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] This invention relates to the fields of biotechnology and cell therapy, specifically to a recombinant construct, CAR-modified cells, and their applications. Background Technology

[0002] Both CAR-NK and CAR-T therapies belong to the category of cell immunotherapy technologies. Currently, CAR-NK cell therapy has shown promising results in early-stage clinical or preclinical trials for various solid tumors, including pancreatic cancer, colorectal cancer, and lung cancer. This makes CAR-NK therapy a potential candidate to fill the gaps in CAR-T therapy for treating solid tumors. Furthermore, its readily available availability can significantly shorten treatment time and reduce costs.

[0003] In the actual construction and modification of CAR-NK cells, the design and modification of extracellular target recognition regions (also known as antigen-binding regions or antigen recognition regions) for different antigens often results in the chimeric antigen receptor (CAR) not being expressed due to the complexity of cell expression. Therefore, how to construct targeted CAR-NK cells or CAR-T cells for different antigens, while simultaneously expressing certain cytokines, is currently a hot topic and a major challenge in the industry. Each successfully constructed CAR-NK cell or CAR-T cell targeting different antigens has enormous value and makes a significant contribution to further conquering and treating various solid tumors in humans. Summary of the Invention

[0004] The purpose of this invention is to provide CAR-modified immune cells, their preparation method, and their applications, in order to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A recombinant construct encoding IL-10, IL-15, and CAR, fragments or variants thereof, wherein the antigen of the CAR comprises: CLDN18.2, CD19, CD5, CD20, CD22, CD23, CD30, CD33, CD34, CD37, CD38, CD43, CD72a, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD319, GPC3, CD3 2b, 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 any one or combination of NKG2DL.

[0006] In one or more embodiments, the amino acid sequence of IL-10 is as shown in SEQ ID NO.5, a fragment thereof, or a variant thereof.

[0007] In one or more embodiments, the amino acid sequence of IL-15 is as shown in SEQ ID NO.6, or a fragment thereof, or a variant thereof.

[0008] In one or more embodiments, the amino acid sequence in the CAR that targets CD19 is the monoclonal antibody scFv sequence shown in Genebank: HM852952.1.

[0009] In one or more embodiments, the amino acid sequence in the CAR that targets the CLDN18.2 portion is shown in SEQ ID NO. 13.

[0010] In one or more embodiments, the amino acid sequence in the CAR that targets the BCMA portion is shown in SEQ ID NO.15.

[0011] In one or more embodiments, the recombinant construct includes one, two, three, or more expression boxes. In one or more embodiments, the recombinant construct includes one expression box encoding IL-10, IL-15, and CAR. In one or more embodiments, the recombinant construct includes two expression boxes encoding any one of IL-10, IL-15, and CAR, and the remaining two. In one or more embodiments, the recombinant construct includes three expression boxes encoding IL-10, IL-15, and CAR, respectively.

[0012] In one or more embodiments, the recombinant construct comprises one, two, three, or more constructs (e.g., plasmids). In one or more embodiments, the recombinant construct comprises one construct (e.g., a plasmid) encoding IL-10, IL-15, and CAR. In one or more embodiments, the recombinant construct comprises two constructs (e.g., plasmids) encoding any one of IL-10, IL-15, and CAR, and the remaining two. In one or more embodiments, the recombinant construct comprises three constructs (e.g., plasmids) encoding IL-10, IL-15, and CAR, respectively.

[0013] In one or more embodiments, 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 thereof or a variant thereof.

[0014] In one or more embodiments, the antigen of the CAR is CD19, and the amino acid sequence encoded by the recombinant construct is shown as SEQ ID NO.1, a fragment thereof, or a variant thereof.

[0015] In one or more embodiments, the antigen of the CAR is CLDN18.2, and the 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; and the nucleic acid sequence of IL-15 in the recombinant construct is shown as SEQ ID NO.12, or a fragment or variant thereof.

[0016] In one or more embodiments, the antigen of the CAR is CLDN18.2, and the amino acid sequence targeting the CLDN18.2 portion in the recombinant construct is shown as SEQ ID NO.13, a fragment thereof, or a variant thereof; the amino acid sequence encoded by IL-10 in the recombinant construct is shown as SEQ ID NO.5, a fragment thereof, or a variant thereof; and the amino acid sequence encoded by IL-15 in the recombinant construct is shown as SEQ ID NO.6, a fragment thereof, or a variant thereof.

[0017] In one or more embodiments, the antigen of the CAR is BCMA, and the partial nucleic acid sequence targeting BCMA in the recombinant construct is shown in SEQ ID NO.16, or a fragment or variant thereof; the nucleic acid sequence of IL-10 in the recombinant construct is shown in SEQ ID NO.11, or a fragment or variant thereof; and the nucleic acid sequence of IL-15 in the recombinant construct is shown in SEQ ID NO.12, or a fragment or variant thereof.

[0018] In one or more embodiments, the antigen of the CAR is BCMA, and the amino acid sequence targeting the BCMA 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; and 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.

[0019] The present invention also provides a CAR-modified cell, comprising any of the recombinant constructs described above.

[0020] In one or more embodiments, the CAR-modified cells are either CAR-NK cells or CAR-T cells.

[0021] In one or more embodiments, the CAR-modified cells are autologous, allogeneic, or xenologous.

[0022] The present invention also provides a pharmaceutical composition comprising a recombinant construct as described in any one of the foregoing claims or a CAR-modified cell as described in any one of the foregoing claims, and at least one pharmaceutically acceptable carrier.

[0023] The present invention also provides the use of a recombinant construct described in any embodiment of the invention, or a CAR-modified cell described in any embodiment of the invention, or a pharmaceutical composition described in any embodiment of the invention, in the preparation of a kit for the diagnosis / treatment of malignant tumors or autoimmune diseases.

[0024] The present invention also provides the use of a recombinant construct as described in any embodiment of the invention, or a CAR-modified cell as described in any embodiment of the invention, or a pharmaceutical composition as described in any embodiment of the invention, in the preparation of drugs for malignant tumors or autoimmune diseases.

