Recombinant construct, CAR (chimeric antigen receptor) modified cell and application

Abstract

The invention relates to the field of biotechnology and cell therapy, in particular to a recombinant construct, a CAR (chimeric antigen receptor) modified cell and application. On the objective basis of different antigen target spots, such as CD19, CLDN18.2 or BCMA and other different antigen target spots corresponding to abnormal expression of tumors, the target spots are used as target spots for clinical treatment; under the condition that the expression of the chimeric antigen is not changed, two cell factors are secreted synergistically; the long-term treatment effect of immune cells is enhanced, and meanwhile, the treatment risk is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and cell therapy, and specifically to a recombinant construct, CAR-modified cells and applications. Background Art

[0002] Both CAR-NK therapy and CAR-T therapy belong to cellular immunotherapy technology. At present, CAR-NK cell therapy has shown certain effects in early clinical or preclinical trials of various solid tumors such as pancreatic cancer, colorectal cancer, and lung cancer, which makes CAR-NK therapy expected to fill the gap of CAR-T therapy in the treatment of solid tumors. In addition, its "spot type" attribute can greatly shorten the treatment time and cost.

[0003] In the actual construction and modification of CAR-NK cells, the design and modification of the extracellular target recognition region (also known as the antigen binding region or antigen recognition region) for different antigens often leads to the non-expression of the chimeric antigen receptor (CAR) due to the complexity of cell expression. Therefore, how to construct targeted CAR-NK cells or CAR-T cells for different antigens and simultaneously express certain cytokines is a hot topic and pain point in current industry research. Each successfully constructed CAR-NK cell or CAR-T cell targeting different antigens has great value and important contributions to further conquering and treating various solid tumors in humans. Summary of the invention

[0004] The purpose of the present invention is to provide a CAR-modified immune cell, a preparation method and an application thereof, so as to solve the problems raised in the above-mentioned background technology.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A recombinant construct encoding IL-10, IL-15 and CAR, fragments or variants thereof, wherein the antigens of the CAR include: CLDN18.2, CD19, CD5, CD20, CD22, CD23, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD319, GPC3, CD Any one or combination of 32b, CD171, CS-1, CLL-1, BCMA, GD2, GD3, PSMA, ROR1, FLT3, FAP, CD44v6, CEA, EPCAM, B7H3, IL-13Ra2, Mesothelin, Her2, MUC1, EGFR, CLDN6, DLL3, NYESO-1, WT1, Nectin-4, GPC3, PDL1 and NKG2DL.

[0007] Preferably, the antigen of the CAR is CD19, and the nucleic acid sequence of the recombinant construct is as shown in SEQ ID NO.7, or a fragment or variant thereof.

[0008] Preferably, the antigen of the CAR is CD19, and the amino acid sequence encoded by the recombinant construct is as shown in SEQ ID NO.1, or a fragment or variant thereof.

[0009] Preferably, the antigen of the CAR is CLDN18.2, and the nucleic acid sequence of the portion of the CLDN18.2 targeted in the recombinant construct is as shown in SEQ ID NO.14, or a fragment or variant thereof; the nucleic acid sequence of IL-10 in the recombinant construct is as shown in SEQ ID NO.11, or a fragment or variant thereof; the nucleic acid sequence of IL-15 in the recombinant construct is as shown in SEQ ID NO.12, or a fragment or variant thereof.

[0010] Preferably, the antigen of the CAR is CLDN18.2, and the amino acid sequence of the CLDN18.2 portion targeted in the recombinant construct is shown in SEQ ID NO.13, or a fragment or variant thereof; the amino acid sequence encoded by IL-10 in the recombinant construct is shown in SEQ ID NO.5, or a fragment or variant thereof; the amino acid sequence encoded by IL-15 in the recombinant construct is shown in SEQ ID NO.6, or a fragment or variant thereof.

[0011] Preferably, the antigen of the CAR is BCMA, and the partial nucleic acid sequence targeting BCMA in the recombinant construct is shown as SEQ ID NO.16, or a fragment or variant thereof; the nucleic acid sequence of IL-10 in the recombinant construct is shown as SEQ ID NO.11, or a fragment or variant thereof; the nucleic acid sequence of IL-15 in the recombinant construct is shown as SEQ ID NO.12, or a fragment or variant thereof.

[0012] Preferably, the antigen of the CAR is BCMA, and the amino acid sequence of the BCMA-targeting portion in the recombinant construct is shown in SEQ ID NO.15, or a fragment or variant thereof; the amino acid sequence encoded by IL-10 in the recombinant construct is shown in SEQ ID NO.5, or a fragment or variant thereof; the amino acid sequence encoded by IL-15 in the recombinant construct is shown in SEQ ID NO.6, or a fragment or variant thereof.

[0013] A CAR-modified cell comprising a recombinant construct as described above.

[0014] Preferably, the CAR-modified cells are any one of CAR-NK cells or CAR-T cells.

[0015] Preferably, the CAR-modified cells are autologous, allogeneic or xenogeneic.

[0016] A pharmaceutical composition comprising a recombinant construct as described in any one of the foregoing or a CAR-modified cell as described in any one of the foregoing and at least one pharmaceutically acceptable carrier.

[0017] Use of a recombinant construct as described in any of the foregoing, a CAR-modified cell as described in any of the foregoing, or a pharmaceutical composition as described in any of the foregoing in the preparation of a kit for diagnosing / treating malignant tumors or autoimmune diseases.

[0018] Use of a recombinant construct as described in any of the foregoing, or a CAR-modified cell as described in any of the foregoing, or a pharmaceutical composition as described in the foregoing in the preparation of a drug for a malignant tumor or an autoimmune disease.

[0019] Preferably, the malignant tumor is any one of gastric adenocarcinoma, B-cell malignancy or myeloma.

[0020] The purpose of the present invention is to provide a method for preparing cells modified with dual cytokine-secreting CAR and its application in the field of cell therapy.

[0021] In the present invention, an expression vector (chimeric antigen receptor expression vector) is provided, which comprises a signal peptide, an extracellular target recognition region (antigen binding region), a hinge region, a transmembrane region, and an intracellular region, and in particular, IL-10 and IL-15 cytokines are co-expressed by the Furin-T2A short peptide, and the expression vector is a lentiviral expression vector.

[0022] For example, a host cell is provided, a preparation of a dual cytokine-secreting chimeric antigen receptor-NK cell (CAR-NK), a CAR expression vector comprising the above-mentioned antigen recognition region, and the host cell is an immune cell, such as a T cell, a tumor infiltrating lymphocyte (TIL), a natural killer cell (NK), and a natural killer T cell (NKT).

[0023] A killing efficiency detection method is provided, which specifically includes: a luciferase-labeled cell killing experiment, a long-term real-time in vitro killing experiment (RTCA experiment), and a CD107a degranulation experiment; the killing function detection method described in the embodiment includes but is not limited to the above methods, such as IF-g detection, granzyme B detection, perforin detection, etc.

[0024] In one or more embodiments, for example, the NK cells can be: NK cells derived from umbilical cord blood, including but not limited to umbilical cord blood sources, such as peripheral blood, IPS-induced, NK cell lines.

[0025] In one or more embodiments, the antigens include, but are not limited to, CLDN18.2, CD19, CD5, CD20, CD22, CD23, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD319, GPC3, CD32b, CD171, CS-1, CLL-1, BCMA, GD2, GD3, PSMA, ROR1, FLT3, FAP, CD44v6, CEA, EPCAM, B7H3, IL-13Ra2, Mesothelin, Her2, MUC1, EGFR, CLDN6, DLL3, NYESO-1, WT1, Nectin-4, GPC3, PDL1, NKG2DL, and any combination of the above antigens.

[0026] Compared with the prior art, the beneficial effects achieved by the present invention are: based on different antigen targets, such as CD19, CLDN18.2 or BCMA, CAR-NK or CAR-T targeting CD19 mainly removes B cells and is applied to B cell malignancies, such as the treatment of B cell leukemia and lymphoma, or autoimmune diseases associated with autoantibodies such as systemic lupus erythematosus. CLDN1 8.2 is mainly expressed in digestive tract tumors such as gastric cancer and pancreatic cancer, and CAR-NK targeting CLDN1 8.2 can be applied to the treatment of corresponding tumors. Targeting BCMA C-NK mainly treats multiple myeloma, and can also be used for autoimmune diseases associated with autoantibodies such as systemic lupus erythematosus. On the objective basis that the above multiple different antigen targets correspond to abnormal expression of different tumors, they are used as targets for clinical treatment; without changing the expression of chimeric antigens, two cytokines are synergistically secreted; the long-term therapeutic effect of immune cells is enhanced, while reducing the risk of treatment.

