLALLLHAARP Anti-CD19 scFv sequence (SEQ ID NO. 8):

[0238] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRF

[0239] SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEV

[0240] KLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL

[0241] KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO. 9):

[0242] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ

[0243] LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK

[0244] GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0245] IL-10 sequence (SEQ ID NO. 10):

[0246] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQ

[0247] LDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLR

[0248] LRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0249] It should be noted that the terms such as first and second, etc., are merely used to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device. Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application, although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent replacements to part of the technical features. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant construct, characterized in that, The recombinant construct encodes IL-10 and CAR, fragment or variant thereof, the antigen of the CAR is CD19.

2. A recombinant construct according to claim 1, wherein, The nucleic acid sequence of the recombinant construct is shown as SEQ ID NO. 1, fragment or variant thereof.

3. The recombinant construct of claim 1, wherein, The amino acid sequence encoded by the recombinant construct is shown as SEQ ID NO. 6, fragment or variant thereof.

4. A CAR-NK cell, characterized in that, The recombinant construct comprises any one of claims 1-3.

5. The CAR-NK cell of claim 4, wherein the CAR comprises an amino acid sequence of SEQ ID NO:

1. The CAR-NK cell is autologous, allogeneic or heterologous.

6. A pharmaceutical composition, characterized by: The recombinant construct comprises any one of claims 1-3 or the CAR-NK cell of any one of claims 4-5.

7. A pharmaceutical composition according to claim 6, wherein: It comprises at least one pharmaceutically acceptable carrier.

8. Use of the recombinant construct of any one of claims 1-3 or the CAR-NK cell of any one of claims 4-5 or the pharmaceutical composition of any one of claims 6-7 in the preparation of a kit for the diagnosis / treatment of malignant tumors or autoimmune diseases.

9. Use of the recombinant construct of any one of claims 1-3 or the CAR-NK cell of any one of claims 4-5 or the pharmaceutical composition of any one of claims 6-7 in the preparation of a drug for malignant tumors or autoimmune diseases.

10. The use according to claim 9, wherein the medicament for malignant tumor or autoimmune disease is prepared for, The malignant tumor is any one of gastric adenocarcinoma, B cell malignant tumor or myeloma.

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