[0025] In one or more embodiments, the malignant tumor is any one of gastric adenocarcinoma, B-cell malignancy, or myeloma.

[0026] The purpose of this invention is to provide a method for preparing dual-cytokine-secreting CAR-modified cells and their application in the field of cell therapy.

[0027] In this invention, an expression vector (chimeric antigen receptor expression vector) is provided, which includes a signal peptide, an extracellular target recognition region (antigen binding region), a hinge region, a transmembrane region, and an intracellular region. In particular, IL-10 and IL-15 cytokines are co-expressed by the short peptide Furin-T2A. The expression vector is a lentiviral expression vector.

[0028] For example, a method for preparing a chimeric antigen receptor-NK cell (CAR-NK) that secretes two cytokines is provided, comprising a CAR expression vector containing the aforementioned antigen recognition region, wherein the host cell is an immune cell, such as a T cell, tumor-infiltrating lymphocyte (TIL), natural killer cell (NK), or natural killer T cell (NKT).

[0029] A method for detecting cell killing efficiency is provided, specifically including: luciferase-labeled cell killing assay, long-term real-time in vitro killing assay (RTCA assay), and CD107a degranulation assay; the cell killing function detection method described in the embodiments includes, but is not limited to, the methods described above, such as IFN-gamma detection, granzyme B detection, perforin detection, etc.

[0030] In one or more embodiments, for example, the NK cells can be: umbilical cord blood-derived NK cells, including but not limited to umbilical cord blood-derived cells such as peripheral blood, iPS-induced NK cell lines.

[0031] 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.

[0032] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: Based on different antigen targets, such as CD19, CLDN18.2, or BCMA, CD19-targeted CAR-NK or CAR-T primarily eliminates B cells and can be applied to the treatment of B-cell malignancies, such as B-cell leukemia and lymphoma, or autoantibody-related autoimmune diseases such as systemic lupus erythematosus. CLDN18.2 is mainly expressed in gastrointestinal tumors such as gastric and pancreatic cancer, and CAR-NK targeting CLDN18.2 can be applied to the treatment of corresponding tumors. BCMA-targeted CAR-NK primarily treats multiple myeloma and can also be used for autoantibody-related autoimmune diseases such as systemic lupus erythematosus. Based on the objective fact that different antigen targets correspond to different abnormal tumor expression, these can be used as targets for clinical treatment; without altering the expression of chimeric antigens, two cytokines are synergistically secreted; enhancing the long-term therapeutic effect of immune cells while reducing treatment risks.

[0033] Interleukin-10 (IL-10) is a multifunctional immunomodulatory cytokine with a particularly complex role in tumor biology, possessing a dual function of promoting and inhibiting tumor growth. Some studies have shown that IL-10 can inhibit tumor growth and metastasis, but it can also promote tumor growth and immune escape through various mechanisms. Given IL-10's dual function of promoting and inhibiting tumor growth, the effects of modifying CAR-NK cells with IL-10 are unpredictable. This application creatively applies IL-10 to the modification of CAR-NK or CAR-T cells, combined with interleukin-15 (IL-15) for coordinated dual-secretion cytokine activity, resulting in unexpected technical effects. Compared to CAR-NK cells expressing IL-10 or IL-15 alone, the dual-secretion CAR-NK cells of this invention exhibit better expansion capacity, minimally promote CD8+ T cell proliferation, minimally induce host anti-graft reactions, improve the safety and survival rate of CAR-NK cell therapy in mice, and demonstrate better in vivo persistence. Attached Figure Description

[0034] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 A schematic diagram of a lentiviral vector targeting CD19 with dual secretion; Figure 2 Identification of dual-secretory CAR-NK expression by flow cytometry; Figure 3 Identification diagram of IL-10 and IL-15 expression in the supernatant of dual-secreting CAR-NK culture medium detected by ELISA; Figure 4 In vitro proliferation diagram of CAR-NK cells; Figure 5 Figure showing the results of a luciferase-labeled cell killing assay; Figure 6 Figure showing the results of a real-time cell killing (RTCA) experiment; Figure 7 Figure showing the results of the CD107a degranulation experiment; Figure 8 Statistical graph of CD107a degranulation experiment; Figure 9 In vivo imaging results of CAR-NK therapy in tumor-bearing mice; Figure 10 The dynamic changes in tumor burden fluorescence signal in CAR-NK treated tumor-bearing mice are shown in the figure. Figure 11 A comparison of survival curves in tumor-bearing mice treated with CAR-NK therapy; Figure 12 Figure 1. Results of flow cytometry analysis of CAR-NK cells in spleen of tumor-bearing mice after dissection. Figure 13 Image showing the results of flow cytometry analysis of CAR-NK cells in bone marrow of tumor-bearing mice after dissection; Figure 14 Figure 1. Results of flow cytometry analysis of CAR-NK cells in peripheral blood of tumor-bearing mice after dissection. Figure 15 Dynamic changes in tumor burden over time in mice treated with different NK cells. Figure 16 Total fluorescence over time in mice; arrows indicate drug administration time. Figure 17 Survival rate of mice over time. Figure 18 The number of CAR-NK cells in the peripheral blood of mice. Figure 19 Evaluation of the ability of two different secretory forms of CD19-CAR-NK to promote CD8+ T cell proliferation after co-culturing with allogeneic PBMCs for 7 days. Figure 20 Peripheral blood CAR-NK cell counts in patients in different treatment groups. Figure 21 Figure 1 shows the results of a cell-killing assay using CLDN1 8.2 dual-secreting CAR-NK luciferase-labeled cells. Figure 22 Figure 1. Results of CD107a degranulation experiment of CLDN18.2 dual-secreting CAR-NK; Figure 23 Figure showing the results of real-time cell killing (RTCA) of dual-secretory CAR-NK cells in CLDN18.2. Figure 24 Figure 1. Results of BCMA dual-secreting CAR-NK luciferase-labeled cell killing assay; Figure 25 Figure 1. Results of CD107a degranulation assay in BCMA dual-secreting CAR-NK; Figure 26 Identification of cytokine secretion by dual-secreting CAR-T cells; Figure 27 Luciferase-labeled cell killing assay; Figure 28 Real-time cell killing (RTCA) experiment; Figure 29 CAR-T cell therapy in tumor-bearing mice. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] In the following embodiments, the main materials are sourced from: RosetteSep TM Cocktail (STEMCELL, 15661); PBS (Gino, GMN20012-5); Ficoll (Tianjin Haoyang, LTS10770125); Red blood cell lysis buffer (DaVen Biotech, WB9510P-500mL); CryoStor CS5 (STEMCELL, 7933); Opti-MEM medium (Thermofisher, 31985070); pLP1, pLP2, and pVSVG and target plasmid (all purchased from Thermofisher, K497500); pLenti-CMV-V5-LUC Blast (purchased from Addgene, 21474); Lipo2000 transfection reagent (Thermofisher, 11668019); CTS TM AIM V TMSFM (Gibco, catalog number 0870112DK); protamine sulfate (Sigma, catalog number P3369-10G); APC anti-FMC63 (Bioswan, catalog number 300402); PE-cy7 anti-hIL-10 (BioLegend, catalog number 501419); 7AAD-Solution for live / dead dyes (Biogems, catalog number 61410-00-200); ELISA kits for IL-10 (Eco-Syne, catalog number EH006-96) and IL-15 (Eco-Syne, catalog number EH057-96); Raji lymphoma cells (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-plate (Agilent, catalog number 300600890); Protein transport inhibitor (BD, catalog number 554724); PE-cy7 anti-hCD107a (BioLegend, catalog number 328618); PE-cy7 anti-hCD56 (BioLegend, catalog number 304628); Pacific Blue anti-hCD45 (BioLegend, catalog number 304029).