[0027] Interleukin 10 (IL-10) is a multifunctional immunomodulatory cytokine. Its role in tumor biology is particularly complex, and it has the dual functions of promoting and inhibiting tumors. Some studies have shown that IL-10 can inhibit tumor growth and metastasis, but IL-10 can also promote tumor growth and immune escape through a variety of mechanisms. Based on the dual functions of IL-10 and promoting and inhibiting tumors, the effect of transforming CAR-NK cells with IL-10 is unpredictable. This application creatively applies IL-10 to CAR-NK or CAR-T cell transformation, and combines interleukin 15 (IL-15) to coordinate dual secretion cytokines, which has unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 Schematic diagram of the lentiviral vector structure of dual-secretionary CAR-NK targeting CD19;

[0030] Figure 2 Expression and identification diagram of dual secretory CAR-NK detected by flow cytometry;

[0031] Figure 3 The expression of IL-10 and IL-15 in the culture supernatant of dual-secretion CAR-NK cells was detected by ELISA;

[0032] Figure 4 In vitro proliferation of CAR-NK cells;

[0033] Figure 5 Luciferase-labeled cell killing experiment results;

[0034] Figure 6 Real-time cell killing (RTCA) experiment results diagram;

[0035] Figure 7 CD107a degranulation experiment results;

[0036] Figure 8 CD107a degranulation experiment statistical graph;

[0037] Fig. 9 In vivo imaging results of CAR-NK therapy in tumor-bearing mice;

[0038] Fig.10 The results of the dynamic changes of fluorescence signals of tumor load in tumor-bearing mice treated with CAR-NK;

[0039] Fig.11Comparison of survival curves of tumor-bearing mice treated with CAR-NK;

[0040] Fig.12 Figure 1. Results of flow cytometric analysis of CAR-NK cells in the spleen of tumor-bearing mice after dissection.

[0041] Fig.13 Figure 1. Results of flow cytometric analysis of CAR-NK cells in the bone marrow of tumor-bearing mice after dissection.

[0042] Fig.14 Figure 1. Results of flow cytometric analysis of CAR-NK cells in peripheral blood of tumor-bearing mice after autopsy;

[0043] Fig.15 CLDN1 8.2 dual secretory CAR-NK luciferase-labeled cell killing experiment results;

[0044] Fig.16 Results of CD107a degranulation experiment of CLDN18.2 dual secretory CAR-NK;

[0045] Fig.17 CLDN18.2 dual secretory CAR-NK real-time cell killing (RTCA) result diagram;

[0046] Fig.18 Results of BCMA dual-secretion CAR-NK luciferase-labeled cell killing experiment;

[0047] Fig.19 Results of CD107a degranulation experiment of BCMA dual secretory CAR-NK;

[0048] Fig. 20 Identification of cytokine secretion by dual-secreting CAR-T cells;

[0049] Fig.21 Luciferase-labeled cell killing assay;

[0050] Fig. 22 Real-time cell killing (RTCA) assay;

[0051] Fig.23 CAR-T cell therapy in tumor-bearing mice. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] In the following examples, the main materials are from: RosetteSep TM Cocktail (STEMCELL, 15661); PBS (Gino, Catalog No. GNM20012-5); Ficoll (Tianjin Haoyang, Catalog No. LTS10770125); Red blood cell lysis buffer (Davern Biotechnology, Catalog No. WB9510P-500mL); CryoStor CS5 (STEMCELL, Catalog No. 7933); Opti-MEM culture medium (Thermofisher, Catalog No. 31985070); pLP1, pLP2 and pVSVG and target plasmids (all purchased from Thermo fisher, Catalog No. K497500); pLenti-CMV-V5-LUC Blast (purchased from Addgene, Catalog No. 21474); Transfection reagent Lipo2000 (Thermo fisher, Catalog No. 11668019); CTS TM AIM V TM SFM (Gibco, catalog number 0870112DK); protamine (Sigma, catalog number P3369-10G); APC anti-FMC63 (Bioswan, catalog number 300402); PE-cy7anti-hIL-10 (BioLegend, catalog number 501419); dead-alive dye 7AAD-Solution (Biogems, catalog number 61410-00-200); ELISA kits for IL-10 (Ecosel, catalog number EH006-96) and IL-15 (Ecosel, catalog number EH057-96); lymphoma cells Raji (purchased from ATCC); JeKo-1 (purchased from the Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); 3T3 (purchased from the Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); E-plate 16 plates (Agilent, Catalog No. 300600890); Protein transport inhibitor (BD, Catalog No. 554724); PE-cy7 anti-hCD 1 07a (BioLegend, Catalog No. 32861 8); PE-cy7 anti-hCD56 (BioLegend, Catalog No. 304628); Pacific Blue anti-hCD45 (BioLegend, Catalog No. 304029).

[0054] Example 1. Preparation of CAR-NK cells

[0055] 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 CD3zeta activation domain was designed and constructed, and Furin-T2A short peptide was used to co-express IL-15 and IL-10; the dual-secretion CAR-NK targeting CD19 formed was 19CAR-IL10-IL15, and the single-secretion CAR-NK was 19CAR-IL15. The schematic diagrams of the two lentiviral vector structures are shown in the figure. Figure 1 As shown. Similarly, the sequence based on targeting CLDN18.2 in Example 6 is shown in SEQ ID NO.14, and the dual secretion CAR-NK targeting CD19 formed is CLDN18.2CAR-IL10-IL15, and the single secretion CAR-NK is CLDN18.2CAR-IL15. The sequence based on targeting BCMA in Example 7 is shown in SEQ ID NO.16, and BCMA dual secretion CAR-NK cells are constructed, and the dual secretion CAR-NK targeting CD19 formed is BCMA-CAR-IL10-IL15, and the single secretion CAR-NK is BCMA-CAR-IL 15.

[0056] 1. The specific steps of CAR-NK preparation are as follows:

[0057] The specific steps for obtaining and freezing umbilical cord blood mononuclear cells and isolating NK cells from umbilical cord blood are as follows:

[0058] 1) Prepare fresh cord blood: After the surface of fresh cord blood is disinfected, take it out from the blood collection bag with a 50mL syringe and transfer it to a sealed T75 culture bottle. At the same time, measure the volume of the cord blood;

[0059] 2) Add RosetteSep to whole blood TM Cocktail (STEMCELL, 15661): Add 50 μL of RosetteSep per mL of umbilical cord blood TM Cocktail reagent, mix well and incubate at room temperature for 20 minutes;

[0060] 3) Dilution of umbilical cord blood: dilute the umbilical cord blood sample with an equal volume of PBS (Gino, catalog number GNM20012-5) and mix well;

[0061] 4) Ficoll (Tianjin Haoyang, catalog number LTS10770125) separation: add 20 mL of Ficoll separation solution to a 50 mL centrifuge tube, slowly add 25 mL of diluted umbilical cord blood to the upper layer of Ficoll separation solution, keep the interface clear, 1200g, 30 minutes, speed up 1, speed down 0;

[0062] 5) Obtaining NK cells: After centrifugation, obvious buffy coat cells can be seen. Discard the plasma, transfer the buffy coat cells to a new centrifuge tube, and add sterile flow cytometry washing buffer (PBS containing 2% fetal bovine serum, i.e., 2% FBS+PBS, hereinafter referred to as FASC Buffer);

[0063] 6) Wash cells with FASC Buffer: 300g, 10 minutes, wash twice, take a small amount of cells to count;

[0064] 7) Red blood cell lysis: Add red blood cell lysis buffer (Davern Biotech, catalog number WB9510P-500mL) according to the total number of cells and the residual red blood cells, at a rate of approximately 2 to 3 × 10 7 Add 30-40 mL of red blood cell lysis buffer to each cell and incubate on ice for 20-30 minutes.

[0065] 8) Cell identification: Wash the cells as in step 6), take a small amount of cells to count, and detect the expression of CD56 / CD3 / CD45 by flow cytometry;

[0066] 9) Cell cryopreservation: The obtained NK cells were cryopreserved using CryoStor CS5 (STEMCELL, Catalog No. 7933) cryopreservation medium at a rate of 5×10 6 Resuspend cells at a density of 5×10 6 The obtained umbilical cord blood NK cells were cryopreserved in a -80℃ liquid nitrogen tank.