[0037] Example 1. Preparation of CAR-NK cells

[0038] Based on the CD19-targeting FMC63 monoclonal antibody scFv sequence (Genebank: HM852952.1), a lentiviral vector with a CAR structure using 4-1BB as a co-stimulatory factor and the CD3zeta activation domain was designed and constructed, and IL-15 and IL-10 were co-expressed using the Furin-T2A short peptide. The resulting CD19-targeting dual-secreting CAR-NK is 19CAR-IL10-IL15, and the single-secreting CAR-NK is 19CAR-IL15. Schematic diagrams of the two lentiviral vector structures are shown below. Figure 1As shown in SEQ ID NO.14, the sequence based on CLDN18.2 in Example 6 resulted in a CD19-targeting dual-secreting CAR-NK cell, CLDN18.2CAR-IL10-IL15, and a single-secreting CAR-NK cell, CLDN18.2CAR-IL15. Similarly, the sequence based on BCMA in Example 7, as shown in SEQ ID NO.16, constructed BCMA dual-secreting CAR-NK cells. The resulting CD19-targeting dual-secreting CAR-NK cell was BCMA-CAR-IL10-IL15, and the single-secreting CAR-NK cell was BCMA-CAR-IL 15.

[0039] 1. The specific steps for CAR-NK preparation are as follows: The specific steps for obtaining and cryopreserving umbilical cord blood mononuclear cells, and isolating NK cells from umbilical cord blood, are as follows: 1) Prepare fresh cord blood: After sterilizing the surface of fresh cord blood, take it out of the blood collection bag with a 50mL syringe and transfer it to a sealed T75 culture bottle, while measuring the volume of cord blood. 2) Add RosetteSep to whole blood TM Cocktail (STEMCELL, 15661): Add 50 μL of RosetteSep per milliliter of cord blood. TM Cocktail reagent, mix well and incubate at room temperature for 20 minutes; 3) Cord blood dilution: Dilute the cord blood sample with an equal volume of PBS (Gino, catalog number GMN20012-5) and mix well; 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, 1200 g, 30 minutes, ramp rate 1, depress rate 0. 5) Obtaining NK cells: After centrifugation, a clear white membrane layer of cells can be seen. Discard the plasma and transfer the white membrane layer cells to a new centrifuge tube. Add sterile flow cytometry washing buffer (PBS containing 2% fetal bovine serum, i.e., 2% FBS+PBS, hereinafter referred to as FASC Buffer). 6) Wash cells with FASC Buffer: 300g, 10 minutes, wash twice, take a small amount of cells for counting; 7) Red blood cell lysis: Add red blood cell lysis buffer (DaVen Biotech, catalog number WB9510P-500mL) according to the total number of cells and the degree of red blood cell residue, at a rate of approximately 2-3 × 10⁻⁶ cells / mL. 7Add 30-40 mL of erythrocyte lysis buffer to each cell and incubate on ice for 20-30 minutes. 8) Cell identification: Wash cells as in step 6), take a small number of cells for counting, and simultaneously detect the expression of CD56 / CD3 / CD45 by flow cytometry; 9) Cell cryopreservation: The obtained NK cells were cryopreserved using CryoStor CS5 (STEMCELL, catalog number 7933) cryopreservation solution at a rate of 5 × 10⁻⁶. 6 Cells were resuspended at a density of 5 × 10⁶ cells / mL. 6 One vial of cells was cryopreserved, and the obtained umbilical cord blood NK cells were cryopreserved in a liquid nitrogen tank at -80°C.

[0040] 2. Preparation of lentiviral vectors: The specific steps for preparing a lentiviral vector for infecting NK cells expressing CAR molecules are as follows: 1) Transfect 293T cells when the confluence is 80-90%. Change the Opti-MEM medium (Thermo Fisher, catalog number 31985070) used for packaging the virus 2 hours before transfection. 2) Lentiviral preparation using a four-plasmid packaging system: This system includes three helper plasmids, specifically pLP1, pLP2, and pVSVG, and the target plasmid (all purchased from Thermo Fisher, catalog number K497500); the transfer plasmid pLenti is a lentiviral expression plasmid with a backbone based on pLenti-CMV-V5-LUC Blast (purchased from Addgene, catalog number 21474). The original CMV promoter of the plasmid was replaced with the EF1α promoter, and the CAR was inserted into the expression framework to obtain 19CAR-IL15 or 19CAR-IL10-IL15, respectively. The four plasmids were mixed in proportion and then added to the transfection reagent Lipo2000 to prepare a transfection reagent mixture. 3) After mixing, incubate at room temperature for 15-25 minutes, then add to 293T cells along the side wall; 4) Collect viral supernatant: Collect viral supernatant after 48 and 72 hours respectively; 5) After filtering cell debris through a 0.45 μm pore size filter membrane, the supernatant was concentrated using ultracentrifugation; 6) Lentiviral titers were detected using NK-92MI cell line (Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and the functional titers were found to be between 0.5 and 1 × 10⁻⁶. 8 Within the TU / mL range, lentiviruses that can effectively transfect cells can be obtained for subsequent experiments.

[0041] 3. Preparation of CAR-NK cells: 1) On day 0, resuscitate and count the mononuclear cells in the umbilical cord blood; 2) Add the trophoblast cells at a ratio of 3:1 (monuclear cells:trophoblast cells, Zhongying Biotechnology, catalog number ZY-NKZ-0104), using CTS culture medium. TM AIM V TM SFM (Gibco, catalog number 08701 12DK, hereinafter referred to as AIM-V) + 10% FBS + 100 IU / mL IL-2; 3) On day 5, calculate the amount of lentivirus added according to the following formula: (cell number × MOI) / virus titer, where MOI is 5, and add protamine sulfate (Sigma, catalog number P3369-10G) as a conversion aid to a final concentration of 10 μg / mL. 4) Add lentivirus and transduction aid to NK cells as described in step 3) above, mix well, and add 1–3 × 10⁶ cells per well. 5 Add 200 μL of the system to a 96-well plate and centrifuge the plate at 1200 g and 37 °C for 90 minutes. 5) After centrifugation, place the cells in an incubator and incubate for 4 hours. Then change the cell culture medium, discard the virus solution, and replace it with fresh NK cell culture medium. 6) On day 7, 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 of NK cells to trophoblast cells and cultured for a period of time. 7) On day 12, proliferated CAR-NK cells were obtained for subsequent functional studies.

[0042] Example 2. Identification of expression in dual-secreting CAR-NK

[0043] CAR-NK cells were prepared according to Example 1. On day 10 of cell culture, CAR-NK cells were used to identify the expression of CAR and IL-10 by flow cytometry. Cell culture supernatant was collected and IL-10 and IL-15 secretion were detected by ELISA.

[0044] 1) CAR-NK cells were prepared according to the above protocol. During the preparation of CAR-NK cells, the number of CAR-NK cells and MOCK cells (NK cells without CAR transduction) was counted on days 6, 8, 10, 13 and 15 respectively.

[0045] 2) Flow cytometry identification of co-expression of CAR and IL-10: (a) On day 10, take 1×10 5 CAR-NK cells and MOCK cells were prepared. The cells were washed twice by centrifugation with FACS Buffer and resuspended in 50 μL of FACS Buffer. (b) Surface Marker staining: APC anti-FMC63 (Bioswan, catalog number 300402) was added to a 50 μL system with antibody at a ratio of 1:500; PE-cy7 anti-hIL-10 (BioLegend, catalog number 501419) was added to a 50 μL system with antibody at a ratio of 1:200. Staining was carried out at 4°C in the dark for 20 minutes. (c) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution (Biogems, catalog number 61410-00-200) to each sample, and perform flow cytometry analysis.

[0046] 3) Collect cell culture supernatant: (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 cell was adjusted to be consistent with the total number of NK cells. (b) Adjusting cell density: 2 × 10⁻⁶ cells from CAR-NK and MOCK cells with consistent CAR% adjustment. 6 The cell density was adjusted to 1 × 10⁶ cells / mL. 6 10 cells / mL were inoculated into a 12-well plate and cultured for three days; (c) Collect cell culture supernatant: Take out the CAR-NK cells and MOCK cells cultured for three days and transfer them to a 15mL centrifuge tube. Centrifuge at 400g for 5 minutes to collect the cell supernatant. Place it in a 1.5mL EP tube. Label each supernatant with the name, CAR%, cell density, culture time, and collection date. Store at -80℃ for later use. (d) The cell culture supernatant stored at -80℃ was used to detect the specific secretion of IL-10 and IL-15 in the cell supernatant using an ELISA kit for IL-10 (Eco-Syne, catalog number EH006-96) and IL-15 (Eco-Syne, catalog number EH057-96).

[0047] 4) ELISA detection of IL-10 and IL-15 secretion: (a) Remove the IL-10 ELISA kit and the IL-15 ELISA kit from the refrigerator and allow them to equilibrate to room temperature; (b) Remove the required strips from the sealed bag that has been equilibrated to room temperature. Put the unused strips and desiccant back into the aluminum foil bag and seal it in a refrigerator at 2–8°C. (c) Preparation of standards and samples: After dissolving the cell culture supernatant stored at -80℃, prepare the original cell culture supernatant, 5-fold dilution and 10-fold dilution respectively. Prepare three replicates for each sample. Dissolve the standard protein IL-10 / IL-15 provided with the ELISA kit in Assay Diluent to a sample concentration of 1000 pg / mL. Let stand for 15 minutes, then perform a two-fold serial dilution to obtain samples of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL and 15.6 pg / mL. Prepare three replicates for each standard. (d) Add 100 μL of standard or sample of different concentrations to the corresponding wells, leaving blank wells as controls. Seal the reaction wells with sealing tape and incubate at 37°C for 90 minutes. (e) Prepare biotinylated antibody working solution: Dilute HumanIL-10 / IL-15 100X Biotin-Antibody 100 times with Assay Diluent according to the amount required for the current experiment to prepare biotinylated antibody working solution. Prepare 30 minutes before use and use only on the same day. (f) Dilute 20X Wash Buffer Concentrate with deionized water to prepare Wash Buffer, 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 at 37°C for 60 minutes. (g) Prepare enzyme conjugation working solution: Dilute 100XHRP-Streptavidin 100 times with Assay Diluent according to the amount required for the current experiment to prepare enzyme conjugation working solution. Prepare 30 minutes before use and use only on the same day. (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 at 37°C for 60 minutes. (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 incubator in the dark for 15 minutes. (j) Add Stop Solution, 100 μL / well, mix well, and immediately detect CD using a microplate reader. 450 value.