[0067] 2. Preparation of Lentiviral Vectors:

[0068] The specific steps for preparing a lentiviral vector for infecting NK cells to express CAR molecules are as follows:

[0069] 1) Transfection was performed when the confluence of 293T cells was 80-90%, and the Opti-MEM medium (Thermo Fisher, Catalog No. 31985070) for packaging virus was replaced 2 hours before transfection;

[0070] 2) Preparation of lentivirus using a four-plasmid packaging system: including three auxiliary plasmids, specifically pLP1, pLP2, and pVSVG and a target plasmid (all purchased from Thermo fisher, catalog number K497500); the transfer plasmid pLenti is a lentiviral expression plasmid, the backbone is based on pLenti-CMV-V5-LUC Blast (purchased from Addgene, catalog number 21474), the original CMV promoter of the plasmid is replaced with the EF1α promoter, and CAR is inserted into the expression framework to obtain 19CAR-IL15 or 19CAR-IL10-IL15 respectively. The four plasmids are mixed in proportion and the transfection reagent Lipo2000 is added to prepare a transfection reagent mixture;

[0071] 3) After mixing, incubate at room temperature for 15-25 minutes and add 293T cells along the side wall;

[0072] 4) Collect virus supernatant: Collect virus supernatant after 48 and 72 hours respectively;

[0073] 5) After filtering the cell debris through a 0.45 μm pore size filter membrane, the supernatant was concentrated by ultracentrifugation; 6) The lentivirus titer was tested using the NK-92MI cell line (Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and the functional titer was measured to be between 0.5 and 1×10 8 Within the TU / mL range, lentivirus that can effectively transfect cells is obtained and subsequent experiments can be carried out.

[0074] 3. Preparation of CAR-NK cells:

[0075] 1) On day 0, umbilical cord blood mononuclear cells were resuscitated and counted;

[0076] 2) Add trophoblast cells (Zhongying Biotechnology, catalog number ZY-NKZ-0104) at a ratio of 3:1, and the culture medium is CTS TM AIM V TM SFM (Gibco, catalog number 08701 12DK, hereinafter referred to as AIM-V) + 10% FBS + 100 IU / mL IL-2;

[0077] 3) On the 5th day, the amount of lentivirus added was calculated according to the following formula: (number of cells × MOI) / virus titer, where MOI was 5, and the transfer agent protamine (Sigma, catalog number P3369-10G) was added at the same time, with a final concentration of 10 μg / mL;

[0078] 4) NK cells were added with lentivirus and transfer agent according to the above step 3) and mixed evenly, with 1-3×10 cells per well. 5 / 200 μL of the system was added to a 96-well flat-bottom plate, and the culture plate was centrifuged at 1200 g and 37°C for 90 min;

[0079] 5) After centrifugation, the cells were placed in an incubator and cultured for 4 hours before changing the medium, discarding the virus medium and replacing it with fresh NK cell culture medium;

[0080] 6) On the 7th day, the proportion of CD56-positive NK cells and the expression of CAR were analyzed by flow cytometry; trophoblast cells were added at a ratio of 1:1 between NK cells and trophoblast cells and continued to be cultured;

[0081] 7) On day 12, the proliferated CAR-NK cells were obtained for subsequent functional research experiments.

[0082] Example 2. Expression and identification of dual secretory CAR-NK

[0083] CAR-NK cells were prepared according to Example 1. On the 10th day of cell culture, CAR-NK cells were collected for flow cytometry identification of CAR and IL-10 expression; cell culture supernatant was collected, and ELISA was used to detect the secretion of IL-10 and IL-15.

[0084] 1) CAR-NK cells were prepared according to the above scheme, and the number of CAR-NK and MOCK cells (NK cells not transduced with CAR) were counted on days 6 / 8 / 10 / 13 / 15 during the preparation of CAR-NK.

[0085] 2) Flow cytometry identification of co-expression of CAR and IL-10:

[0086] (a) On the 10th day, 1×10 5 CAR-NK cells and MOCK cells were centrifuged and washed twice with FACS Buffer, and the cells were resuspended with 50 μL FACS Buffer;

[0087] (b) Surface marker staining: APC anti-FMC63 (Bioswan, Catalog No. 300402) was added to 50 μL of the system at a ratio of 1:500; PE-cy7 anti-hIL-10 (BioLegend, Catalog No. 501419) was added to 50 μL of the system at a ratio of 1:200, and the staining was carried out at 4°C in the dark for 20 minutes.

[0088] (c) After washing twice with FACS buffer, the cells were resuspended in 200 μL FACS buffer, and 1 μL of the dead-alive dye 7AAD-Solution (Biogems, catalog number 61410-00-200) was added to each sample and detected on a flow cytometer.

[0089] 3) Collecting cell culture supernatant:

[0090] (a) Adjusting CAR%: On day 10, 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells were collected, and the CAR% of each CAR-NK cell was detected by flow cytometry. The CAR-NK cells were resuspended in preheated NK medium, and the CAR% of each CAR-NK was adjusted to be consistent with the total number of NK cells;

[0091] (b) Adjust cell density: Take 2×10 cells of CAR-NK and MOCK cells with consistent CAR% adjustment 6 The cell density was adjusted to 1×10 6 / mL, inoculated into 12-well plates and cultured for three days;

[0092] (c) Collecting cell culture supernatant: Take out the CAR-NK cells and MOCK cells cultured for three days and transfer them to a 15 mL centrifuge tube. Centrifuge at 400 g for 5 minutes to obtain the cell supernatant, place it in a 1.5 mL EP tube, label each supernatant with the name, CAR%, cell density, culture time, collection date, and store it at -80°C for later use;

[0093] (d) The cell culture supernatant stored at -80°C was used to detect the specific secretion levels of IL-10 and IL-15 in the cell supernatant using ELISA kits for IL-10 (Ekosai, catalog number EH006-96) and IL-15 (Ekosai, catalog number EH057-96).

[0094] 4) ELISA to detect the secretion of IL-10 and IL-15:

[0095] (a) taking out the IL-10 ELISA detection kit and the IL-15 ELISA detection kit from the refrigerator and equilibrating them to room temperature;

[0096] (b) Take out the strips needed for the experiment from the sealed bag that has been equilibrated to room temperature, and put the unused strips and desiccant back into the aluminum foil bag and seal it in a refrigerator at 2-8°C;

[0097] (c) Prepare standards and samples: After dissolving the cell culture supernatant stored at -80°C, prepare the cell culture supernatant stock solution, 5-fold dilution and 10-fold dilution, respectively, and prepare three replicate wells for each sample; dissolve the standard protein IL-10 / IL-15 included in the ELISA kit in the sample dissolving solution Assay Diluent to a sample of 1000 pg / mL, let it stand for 15 minutes, and then dilute it two-fold to 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.6 pg / mL samples, and prepare three replicate wells for each standard;

[0098] (d) Add different concentrations of standard or sample into the corresponding wells, 100 μL / well, leave blank wells as controls, seal the wells with sealing tape, and incubate in a 37°C constant temperature incubator for 90 min;

[0099] (e) Prepare biotinylated antibody working solution: Dilute Human IL-10 / IL-15100X Biotin-Antibody 100 times with Assay Diluent according to the amount required for the experiment to prepare the biotinylated antibody working solution. Prepare it 30 minutes before use and use it only on the same day.

[0100] (f) Dilute 20X Wash Buffer Concentrate with deionized water to make a washing working solution (WashBuffer), discard the liquid in the well plate, wash the plate five times with Wash Buffer, add biotinylated antibody working solution, 100 μL / well, seal the reaction wells with sealing tape, and incubate in a 37°C constant temperature incubator for 60 min;

[0101] (g) Prepare enzyme conjugate working solution: Dilute 100X HRP-Streptavidin 100 times with Assay Diluent according to the amount required for the experiment to prepare the enzyme conjugate working solution. Prepare it 30 minutes before use and use it only on the same day.

[0102] (h) Discard the liquid in the well plate, wash the plate five times with Wash Buffer, add enzyme conjugate working solution, 100 μL / well, and incubate in a 37°C constant temperature incubator for 60 min;

[0103] (i) Discard the liquid in the well plate, wash the plate five times with Wash Buffer, add Substrate Solution, 100 μL / well, and incubate in a 37°C constant temperature incubator in the dark for 15 min;

[0104] (i) Add Stop Solution, 100 μL / well, mix well and immediately detect CD using an ELISA reader. 450 value.

[0105] The results are as follows Figure 2 and Figure 3 As shown, the dual-secretion CAR-NK cell CAR and IL-10 were co-expressed on the surface of NK cells; at the same time, ELISA results showed that 19CAR-IL10-IL15 secreted a large amount of IL-10 and IL-15, while 19CAR-IL15 only secreted IL-15 cytokine, indicating that the dual-secretion CAR-NK structure we constructed can normally express and secrete two cytokines without affecting the expression of CAR; Figure 4 The results showed that after transduction of CAR-NK lentivirus, umbilical cord blood NK cells could still proliferate in large numbers.