[0048] The results are as follows Figure 2 and Figure 3As shown, CAR and IL-10 are co-expressed on the surface of NK cells in dual-secreting CAR-NK cells; at the same time, ELISA results showed that 19CAR-IL10-IL15 secreted large amounts of IL-10 and IL-15, while 19CAR-IL15 only secreted IL-15 cytokine, indicating that the dual-secreting CAR-NK structure we constructed can normally express and secrete both cytokines without affecting CAR expression. Figure 4 The results showed that NK cells in umbilical cord blood could still proliferate in large quantities after transduction of CAR-NK lentivirus.

[0049] Example 3. In vitro functional verification of dual-secreting CAR-NK cells

[0050] We constructed two cell lines, Raji-CBR-Luc-GFP and JeKo-1-CBR-Luc-GFP, co-expressing luciferase CBR and green fluorescent protein GFP, using Raji lymphoma cells (purchased from ATCC) and JeKo-1 cells (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). CBR stands for click beetlered luciferase, which catalyzes the excitation of a red spectrum from a luciferin substrate; CBR and GFP were simultaneously introduced into Raji and JeKo-1 cells. These cell lines were used as target cells for short-term in vitro CAR-NK cell killing and degranulation experiments. Simultaneously, we constructed a cell line overexpressing human CD19 antigen, 3T3-CD19-Luc-GFP, using mouse fibroblast 3T3 cells (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). This cell line was used as target cells for long-term in vitro CAR-NK cell killing function verification.

[0051] 1. Luciferase-labeled cell killing assay: 1) Target cell plating: Collect Raji and JeKo-1 tumor cells in logarithmic growth phase, centrifuge, count, and adjust cell density to 1×10⁻⁶. 5 The sample was injected at a rate of 100 μL / well into a 96-well U-plate, i.e., 1 × 10⁻⁶ μL / well. 4 One target cell; 2) Adjusting CAR%: Collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells at the CAR-NK cell concentration required for the most effective target ratio (calculate cell density at 100 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. max Using Triton-100 and a negative control (K) min (using NK medium); 4) Gently mix the cell mixture in each well and incubate in an incubator for 4-6 hours. Preheat the Luciferin substrate. After washing 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. Use a microplate reader in chemiluminescence mode to read the data. 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0052] 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-to-target ratio conditions. However, the killing function of dual-secreting CAR-NK and single-secreting CAR-NK did not show significant difference in in vitro time.

[0053] 2. Long-term real-time in vitro killing experiment (RTCA experiment): 1) Take out the E-plate 16 (Agilent, catalog number 300600890), preheat the culture medium to 37°C, 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; 2) Target cell plating: Collect 3T3-CD19-Luc-GFP cells and adjust the cell density to 10,000 cells per well with a volume of 150 μL; 3) Add 150 μL of cell suspension to each well of the E-plate 16, 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; 4) When the OD value reaches approximately 1.0, centrifuge and wash the CAR-NK cells, resuspend them in preheated culture medium, count them, adjust the CAR% to be consistent, and adjust the CAR density to 1.25 × 10⁻⁶. 4 cells / mL; 5) Effector cell plating: Interrupt the RTCA experiment, remove the E-plate 16, discard the original culture medium, add 200 μL of CAR-NK cells of adjusted density to each well according to the group, and retain the negative control (only target cells); 6) Place the E-plate 16 back onto the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-NK cells against target cells.

[0054] The results are as follows Figure 6 As shown, CAR-NK can significantly kill 3T3 cells expressing CD19 antigen under a 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-secreting CAR-NK is slightly stronger than that of single-secreting CAR-NK.

[0055] 3. CD107a degranulation experiment: 1) Target cell plating: Collect Raji and JeKo-1 lymphoma cells in logarithmic growth phase, centrifuge, count, and plate at 1×10⁶ cells per well. 5 One cell was inoculated into a 96-well U-plate, 100 μL per well; 2) Adjusting CAR%: Collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: Adjust the cell density of each CAR-NK cell at an effector-to-target ratio of 1:1 and inoculate them into 96-well U-plates. Add Protein transport inhibitor (BD, catalog number 554724) at a ratio of 1:1500 of the total volume to each well, and add PE-cy7 anti-hCD107a antibody (BioLegend, catalog number 328618) at a ratio of 1:200. Mix with target cells and co-culture for 4-6 hours. Leave tumor-free NK cells in each group as controls. 4) Wash twice with FACS buffer according to the flow cytometry method, stain the surface with marker antibody, add APC anti-FMC63 antibody at a ratio of 1:500 in 100μL system, and stain at 4℃ for 20 minutes in the dark. 5) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution to each sample, and then perform flow cytometry analysis.

[0056] The results are as follows Figure 7 and Figure 8As shown, CAR-NK cells undergo significant CD107a degranulation after contact with target cells, with the degranulation rate of dual-secreting CAR-NK cells being slightly higher than that of single-secreting CAR-NK cells.

[0057] Example 4. Validation of the in vivo antitumor function of dual-secreting CAR-NK cells in a mouse xenograft model 1

[0058] JeKo-1-CBR-Luc-GFP cells, which were constructed with luciferase, were injected into severely immunodeficient mice (NSG) via the tail vein to prepare CAR-NK cells, which were then used to treat tumor-burdened mice (NSG) intravenously.

[0059] 1) Venous tumorigenesis of lymphoma cells: Collect logarithmic growth phase JeKo-1-CBR-Luc-GFP cells, centrifuge at 400g for 5 minutes and discard the supernatant, wash twice with serum-free PBS to ensure no serum residue; 2) Count the cells, resuspend them in pre-cooled PBS, and administer 1×10⁶ cells per NSG mouse. 4 One tumor cell, 300 μL / cell, was used to adjust the tumor cell density to 3.34 × 10⁻⁶. 4 1 / mL, ready for tail vein injection; 3) Tumor cells were injected into each mouse via the tail vein at a volume of 300 μL to establish the model. Two mice were reserved as blank controls without any treatment. 4) Small animal in vivo imaging was performed 48 hours later. Tumor formation was assessed based on the imaging results, and animals were randomly grouped as follows: (a) MOCK group: injected with untransduced CAR ordinary cord blood NK cells (5 animals); (b) 19CAR-IL15 group: umbilical cord blood NK cells transduced with single secretory CAR-NK (5 animals); (c) 19CAR-IL10-IL15 group: umbilical cord blood NK cells transduced with dual secretory CAR-NK (5 animals); (d) Blank group: blank control group, no treatment was given (2 animals); 5) NK cell tail vein therapy 72 hours later: (a) Collect NK cells in the logarithmic growth phase and detect CAR% by flow cytometry. Adjust the CAR% of 19CAR-IL15, 19CAR-IL10-IL15 and MOCK cells to be consistent, and ensure that the total number of NK cells is consistent. (b) Wash twice with pre-cooled PBS without serum to ensure no serum residue remains; (c) Counting: Adjust the CAR density to 1.667 × 10⁻⁶ using pre-chilled PBS without serum. 61 / mL, in preparation for tail vein treatment; (d) In accordance with the grouping in step 4), each mouse was injected with 300 μL of the corresponding MOCK and CAR-NK cells via the tail vein. (e) Animal in vivo imaging was performed twice a week during the first week of intravenous treatment, followed by weekly animal in vivo imaging to assess tumor progression. 6) Analyze fluorescence curves and survival curves based on tumor burden and mouse survival status.

[0060] The results are as follows Figure 9 , Figure 10 and Figure 11 As shown, with the increase of tumor burden time, the tumor burden in the MOCK group increased sharply, while the tumors in the 19CAR-IL15 group and the 19CAR-IL10-IL15 group were almost completely cleared without an increase in tumor burden. At the same time, the 19CAR-IL15 group was the first to show NSG mouse death, followed by a large number of deaths in the MOCK and 19CAR-IL15 groups. One mouse in the 19CAR-IL10-IL15 group died after one month ("X" indicates death). The fluorescence curve of NSG mice shows that both types of CAR-NK cells have significant effects in treating lymphoma in vivo, but the dual-secreting CAR-NK greatly improves the survival rate of NSG mice and improves the safety of CAR-NK cell therapy. The comprehensive evaluation shows that the dual-secreting CAR-NK has the best effect in treating tumors in vivo.

[0061] Example 5. Distribution of CAR-NK cells in mice 1

[0062] The NSG mice in Example 4 were dissected to analyze the distribution of CAR-NK cells in the animals.

[0063] 1) On day 49 of NSG treatment in Example 4, the surviving mice were brought back to the laboratory for dissection. All of the surviving mice in the 19CAR-IL15 group died. 2) Dissection and tissue collection: Peripheral blood was collected from animals in the Bank / MOCK / 19CAR-IL10-IL15 group. The animals were then euthanized by cervical dislocation. The spleen and bone marrow of the mice were obtained by dissection, and the corresponding cells were obtained by grinding and filtering. 3) Wash twice with FACS buffer according to the flow cytometry method, stain the surface with marker antibodies, add APC anti-FMC63 antibody at a ratio of 1:500 in 50μL system, add PE-cy7 anti-hCD56 (BioLegend, catalog number 304628) antibody at a ratio of 1:200 in 50μL system, add Pacific Blue anti-hCD45 (BioLegend, catalog number 304029) antibody at a ratio of 1:200 in 50μL system, stain at 4℃ in the dark for 20 minutes; 4) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution to each sample, and then perform flow cytometry analysis.

[0064] The results are as follows Figure 12 , Figure 13 and Figure 14 As shown, on day 49, a large number of 19CAR-IL10-IL15 cells were still present in the peripheral blood, spleen, and bone marrow of NSG mice, indicating that dual-secreting CAR-NK cells can survive in animals for at least 45 days and achieve long-term therapeutic effects.

[0065] Example 6. Validation of the in vivo antitumor function of dual-secreting CAR-NK cells in a mouse xenograft model 2

[0066] Raji-CBR-Luc-GFP cells, which were constructed with luciferase, were injected via the tail vein into severely immunodeficient mice (NSG) to prepare CAR-NK cells, which were then used to treat tumor-burdened mice (NSG) intravenously.

[0067] 1) Venous tumorigenesis of lymphoma cells: Collect Raji-CBR-Luc-GFP cells in logarithmic growth phase, centrifuge at 400g for 5 minutes and discard the supernatant, wash twice with serum-free PBS to ensure no serum residue; 2) Count the cells, resuspend them in pre-cooled PBS, and administer 8 × 10⁶ cells per NSG mouse. 4 One tumor cell, 100 μL / cell, with the tumor cell density adjusted to 8 × 10⁻⁶. 5 1 / mL, ready for tail vein injection; 3) Tumor cells were injected via tail vein into each mouse to model tumor cells; 4) Small animal in vivo imaging was performed 72 hours later. Tumor formation was assessed based on the imaging results, and animals were randomly grouped as follows: (a) 19CAR group: 4 animals were injected with umbilical cord blood NK cells transduced with CAR. (b) 19CAR-IL10 group: umbilical cord blood NK cells transduced with single secretory CAR-NK (4 animals). (c) 19CAR-IL15 group: umbilical cord blood NK cells transduced with single secretory CAR-NK cells were injected (4 animals). (d) 19CAR-IL10-IL15 group: umbilical cord blood NK cells transduced with dual secretory CAR-NK (4 animals). 5) NK cell tail vein therapy 96 hours later (a) Collect NK cells in the logarithmic growth phase and detect CAR% by flow cytometry. Adjust the CAR% of 19CAR, 19CAR-IL10, 19CAR-IL15 and 19CAR-IL10-IL15 and MOCK cells to be consistent, and ensure that the total number of NK cells is consistent. (b) Wash twice with pre-cooled PBS without serum to ensure no serum residue remains; (c) Counting: Adjust the CAR density to 2.4 × 10⁻⁶ using pre-cooled PBS without serum. 7 1 / mL, in preparation for tail vein treatment; (d) In accordance with the grouping in step 4), each mouse was injected with 100 μL of the corresponding MOCK and CAR-NK cells via the tail vein; (e) Animal in vivo imaging was performed twice a week during the first week of intravenous treatment, followed by weekly animal in vivo imaging to assess tumor progression. 6) Analyze fluorescence curves and survival curves based on tumor burden and mouse survival status.