[0106] Example 3. In vitro functional verification of dual-secreting CAR-NK cells

[0107] Lymphoma cells Raji (purchased from ATCC) and JeKo-1 (purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct cell lines Raji-CBR-Luc-GFP and JeKo-1-CBR-Luc-GFP (CBR is click beetlered luciferase, a beetle luciferase that can catalyze the excitation of luciferin substrate to the red spectrum; CBR and GFP were simultaneously introduced into Raji and JeKo-1 cells), which co-express luciferase CBR and green fluorescent protein GFP. These cell lines were used as target cells for short-term in vitro killing and degranulation experiments of CAR-NK cells. At the same time, mouse fibroblasts 3T3 (purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct the cell line 3T3-CD19-Luc-GFP overexpressing human CD19 antigen, which was used as target cells for long-term in vitro killing function verification of CAR-NK cells.

[0108] 1. Luciferase-labeled cell killing experiment:

[0109] 1) Target cell plating: Collect tumor cells Raji and JeKo-1 in the logarithmic growth phase, count after centrifugation, and adjust the cell density to 1×10 5 100 μL / well was added to a 96-well U-bottom plate, i.e. 1×10 4 target cells;

[0110] 2) Adjust CAR%: collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0111] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells according to the CAR-NK cell concentration required for the highest effect-target ratio (calculate the cell density based on 100 μL per well), and perform gradient dilution plating for the remaining effect-target ratios. At the same time, set up a positive control (K max , with Triton-100) and negative control (K min , with NK medium);

[0112] 4) Gently mix the cell mixture in each well and incubate in an incubator for 4 to 6 hours. Preheat the Luciferin substrate in advance, wash the cells twice with FACS Buffer by centrifugation, add 0.5 mM luciferase 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;

[0113] 5) Calculate the killing efficiency % = (K min -K) / (K min -K max )×100%.

[0114] The results are as follows Figure 5 As shown, compared with MOCK, CAR-NK cells of 19CAR-IL15 and 19CAR-IL10-IL15 showed significantly enhanced killing function against Raji or JeKo-1 cells expressing CD19 antigen under different effector-target ratios, but there was no significant difference in the killing function between dual-secretion CAR-NK and single-secretion CAR-NK in vitro within a short period of time.

[0115] 2. Long-term real-time in vitro killing experiment (RTCA experiment):

[0116] 1) Take out the E-plate 16 plate (Agilent, catalog number 300600890), preheat the culture medium at 37°C in advance, add 50 μL of culture medium to each well, measure the baseline value, and ensure that the optical density (OD) value of each well is the same;

[0117] 2) Target cell plating: Collect 3T3-CD19-Luc-GFP cells and adjust the cell density to 10,000 cells per well in a volume of 150 μL;

[0118] 3) Add 150 μL of cell suspension to each well of the E-plate 16 plate, let it stand at room temperature for 30 minutes, and then place it in the xCELLigence RTCA instrument to detect the OD value of the cells in real time;

[0119] 4) When the OD value reaches about 1.0, the CAR-NK cells are washed by centrifugation and resuspended in preheated culture medium. After counting, the CAR% is adjusted to be consistent, and the CAR density is adjusted to 1.25×10 4 Pieces / mL;

[0120] 5) Effector cell plating: interrupt the RTCA experiment, take out the E-plate 16 plate, discard the original culture medium, add 200 μL of CAR-NK cells with adjusted density to each well according to the group, and keep the negative control (only target cells);

[0121] 6) Place the E-plate 16 back on the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-NK cells on target cells.

[0122] The results are as follows Figure 6 As shown in the figure, CAR-NK can significantly kill 3T3 cells expressing CD19 antigen under the condition of 1:4 effector-target ratio; at the same time, it can be seen that in long-term real-time killing, the killing function of dual-secretion CAR-NK is slightly stronger than that of single-secretion CAR-NK.

[0123] 3.CD107a degranulation experiment:

[0124] 1) Target cell plating: Raji and JeKo-1 lymphoma cells in the logarithmic growth phase were collected and counted after centrifugation. 1×10 5 The cells were inoculated into 96-well U-bottom plates, 100 μL per well;

[0125] 2) Adjust CAR%: collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0126] 3) Effector cell plating: The cell density of each CAR-NK cell was adjusted according to the effector-target ratio of 1:1 and inoculated into a 96-well U-bottom plate. Protein transport inhibitor (BD, Catalog No. 554724) was added to each well at a total volume of 1:1500, and PE-cy7 anti-hCD107a (BioLegend, Catalog No. 328618) antibody was added at a total volume of 1:200. The cells were mixed and co-cultured with the target cells for 4 to 6 hours. Tumor-free NK cells were kept in each group as a control.

[0127] 4) Wash twice with FACS buffer according to the flow cytometry method, stain with surface marker antibody, add APC anti-FMC63 antibody at a ratio of 1:500 in 100 μL system, and stain for 20 minutes at 4°C in the dark;

[0128] 5) After washing twice with FACS buffer, resuspend the cells in 200 μL FACS buffer, add 1 μL of the dead and alive dye 7AAD-Solution to each sample, and detect on a flow cytometer.

[0129] The results are as follows Figure 7 and Figure 8 As shown, after CAR-NK cells contact target cells, they undergo a significant CD107a degranulation reaction, and the degranulation of dual-secretion CAR-NK is slightly higher than that of single-secretion CAR-NK.

[0130] Example 4. Mouse transplant tumor model verifies the anti-tumor function of dual-secreting CAR-NK cells in vivo

[0131] The self-constructed luciferase-carrying cells JeKo-1-CBR-Luc-GFP were injected into the tail vein of severely immunodeficient mice (NSG) to prepare CAR-NK cells and intravenously treat tumor-bearing mice (NSG).

[0132] 1) Intravenous tumorigenesis of lymphoma cells: JeKo-1-CBR-Luc-GFP cells in logarithmic growth phase were collected, centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cells were washed twice with serum-free PBS to ensure that no serum was left.

[0133] 2) Count and resuspend the cells in pre-cooled PBS. Inoculate 1×10 cells per NSG mouse. 4 Tumor cells were collected at a volume of 300 μL per cell and the tumor cell density was adjusted to 3.34×10 4 / mL, prepared for tail vein injection;

[0134] 3) Tumor cells were injected into the tail vein of each mouse at a volume of 300 μL to model the disease, and 2 mice were left untreated as blank controls;

[0135] 4) After 48 hours, the animals were imaged in vivo, and the tumor formation was evaluated based on the imaging results. The animals were randomly grouped as follows:

[0136] (a) MOCK group: injection of normal cord blood NK cells without CAR transduction (5 animals);

[0137] (b) 19CAR-IL15 group: injection of umbilical cord blood NK cells transduced with a single secretory CAR-NK (5 animals);

[0138] (c) 19CAR-IL10-IL15 group: injection of umbilical cord blood NK cells transduced with dual secretory CAR-NK (5 animals);

[0139] (d) Blank group: blank control group, without any treatment (2 animals);

[0140] 5) NK cell tail vein treatment after 72 hours:

[0141] (a) NK cells in the logarithmic growth phase were collected, and CAR% was detected by flow cytometry. The CAR% of 19CAR-IL15, 19CAR-IL10-IL15 and MOCK cells were adjusted to be consistent, and the total number of NK cells was consistent;

[0142] (b) Wash twice with pre-cooled PBS without serum to ensure that there is no serum residue;

[0143] (c) Counting, adjust the CAR density to 1.667×10 using pre-cooled PBS without serum. 6 / mL, prepared for tail vein treatment;

[0144] (d) According to the grouping in step 4), each mouse was injected with corresponding MOCK and CAR-NK cells via tail vein injection at a volume of 300 μL;

[0145] (e) Live imaging of animals was performed twice a week during the first week of intravenous treatment, and then once a week to assess tumor progression;

[0146] 6) Fluorescence curves and survival curves were calculated based on tumor burden and mouse survival.

[0147] The results are as follows Fig. 9 , Fig.10 and Fig.11 As shown in the figure, with the increase of tumor load time, the tumor load in the MOCK group increased sharply, and the tumors in the 19CAR-IL15 group and the 19CAR-IL10-IL15 group were almost completely cleared, and the tumor load did not increase; at the same time, the death of NSG mice occurred first in the 19CAR-IL15 group, followed by a large number of NSG deaths in the MOCK and 19CAR-IL15 groups, and one death occurred in the 19CAR-IL10-IL15 group one month later ("X" indicates death); from the fluorescence curve graph of NSG mice, it can be seen that the effects of both CAR-NK cells in the treatment of lymphoma in vivo are significant, but the dual-secretory CAR-NK greatly improves the survival rate of NSG mice and improves the safety of CAR-NK cell therapy. Comprehensive evaluation shows that the dual-secretory CAR-NK has the best effect in the treatment of tumors in vivo.

[0148] Example 5. Distribution of CAR-NK cells in mice

[0149] The NSG mice in Example 4 were dissected to analyze the distribution of CAR-NK cells in the animals.

[0150] 1) On the 49th day of treatment of NSG mice in Example 4, the surviving mice were brought back to the laboratory for autopsy, and all the surviving mice in the 19CAR-IL1 5 group died;

[0151] 2) Anatomy: After collecting peripheral blood from the animals in the B1ank / MOCK / 19CAR-IL10-IL15 group, the animals were killed by cervical dislocation, and the spleen and bone marrow of the mice were obtained by dissection, and the corresponding cells were obtained by grinding and filtering;

[0152] 3) Wash twice with FACS buffer according to the flow staining method, stain with surface marker antibodies, add APC anti-FMC63 at a ratio of 1:500 in 50 μL, add PE-cy7 anti-hCD56 (BioLegend, Catalog No. 304628) at a ratio of 1:200 in 50 μL, and add Pacific Blue anti-hCD45 (BioLegend, Catalog No. 304029) at a ratio of 1:200 in 50 μL, and stain for 20 minutes at 4°C in the dark;

[0153] 4) After washing twice with FACS buffer, resuspend the cells in 200 μL FACS buffer, add 1 μL of the dead and alive dye 7AAD-Solution to each sample, and detect on a flow cytometer.

[0154] The results are as follows Fig.12 , Fig.13 and Fig.14 As shown, on the 49th day, a large amount of 19CAR-IL10-IL15 was still present in the peripheral blood, spleen, and bone marrow of NSG mice, indicating that dual-secretionary CAR-NK cells can survive in the animal body for at least 45 days and achieve long-term therapeutic effects.

[0155] Example 6. In vitro functional verification of CLDN18.2 dual-secreting CAR-NK cells

[0156] Human gastric adenocarcinoma cells AGS (purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee / Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct a cell line co-expressing luciferase CBR (CBR is clickbeetle red luciferase, a beetle luciferase that can catalyze the luciferin substrate to excite the red spectrum; CBR and GFP were simultaneously introduced into AGS cells), and this cell line was used as a target cell for a short-term in vitro killing experiment of CAR-NK cells. 1. Luciferase-labeled cell killing experiment:

[0157] 1) Target cell plating: Collect tumor cells AGS in the logarithmic growth phase, count after centrifugation, and adjust the cell density to 2×10 5 / mL, and 50 μL / well was added to a 96-well U-bottom plate, i.e. 1×10 4 target cells;

[0158] 2) Adjust CAR%: Collect CLDN18.2CAR-IL15 / CLDN18.2CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0159] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells according to the CAR-NK cell concentration required for the highest effect-target ratio (calculate the cell density based on 50 μL per well), and perform gradient dilution plating for the remaining effect-target ratios. At the same time, set up a positive control (K max , with Triton-100) and negative control (K min , with NK medium);

[0160] 4) Gently mix the cell mixture in each well and incubate in an incubator for 4 hours. Preheat the Luciferin substrate in advance, wash the cells twice with FACS Buffer by centrifugation, add 0.5 mM luciferase substrate, and incubate at 37°C in the dark for 1 minute. Select the chemiluminescence mode on the microplate reader to read the data K;

[0161] 5) Calculate the killing efficiency % = (K min -K) / (K min -K max )×100%.

[0162] The results are as follows Fig.15 As shown, compared with MOCK, CLDN18.2CAR-IL15 and CLDN18.2CAR-IL10-IL15 CAR-NK cells showed significantly enhanced killing function against AGS cells expressing CLDN18.2 antigen under different effector-target ratios, but there was no significant difference in the killing function between dual-secretion CAR-NK and single-secretion CAR-NK in vitro within a short period of time.

[0163] 2.CD107a degranulation experiment:

[0164] 1) Target cell plating: Collect lymphoma AGS cells in the logarithmic growth phase, count them after centrifugation, and plate 1×10 5 The cells were inoculated into a 96-well U-bottom plate, with 50 μL per well;

[0165] 2) Adjust CAR%: Collect CLDN18.2CAR-IL15 / CLDN18.2CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0166] 3) Effector cell plating: The cell density of each CAR-NK cell was adjusted according to the effector-target ratio of 1:1 and inoculated into a 96-well U-bottom plate. Protein transport inhibitor (BD, Catalog No. 554724) was added to each well at a total volume of 1:1500, and PE-Cy7 anti-hCD107a (BioLegend, Catalog No. 328618) antibody was added at a total volume of 1:200. The cells were mixed and co-cultured with the target cells for 4 hours. Tumor-free NK cells were kept in each group as a control.

[0167] 4) Wash twice with FACS buffer according to the flow cytometry method, stain with surface marker antibody, add APC anti-NGFR antibody at a ratio of 1:500 in 100 μL system, and stain for 20 minutes at 4°C in the dark;

[0168] 5) After washing twice with FACS buffer, resuspend the cells in 200 μL FACS buffer, add 1 μL of the dead and alive dye 7AAD-Solution to each sample, and detect on a flow cytometer.

[0169] The results are as follows Fig.16 As shown in the figure, after CAR-NK cells contacted target cells, the CD107a degranulation reaction was significantly enhanced, and the degranulation of dual-secretion CAR-NK was slightly higher than that of single-secretion CAR-NK.

[0170] 3. Long-term real-time in vitro killing experiment (RTCA experiment):

[0171] 1) Take out the E-plate 16 plate (Agilent, catalog number 300600890), preheat the culture medium at 37°C in advance, add 50 μL of culture medium to each well, measure the baseline value, and ensure that the optical density (OD) value of each well is the same;

[0172] 2) Target cell plating: AGS-CLDN18.2-Luc-GFP cells were collected and the cell density was adjusted to 10,000 cells per well in a volume of 150 μL;

[0173] 3) Add 150 μL of cell suspension to each well of the E-plate 16 plate, let it stand at room temperature for 30 minutes, and then place it in the xCELLigence RTCA instrument to detect the OD value of the cells in real time;

[0174] 4) When the OD value reaches about 2.0, the CAR-NK cells are washed by centrifugation and resuspended in preheated culture medium. After counting, the CAR% is adjusted to be consistent, and the CAR density is adjusted to 1.25×10 4 / mL;

[0175] 5) Effector cell plating: interrupt the RTCA experiment, take out the E-plate 16 plate, discard the original culture medium, add 200 μL of CAR-NK cells with adjusted density to each well according to the group, and keep the negative control (only target cells);

[0176] 6) Place the E-plate 16 back on the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-NK cells on target cells.

[0177] The results are as follows Fig.17 As shown in the figure, CAR-NK can significantly kill AGS cells expressing CLDN18.2 antigen under the condition of 1:4 effector-target ratio; at the same time, it can be seen that in long-term real-time killing, the killing function of dual-secretion CAR-NK is significantly stronger than that of single-secretion CAR-NK.

[0178] Example 7. In vitro functional verification of BCMA dual-secreting CAR-NK cells

[0179] Human multiple myeloma cells MM.1S (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct a cell line co-expressing luciferase CBR (CBR is click beetle red luciferase, a beetle luciferase that can catalyze the luciferin substrate to excite the red spectrum; CBR and GFP were simultaneously introduced into MM.1S cells), and this cell line was used as a target cell for a short-term in vitro killing experiment of CAR-NK cells.

[0180] 1. Luciferase-labeled cell killing experiment:

[0181] 1) Target cell plating: Collect tumor cells MM.1S in the logarithmic growth phase, count them after centrifugation, and adjust the cell density to 2×10 5 / mL, and 50 μL / well was added to a 96-well U-bottom plate, i.e. 1×10 4 target cells;

[0182] 2) Adjust CAR%: Collect BCMA-CAR-IL15 / BCMA-CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0183] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells according to the CAR-NK cell concentration required for the highest effect-target ratio (calculate the cell density based on 50 μL per well), and perform gradient dilution plating for the remaining effect-target ratios. At the same time, set up a positive control (K max , with Triton-100) and negative control (K min , with NK medium);

[0184] 4) Gently mix the cell mixture in each well and incubate in an incubator for 4 hours. Preheat the Luciferin substrate in advance, wash the cells twice with FACS Buffer by centrifugation, add 0.5 mM luciferase 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;

[0185] 5) Calculate the killing efficiency % = (K min -K) / (K min -K max )×100%.

[0186] The results are as follows Fig.18 As shown, compared with MOCK, BCMA-CAR-IL15 and BCMA-CAR-IL10-IL15 CAR-NK cells showed significantly enhanced killing function against MM.1S cells expressing BCMA antigen under different effector-target ratios, and the killing ability of dual-secretion CAR-NK was stronger than that of single-secretion CAR-NK.