[0068] The results are as follows Figure 15 , Figure 16 and Figure 17 As shown, with the increase of tumor burden time, the tumor burden in the 19CAR group increased sharply, while the tumor burden in the 19CAR-IL10, 19CAR-IL15, and 19CAR-IL10-IL15 groups decreased significantly. Meanwhile, the MOCK group showed the earliest NSG mouse death, followed by a large number of deaths in the 19CAR-IL10 and 19CAR-IL15 groups, with the 19CAR-IL10-IL15 group showing the latest death ("X" indicates death). The fluorescence curves of the NSG mice show that both CAR-NK cells are effective in treating lymphoma in vivo, but the dual-secreting CAR-NK significantly improved the survival rate of NSG mice and enhanced the safety of CAR-NK cell therapy. Overall, the dual-secreting CAR-NK showed the best efficacy in treating tumors in vivo.

[0069] Example 7. Distribution of CAR-NK cells in mice 2

[0070] The NSG mice in Example 6 were dissected to analyze the distribution of CAR-NK cells in the animals.

[0071] 1) On the 8th day of NSG treatment in Example 6, 100 μL of peripheral blood was randomly collected from 3 mice. 2) Wash twice with FACS buffer according to the flow cytometry method, stain the surface marker antibody, add Percep anti-mCD45 antibody at a ratio of 1:200 in 50μL system, add PE-cy7 anti-hCD56 (BioLegend, catalog number 304628) antibody at a ratio of 1:200 in 50μL system, add Pacific Blue anti-hCD45 (BioLegend, catalog number 304029) antibody at a ratio of 1:200 in 50μL system, add PE-anti-FMC63 antibody at a ratio of 1:200 in 50μL system, stain at 4℃ in the dark for 20 minutes; 3) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer and analyze them using a flow cytometer.

[0072] The results are as follows Figure 18 As shown, on day 8, the number of NK cells (19CAR-IL10-IL15) and CAR-NK cells in the peripheral blood of NSG mice were significantly higher than those in the control group, indicating better cell proliferation capacity.

[0073] Example 8. Enhanced immunocompatibility of CAR-NK in vitro

[0074] 1. NK cells infected with two different lentiviruses

[0075] 1.1 Flow cytometry analysis of the proportion of CD3 in NK

[0076] 1) Take 100 μl of cells, add 1 ml of PBS, centrifuge at 400 g for 5 min; 2) Discard the supernatant, reselect with 100 μl PBS, and add APC / Cy7 anti-human CD8 Antibody (biolegend, 300318) and PE / Cyanine7 anti-human CD56 (NCAM) Antibody (biolegend, 318318), and incubate at 4℃ for 20 min; 3) Add 1 ml of PBS, centrifuge at 400 g for 5 min; 4) Discard the supernatant, resuspend in 100 μl PBS, and perform flow cytometry analysis.

[0077] 1.2 NK Lentiviral Infection

[0078] 1) Adjust the cell density to 3e5 / 50μl, and add different lentiviruses (MOI=5) and protamine (final concentration 8pg / ml) to a 96-well plate, 100μl / well; 2) Centrifuge at 32℃, 1200g for 90 minutes. 3) After centrifugation, remove the 96-well plate and incubate it in a 37°C incubator for 4 hours; 4) Collect cells, 400g, centrifuge for 5 minutes; 5) Discard the supernatant, adjust the cell density to 1e6 / ml, and incubate overnight in an incubator.

[0079] 2. CAR-NK cells undergo a third nourishment process.

[0080] 2.1 Proportion of CAR detected by flow cytometry

[0081] 1) Take 100 μl of cells, add 1 ml of PBS, centrifuge at 400 g for 5 min; 2) Discard the supernatant, reselect with 100 μl PBS, and add Mouse Anti-Mouse FMC63 scFv Monoclonal Antibody (BioSwan, 300402), incubate at 4℃ for 20 min; 3) Add 2ml of PBS, centrifuge at 400g for 5 minutes; 4) Discard the supernatant, resuspend in 100 μl PBS, and perform flow cytometry analysis.

[0082] 2.2 CAR-NK Third Nourishment

[0083] 1) Collect cells and place them in a 15ml centrifuge tube. Centrifuge at 400g for 5 minutes. 2) Discard the supernatant, resuspend in 10ml PBS, take 100μl of cells for flow cytometry counting, and centrifuge the remaining cells at 400g for 5min; 3) Resuscitate trophoblast cells, 400g, centrifuge for 5 minutes; 4) Discard the supernatant, resuspend in 30ml PBS, take 100μl for flow cytometry counting, and centrifuge the remaining cells at 400g for 5min. 5) Culture the cells in 24-well plates at a CAR-NK:feeder cells ratio of 1:1 and incubate overnight at 37°C in a 5% CO2 cell culture incubator.

[0084] 3. Co-culture of CAR-NK cells and PBMCs

[0085] 3.1 PBMC CFSE staining

[0086] 1) Resuscitate PBMCs and count them using a flow cytometer (100 μl). 2) CFSE staining: Adjust the cell density of the PBMCs after counting to 1e7 / ml, add CFSE dye (Invitrogen, C34554) at a ratio of 1:1000, and incubate at 37℃ for 20 min. 3) Stop staining: Add 10 times the volume of complete culture medium to the cells and incubate at 37°C for 10 min; 4) Washing: Wash twice with complete culture medium, 400g, 5min; 5) Counting: Count the stained PBMCs and adjust the cell density to 2e5 / 100μl for subsequent experiments; 6) Antibody staining: Incubate PBMC-CFSE with APC anti-human CD3 Antibody (Biolegend, 300312) and APC / Cy7 anti-human CD8 Antibody (Biolegend, 344714) at 37°C for 20 min. 7) Add 2ml of PBS, centrifuge at 400g for 5 minutes; 8) Discard the supernatant, resuspend in 100 μl PBS, and perform flow cytometry analysis.