[0187] 2.CD107a degranulation experiment:

[0188] 1) Target cell plating: Myeloma cells MM.1S in the logarithmic growth phase were collected and counted after centrifugation. 1×10 5 The cells were inoculated into a 96-well U-bottom plate, with 50 μL per well;

[0189] 2) Adjust CAR%: Collect BCMA-CAR-IL15 / BCMA-CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK by flow cytometry, resuspend the CAR-NK cells in preheated NK culture medium, and adjust the CAR% of each CAR-NK to be consistent with the total number of NKs;

[0190] 3) Effector cell plating: The cell density of each CAR-NK cell was adjusted according to the effector-target ratio of 1:1 and inoculated into a 96-well U-bottom plate. Protein transport inhibitor (BD, Catalog No. 554724) was added to each well at a total volume of 1:1500, and PE-Cy7 anti-hCD107a (BioLegend, Catalog No. 328618) antibody was added at a total volume of 1:200. The cells were mixed and co-cultured with the target cells for 4 hours. Tumor-free NK cells were kept in each group as a control.

[0191] 4) Wash twice with FACS buffer according to the flow cytometry method, stain with surface marker antibody, add APC anti-NGFR antibody at a ratio of 1:500 in 100 μL system, and stain for 20 minutes at 4°C in the dark;

[0192] 5) After washing twice with FACS buffer, resuspend the cells in 200 μL FACS buffer, add 1 μL of the dead and alive dye 7AAD-Solution to each sample, and detect on a flow cytometer.

[0193] The results are as follows Fig.19 As shown in the figure, after CAR-NK cells contacted target cells, the CD107a degranulation reaction was significantly enhanced, and the degranulation of dual-secretion CAR-NK was slightly higher than that of single-secretion CAR-NK.

[0194] Example 8. Preparation of CAR-T cells

[0195] Based on the FMC63 monoclonal antibody scFv sequence targeting CD19, a CAR structure lentiviral vector using 4-1BB as a co-stimulatory factor and CD3zeta activation domain was designed and constructed, and Furin-2A short peptide was used to co-express IL-15 and IL-10; the dual-secretion CAR-T targeting CD19 was 19CAR-IL10-IL15, and the single-secretion CAR-T was 19CAR-IL15. The specific steps of CAR-T preparation are as follows:

[0196] 1. Acquisition and cryopreservation of human peripheral blood mononuclear cells

[0197] Mononuclear cells (PBMC) were isolated from peripheral blood. The specific steps are as follows:

[0198] 1) Prepare healthy human peripheral blood: Draw 10-15 mL of healthy human peripheral blood into a blood collection tube (BD, Catalog No. 365974), shake evenly, transport to the laboratory at low temperature, and after the surface is disinfected, remove the blood from the blood collection tube with a 20 mL syringe and transfer it to a sealed 50 mL centrifuge tube. At the same time, measure the volume of peripheral blood;

[0199] 2) Dilution of human peripheral blood: dilute peripheral blood with an equal volume of PBS (Gino, catalog number GNM20012-5);

[0200] 3) Ficoll (Tianjin Haoyang, catalog number LTS10770125) separation: add 20 mL of Ficoll separation solution to a 50 mL centrifuge tube, slowly add 25 mL of diluted human peripheral blood to the upper layer of Ficoll separation solution, keep the interface clear, 800g, 30 minutes, breakoff (up 1, down 0);

[0201] 4) Obtaining mononuclear cells: After centrifugation, obvious buffy coat cells can be seen. Discard the plasma, transfer the buffy coat cells to a new centrifuge tube, and add sterile flow cytometry washing buffer (PBS containing 2% fetal bovine serum, i.e., 2% FBS+PBS, hereinafter referred to as FASC Buffer);

[0202] 5) Wash cells with FASC Buffer: 300g, 10 minutes, wash twice, take a small amount of cells to count;

[0203] 6) Cell identification: Take a small amount of cells from step 5) and perform flow cytometry to detect the expression of CD3 / CD45;

[0204] 7) Cell cryopreservation: The obtained mononuclear cells were cryopreserved, and the cryopreserved number was calculated as total number of cells × CD45%; CryoStor CS5 (STEMCELL, Catalog No. 7933) was used as cryopreservation medium, and the cryopreserved number was calculated as 2 × 10 6 Resuspend cells at a density of 2×10 6 The mononuclear cells were cryopreserved in a -80℃ liquid nitrogen tank.

[0205] 2. Preparation of Lentiviral Vectors

[0206] The specific steps for preparing a lentiviral vector for infecting T cells to express CAR molecules are as follows:

[0207] 1) Transfection was performed when the confluence of 293T cells reached 80-90%, and the virus packaging medium (10% FBS Opti-MEM) was replaced 2 hours before transfection;

[0208] 2) Preparation of lentivirus using a four-plasmid packaging system: including three helper plasmids and target plasmids (19CAR-IL15 and 19CAR-IL10-IL15), and adding transfection reagents such as Lipo2000 to prepare a transfection reagent mixture;

[0209] 3) After mixing, incubate at room temperature for 15-25 minutes and add 293T cells along the side wall;

[0210] 4) Collect virus supernatant: Collect virus supernatant after 48 and 72 hours respectively;

[0211] 5) After filtering the cell debris through a 0.45 μm pore size filter membrane, the lentivirus was concentrated by ultracentrifugation; 6) The lentivirus titer was tested using the NK-92MI cell line, and the functional titer was measured to be between 0.5 and 1×10 8 Within the TU / mL range, lentivirus that can effectively transfect cells is obtained and subsequent experiments can be carried out.

[0212] 3. Preparation of CAR-T cells

[0213] 1) On day 0, human mononuclear cells were resuscitated and counted;

[0214] 2) Day 1, T cell activation: According to mononuclear cells: Dynabeads TM Human T-Expander CD3 / CD28 (Gibco, catalog number 11141D) was added to T cell activation antibody magnetic beads in a 1:1 ratio, and the culture medium was MOCKS TM AIMV TM SFM (Gibco, Catalog No. 08701 12DK, hereinafter referred to as AIM-V) + 10% FBS + 300 IU / mL IL-2 + 50 ng / mL IL-7 + 50 ng / mL IL-15 + 3% L-glutamine (200 mM) (Gibco, Catalog No. 25030-081) + 1% HEPES (1 M) (Gibco, Catalog No. 15630080) + 1% MEM non-essential amino acid solution (100×) (Gibco, Catalog No. 11140050) + 1% sodium pyruvate (100 mM) (Gibco, Catalog No. 11360-070);

[0215] 3) Day 2 lentiviral transduction: The number of cells was calculated based on the total number of counts on the previous day, and the amount of lentivirus added was calculated according to the following formula: (number of cells × MOI) / virus titer, where MOI was 5, and the transfer agent Polybrene (Biyuntian, catalog number C0351-50mg) was added at the same time, with a final concentration of 8 μg / mL;

[0216] 4) T cells were added with lentivirus and transfer agent according to the above step 3) and mixed evenly, with 1-3×10 per well. 5 / 200 μL of the system was added to a 96-well flat-bottom plate, and the plate was centrifuged at 1200 g at 37 °C for 90 min;

[0217] 5) After centrifugation, the cells were placed in an incubator and cultured for 4 hours, and the cell medium was changed. The virus medium was discarded and replaced with fresh T cell culture medium to continue the culture;

[0218] 6) On the 4th day, remove the activated antibody magnetic beads: disinfect the surface of the DynaMag-15 magnetic stand and place it in the clean bench for use; transfer the mixture of CAR-T cells and antibody magnetic beads to a 15mL centrifuge tube, blow it repeatedly to separate the cells from the antibodies and place it on the DynaMag-15 magnetic stand, let it stand for 1-3 minutes, and gently take out the cell suspension on the magnetic stand and transfer it to a new 15mL centrifuge tube. Be careful not to touch the wall of the centrifuge tube when taking the cell suspension; centrifuge the cell suspension without antibody magnetic beads and continue to culture, and keep the cell density at 2×10 6 / mL;

[0219] 7) On days 12-16, obtain the proliferated CAR-T cells for subsequent functional research experiments.

[0220] Example 9. Identification of cytokine secretion by dual-secreting CAR-T

[0221] CAR-T cells were prepared according to Example 1. On the 13th day of cell culture, CAR-T cells were taken for flow cytometry detection of CAR expression, CAR%, total cell number, and cell density were adjusted to be consistent, and cultured for three days. The cell culture supernatant was collected, and the secretion of IL-10 and IL-15 was detected by ELISA.

[0222] 1) CAR-T cells were prepared according to the above scheme. During the preparation of CAR-T cells, the number of CAR-T and MOCK cells was counted on days 10 / 13 / 15 / 17 / 20 respectively;

[0223] 2) Flow cytometry detection of CAR expression

[0224] (a) On the 13th day, 1×10 5 CAR-T cells and MOCK cells were centrifuged and washed twice with FACS Buffer, and the cells were resuspended with 50 μL FACS Buffer;

[0225] (b) Surface marker staining: APC anti-FMC63 (Bioswan, Cat. No. 300402) was added to 50 μL of the system at a dilution of 1:500 and stained at 4°C in the dark for 20 minutes;

[0226] (c) After washing twice with FACS buffer, the cells were resuspended in 200 μL FACS buffer, and 1 μL of the dead-alive dye 7AAD-Solution (Biogems, catalog number 61410-00-200) was added to each sample and detected on a flow cytometer.

[0227] 3) Collect cell culture supernatant

[0228] (a) Adjusting CAR%: On day 13, 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells were collected, and the CAR% of each CAR-T cell was detected by flow cytometry. The CAR-T cells were resuspended in preheated T cell culture medium, and the CAR% of each CAR-T was adjusted to be consistent with the total number of T cells;

[0229] (b) Adjust cell density: 2×10 CAR-T and T cells with consistent CAR% were taken 6 The cell density was adjusted to 1×10 6 / mL, inoculated into 12-well plates and cultured for three days

[0230] (c) Collecting cell culture supernatant: Take out the CAR-T cells and MOCK cells cultured for three days and transfer them to a 15 mL centrifuge tube. Centrifuge at 400 g for 5 minutes to obtain the cell supernatant, place it in a 1.5 mL EP tube, label each supernatant with the name, CAR%, cell density, culture time, collection date, and store it at -80°C for later use;

[0231] (d) The cell culture supernatant stored at -80°C was used to detect the specific secretion levels of IL-1v and IL-15 in the cell supernatant using ELISA kits for IL-10 (Ekosai, catalog number EH006-96) and IL-15 (Ekosai, catalog number EH057-96).

[0232] 4) ELISA to detect the secretion of IL-10 and IL-15

[0233] (a) Take out the ELISA detection kits of IL-10 and IL-15 from the refrigerator and equilibrate to room temperature;

[0234] (b) Take out the strips needed for the experiment from the sealed bag that has been equilibrated to room temperature, and put the unused strips and desiccant back into the aluminum foil bag and seal it in a refrigerator at 2-8°C;

[0235] (c) Preparation of standards and samples: After dissolving the cell culture supernatant stored at -80°C, prepare the original cell culture supernatant solution, 25-fold dilution solution and 50-fold dilution solution, respectively, and prepare three replicate wells for each sample; dissolve the standard protein IL-10 / IL-15 included in the ELISA kit into a sample of 1000 pg / mL using the sample dissolving solution Assay Diluent, let it stand for 15 minutes, and then dilute it two-fold to 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.6 pg / mL samples, and prepare three replicate wells for each standard;

[0236] (d) Add different concentrations of standard or sample into the corresponding wells, 100 μL / well, leave blank wells as controls, seal the wells with sealing tape, and incubate in a 37°C constant temperature incubator for 90 min;

[0237] (e) Prepare biotinylated antibody working solution: Dilute Human IL-10 / IL-15100X Biotin-Antibody 100 times with Assay Diluent according to the amount required for the experiment to prepare the biotinylated antibody working solution. Prepare it 30 minutes before use and use it only on the same day.

[0238] (f) Dilute 20X Wash Buffer Concentrate with deionized water to make a washing working solution, discard the liquid in the well plate, wash the plate five times with Wash Buffer, add biotinylated antibody working solution, 100 μL / well, seal the reaction wells with sealing tape, and incubate in a 37°C constant temperature incubator for 60 min;

[0239] (g) Prepare enzyme conjugate working solution: Dilute 100X HRP-Streptavidin 100 times with Assay Diluent according to the amount required for the experiment to prepare the enzyme conjugate working solution. Prepare it 30 minutes before use and use it only on the same day.

[0240] (h) Discard the liquid in the well plate, wash the plate five times with Wash Buffer, add enzyme conjugate working solution, 100 μL / well, and incubate in a 37°C constant temperature incubator for 60 min;

[0241] (i) Discard the liquid in the well plate, wash the plate five times with Wash Buffer, add Substrate Solution, 100 μL / well, and incubate in a 37°C constant temperature incubator in the dark for 15 min;

[0242] (i) Add Stop Solution, 100 μL / well, mix well and immediately detect CD using an ELISA reader. 450 value

[0243] The results are as follows Fig. 20 As shown, 19CAR-IL10-IL15 secretes a large amount of IL-10 and IL-15, while 19CAR-IL15 only secretes IL-15 cytokine, and the IL-10 secretion of dual-secreting CAR-T cells can reach more than 3ng / mL.

[0244] Example 10. In vitro functional verification of dual-secreting CAR-T cells

[0245] Lymphoma cells Raji (purchased from ATCC) and JeKo-1 (purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct cell lines Raji-CBR-GFP and JeKo-1-CBR-GFP (CBR is click beetle redluciferase, a beetle luciferase that can catalyze the excitation of luciferin substrate to the red spectrum; CBR and GFP were simultaneously introduced into Ra" and JeKo-1 cells) that co-express luciferase CBR and green fluorescent protein GFP. These cell lines were used as target cells for CAR-T cell in vitro killing, degranulation experiments, and IFN-γ / TNF-α cytokine secretion experiments; at the same time, mouse fibroblasts 3T3 (purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used to construct the cell line 3T3-CD19 that overexpressed the human CD19 antigen, and these cell lines were used as target cells for long-term real-time in vitro killing function monitoring of CAR-T cells.

[0246] 1. Luciferase-labeled cell killing assay

[0247] 1) Target cell plating: Collect tumor cells Raji and JeKo-1 in the logarithmic growth phase, count after centrifugation, and adjust the cell density to 1×10 5 / mL, and inoculated into 96-well U-bottom plates at 100 μL / well, i.e., 1×10 4 target cells;

[0248] 2) Adjust CAR%: Collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-T by flow cytometry, preheat T cell culture medium to resuspend CAR-T cells, and adjust the CAR% of each CAR-T to be consistent with the total number of T cells; 3) Effector cell plating: Calculate the number of CAR-T cells required for plating, resuspend the cells according to the CAR-T cell concentration required for the highest efficiency-target ratio (calculate the cell density according to 100 μL per well), and perform gradient dilution plating for the remaining effect-target ratios, and set up a positive control (K max , with Triton-100) and negative control (K min , using T cell culture medium)

[0249] 4) Gently mix the cell mixture in each well and incubate in an incubator for 4-6 hours. Preheat the Luciferin substrate in advance, wash the cells twice with FACS Buffer by centrifugation, add 0.5mM luciferase 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;

[0250] 5) Calculate the killing efficiency % = (Kmin -K) / (K min -K max )×100%.

[0251] The results are as follows Fig.21 As shown in the figure, compared with MOCK, 19CAR-IL15 and 19CAR-IL10-IL15 CAR-T cells showed significantly enhanced killing function against Raji or JeKo-1 cells expressing CD19 antigen under different effector-target ratio conditions. At the same time, the in vitro killing function of dual-secretion CAR-T cells was stronger than that of single-secretion CAR-T cells, with significant differences. 2. Long-term real-time in vitro killing experiment (RTCA experiment)

[0252] 1) Take out the E-plate 16 plate (Agilent, catalog number 300600890), preheat the culture medium at 37°C in advance, add 50 μL of culture medium to each well, measure the baseline value, and ensure that the optical density (OD) value of each well is the same;

[0253] 2) Target cell plating: Collect 3T3-CD19 cells and adjust the cell density to 10,000 cells per well in a volume of 150 μL;

[0254] 3) Add 150 μL of cell suspension to each well of the E-plate 16 plate, let it stand at room temperature for 30 minutes, and then place it in the xCELLigence RTCA instrument to detect the OD value of the cells in real time;

[0255] 4) When the OD value reaches about 1.0, the CAR-T cells are centrifuged and washed, and then resuspended in preheated T cell culture medium. After counting, the CAR% is adjusted to be consistent, and the CAR density is adjusted to 1.667×10 4 / mL;

[0256] 5) Effector cell plating: interrupt the RTCA experiment, take out the E-plate 16 plate, discard the original culture medium, add 200 μL of CAR-T cells with adjusted density to each well according to the group, and keep the negative control (only target cells);

[0257] 6) Place the E-plate 16 back on the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-T cells on target cells.

[0258] The results are as follows Fig. 22 As shown in the figure, CAR-T cells can significantly kill 3T3 cells expressing CD19 antigen under the condition of 1:3 effector-target ratio; at the same time, it can be seen that in long-term real-time killing, the killing function of dual-secretionary CAR-T cells is stronger than that of single-secretionary CAR-T cells, and they can quickly kill target cells in a short time, and the killing advantage exists for a long time.

[0259] Example 11. Mouse transplant tumor model verifies the anti-tumor function of dual-secreting CAR-T cells in vivo

[0260] The self-constructed luciferase-carrying cells JeKo-1-CBR-GFP were injected into the tail vein of severely immunodeficient mice (NSG) to prepare CAR-T cells and intravenously treat tumor-bearing mice (NSG).

[0261] 1) Intravenous tumorigenesis of lymphoma cells

[0262] (a) Cell collection: JeKo-1-CBR-GFP cells in logarithmic growth phase were collected, centrifuged at 400 g for 5 min, supernatant discarded, and washed twice with serum-free PBS to ensure that no serum remained;

[0263] (b) Adjust the cell density: resuspend the cells in pre-cooled PBS and count them. Inoculate 1×10 cells per NSG mouse. 4 Tumor cells were collected at a volume of 300 μL per cell and the tumor cell density was adjusted to 3.34×10 4 / mL, prepared for tail vein injection;

[0264] (c) Tumor cells were injected into the tail vein of each mouse at a volume of 300 μL to model the tumor, and 2 mice were left untreated as blank controls.

[0265] 2) Animal in vivo imaging grouping: Animal in vivo imaging was performed 48 hours after intravenous tumor formation. The tumor formation was evaluated based on the imaging results and the animals were randomly grouped. The specific groups are as follows:

[0266] (a) MOCK group: injection of common T cells without CAR transduction (6 animals);

[0267] (b) 19CAR-IL15 group: injection of single secretory CAR-T cells (6 animals);

[0268] (c) 19CAR-IL10-IL15 group: injection of dual-secreting CAR-T cells (6 animals);

[0269] (d) Blank group: blank control group, without any treatment (2 animals);

[0270] 3) CAR-T cell tail vein treatment 72 hours after intravenous tumor formation

[0271] (a) CAR-T cells in the logarithmic growth phase were collected, and CAR% was detected by flow cytometry. The CAR% of 19CAR-IL15, 19CAR-IL10-IL15 and MOCK cells were adjusted to be consistent, and the total number of T cells was consistent;

[0272] (b) Wash twice with pre-cooled PBS without serum to ensure that there is no serum residue;

[0273] (c) Resuspend the CAR-T cells in pre-cooled PBS without serum and count them. The CAR density is adjusted to 3.34×10 5 / mL, prepared for tail vein treatment;

[0274] (d) According to the grouping in step 2), each mouse was injected with the corresponding MOCK and CAR-T cells via the tail vein in a volume of 300 μL;

[0275] (e) Live imaging of animals was performed twice a week during the first week of intravenous treatment, and then once a week to assess tumor progression;

[0276] 4) Fluorescence curves and survival curves were calculated based on tumor burden and mouse survival.

[0277] The results are as follows Fig.23 As shown in the figure, with the increase of tumor load time, the tumor load in the MOCK group increased sharply; in the early stage, the tumor load in the 19CAR-IL15 group and 19CAR-IL10-IL15 group increased in a short period of time and then was almost completely cleared, and the tumor load did not increase significantly; after one week of treatment, the tumor load in the single secretory CAR-T cell group increased significantly, and the tumor load in the dual secretory CAR-T group did not increase, and the two groups had the same degree of animal death; from the fluorescence curve of NSG mice, it can be seen that the therapeutic effect of dual secretory CAR-T is significantly stronger than that of single secretory CAR-T cells.

[0278] Amino acid sequence of 19CAR-IL10-IL15 CAR molecule (SEQ ID NO.1):

[0279]

[0280]

[0281] in,

[0282] Signal peptide sequence (SEQ ID NO.2): MALPVTALLLPLALLLHAARP

[0283] Anti-CD19 scFv sequence (SEQ ID NO.3):

[0284]

[0285] 4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO.4):

[0286]

[0287] IL-10 sequence (SEQ ID NO.5):

[0288]

[0289] IL-15 sequence (SEQ ID NO.6):

[0290]

[0291] DNA sequence of 19CAR-IL10-IL15 CAR molecule (SEQ ID NO.7):

[0292]

[0293]

[0294] in,

[0295] Signal peptide sequence (SEQ ID NO.8):

[0296]

[0297] Anti-CD19 scFv sequence (SEQ ID NO.9):

[0298]

[0299] 4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO.10):

[0300]

[0301]

[0302] IL-10 sequence (SEQ ID NO.11):

[0303]

[0304] IL-15 sequence (SEQ ID NO.12):

[0305]

[0306] Targeting CLDN18.2 CAR binding domain amino acid sequence (SEQ ID NO.13):

[0307]

[0308] The amino acid sequence of the CAR binding domain targeting CLDN18.2 corresponds to the DNA sequence (SEQ ID NO.14):

[0309]

[0310] Targeting BCMACAR binding domain amino acid sequence (SEQ ID NO.15):

[0311]

[0312] Targeting BCMACAR binding domain amino acid sequence corresponding to the DNA sequence (SEQ ID NO.16):

[0313]

[0314] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" 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 includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions recorded in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant construct, characterized in that Simultaneously encodes IL-10, IL-15 and CAR, fragments or variants thereof, wherein the antigens of the CAR include any one or combination of CLDN18.2, CD19, CD5, CD20, CD22, CD23, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD319, GPC3, CD32b, CD171, CS-1, CLL-1, BCMA, GD2, GD3, PSMA, ROR1, FLT3, FAP, CD44v6, CEA, EPCAM, B7H3, IL-13Ra2, Mesothelin, Her2, MUC1, EGFR, CLDN6, DLL3, NYESO-1, WT1, Nectin-4, GPC3, PDL1 and NKG2DL.

2. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is CD19, and the nucleic acid sequence of the recombinant construct is shown in SEQ ID NO.7, or a fragment or variant thereof.

3. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is CD19, and the amino acid sequence encoded by the recombinant construct is shown in SEQ ID NO.1, or a fragment or variant thereof.

4. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is CLDN18.2, and the partial nucleic acid sequence targeting CLDN18.2 in the recombinant construct is shown as SEQ ID NO.14, or a fragment or variant thereof; the nucleic acid sequence of IL-10 in the recombinant construct is shown as SEQ ID NO.11, or a fragment or variant thereof; the nucleic acid sequence of IL-15 in the recombinant construct is shown as SEQ ID NO.12, or a fragment or variant thereof.

5. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is CLDN18.2, and the amino acid sequence of the CLDN18.2-targeting portion in the recombinant construct is shown in SEQ ID NO.13, or a fragment or variant thereof; the amino acid sequence encoded by IL-10 in the recombinant construct is shown in SEQ ID NO.5, or a fragment or variant thereof; the amino acid sequence encoded by IL-15 in the recombinant construct is shown in SEQ ID NO.6, or a fragment or variant thereof.

6. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is BCMA, and the partial nucleic acid sequence targeting BCMA in the recombinant construct is shown as SEQ ID NO.16, or a fragment or variant thereof; the nucleic acid sequence of IL-10 in the recombinant construct is shown as SEQ ID NO.11, or a fragment or variant thereof; the nucleic acid sequence of IL-15 in the recombinant construct is shown as SEQ ID NO.12, or a fragment or variant thereof.

7. A recombinant construct according to claim 1, characterized in that The antigen of the CAR is BCMA, and the amino acid sequence of the BCMA-targeting portion in the recombinant construct is shown in SEQ ID NO.15, or a fragment or variant thereof; the amino acid sequence encoded by IL-10 in the recombinant construct is shown in SEQ ID NO.5, or a fragment or variant thereof; the amino acid sequence encoded by IL-15 in the recombinant construct is shown in SEQ ID NO.6, or a fragment or variant thereof.

8. A CAR-modified cell, characterized in that: It comprises a recombinant construct as described in any one of claims 1 to 7.

9. A CAR-modified cell according to claim 8, characterized in that: The CAR-modified cells are any one of CAR-NK cells or CAR-T cells.

10. A CAR-modified cell according to claim 8, characterized in that: The CAR-modified cells are autologous, allogeneic or xenogeneic.

11. A pharmaceutical composition, characterized in that: It comprises a recombinant construct as described in any one of claims 1 to 7 or a CAR-modified cell as described in any one of claims 8 to 10 and at least one pharmaceutically acceptable carrier.

12. Use of a recombinant construct as described in any one of claims 1 to 7, a CAR-modified cell as described in any one of claims 8 to 10, or a pharmaceutical composition as described in claim 11 in the preparation of a kit for diagnosing / treating malignant tumors or autoimmune diseases.

13. Use of a recombinant construct as described in any one of claims 1 to 7, a CAR-modified cell as described in any one of claims 8 to 10, or a pharmaceutical composition as described in claim 11 in the preparation of drugs for malignant tumors or autoimmune diseases.

14. The use of claim 13 in preparing a drug for a malignant tumor or autoimmune disease, characterized in that: The malignant tumor is any one of gastric adenocarcinoma, B cell malignant tumor or myeloma.

Images