[0087] 3.2 Co-culture of 19CAR-NK with stained PBMCs

[0088] 1) Adjusting the CAR ratio: Flow cytometry was used to detect the expression of CAR in two types of 19CAR-NK cells, and NK cells were used to adjust the expression of CAR in different CAR-NK cells to be consistent. 2) Two types of 19CAR-NK cells were co-cultured with PBMC-CFSE (E:T=4:1) in 24-well plates; 3) A negative control group was also set up. 4) Incubate in a 37℃ incubator for 7 days.

[0089] 3.3 Flow cytometry detection of CD8+ co-cultured with 19CAR-NK + T cell proliferation

[0090] 1) On day 7, collect all cells, 400g, and centrifuge for 5 minutes; 2) Discard the supernatant, resuspend in PBS, and use 100 μl for flow cytometry counting; 3) Take 100 μl of cells and incubate them with APC anti-human CD3 Antibody (Biolegend, 300312) and APC / Cy7 anti-human CD8 Antibody (Biolegend, 344714) at 4℃ for 20 min; 4) Add 2 ml of PBS, centrifuge at 400 g for 5 min; 5) Discard the supernatant, resuspend in 100 μl PBS, and perform flow cytometry analysis.

[0091] like Figure 19 As shown, compared with the negative control, 19CAR-NK-IL-15 promotes CD8 + T cell proliferation is somewhat enhanced. However, the 19CAR-NK-IL-10-IL-15 pathway, which secretes IL-10 and IL-15, does not significantly promote CD8 cell proliferation. + T proliferation was similar to that of the negative control. This demonstrates the safety of 19CAR-NK-IL-10-IL-15, which essentially does not induce host resistance to grafts.

[0092] Example 9. Dual-secreting CAR-NK showed better persistence in patients.

[0093] Starting in July 2022, the Department of Hematology at the Second Affiliated Hospital of Zhejiang University School of Medicine initiated an investigator-initiated clinical trial of 19CAR-IL15 for the treatment of relapsed and refractory B-cell lymphoma (Clinical Trial Registration No. NCT05472558). Starting in December 2024, another investigator-initiated clinical trial of 19CAR-IL10-IL15 for the treatment of relapsed and refractory B-cell lymphoma was launched (Clinical Trial Registration No. NCT06707259). The starting dose for both trials was 2x10-10. 6 / kg (cell count / body weight).

[0094] Flow cytometry analysis of CAR-NK cells in each patient: 1) On the 7th day after treatment, approximately 1 mL of peripheral blood was collected from the anticoagulant tube; 2) Add 400g of isodense centrifugal liquid and centrifuge for 10 minutes; 3) Obtain PBMCs, add PE-cy7 anti-hCD56 (BioLegend, catalog number 304628), Pacific Blue anti-hCD45 (BioLegend, catalog number 304029), APC-anti-FMC63 CAR (BioSwan, catalog number 300402), and FITC anti-hCD3 (Biolegend, catalog number 981002), add antibodies, and stain at 4°C in the dark for 20 minutes. 4) Add 2 ml of PBS, centrifuge at 400 g for 5 min; 5) Discard the supernatant, resuspend in 100 μl PBS, and perform flow cytometry analysis.

[0095] like Figure 20 As shown, Pt1-Pt3 patients used 2x10 6 The 19CAR-IL15 group secreted IL15 at a rate of / kg (cells / body weight), while Pt4-Pt6 used the 19CAR-NK-IL-10-IL-15 group, which secretes both IL-10 and IL-15. After day 7 of treatment, the 19CAR-NK-IL-10-IL-15 treatment group showed significantly higher peripheral blood CAR-NK cell counts, demonstrating better durability.

[0096] Example 10. In vitro functional validation of CLDN18.2 dual-secreting CAR-NK cells

[0097] A cell line co-expressing luciferase CBR (clickbeetle red luciferase, which catalyzes the excitation of a red spectrum by luciferin substrates) was constructed using human gastric adenocarcinoma cells AGS (purchased from the Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). This cell line was used as target cells for short-term in vitro CAR-NK cell killing experiments. 1. Luciferase-labeled cell killing experiment: 1) Target cell plating: Collect AGS tumor cells in logarithmic growth phase, centrifuge, count, and adjust cell density to 2×10⁻⁶. 5 / mL, add 50μL / well to the 96-well U-type substrate, i.e., 1×10⁶ μL / well. 4 One target cell; 2) Adjusting CAR%: Collect CLDN18.2CAR-IL15 / CLDN18.2CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells at the CAR-NK cell concentration required for the most effective target ratio (calculate cell density at 50 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. max Using Triton-100 and a negative control (K) min (using NK medium); 4) Gently mix the cell mixture in each well, incubate in an incubator for 4 hours, preheat the Luciferin substrate, 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; read the data using a microplate reader in chemiluminescence mode. 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0098] The results are as follows Figure 21 As shown, compared with MOCK, CLDN18.2CAR-IL15 and CLDN18.2CAR-IL10-IL15 CAR-NK cells both showed significantly enhanced killing function against AGS cells expressing CLDN18.2 antigen under different effector-to-target ratio conditions. However, there was no significant difference in killing function between dual-secreting CAR-NK and single-secreting CAR-NK cells in vitro over a short period of time.

[0099] 2. CD107a degranulation experiment: 1) Target cell plating: Collect AGS lymphoma cells in the logarithmic growth phase, centrifuge, count, and divide into groups of 1×10⁶ cells per well. 5 50 μL of cells were inoculated into a 96-well U-plate; 2) Adjusting CAR%: Collect CLDN18.2CAR-IL15 / CLDN18.2CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: The cell density of each CAR-NK cell was adjusted to a target-effect ratio of 1:1 and the cells were inoculated into 96-well U plates. Protein transport inhibitor (BD, catalog number 554724) was added to each well at a ratio of 1:1500 of the total volume, and PE-Cy7 anti-hCD107a antibody (BioLegend, catalog number 328618) was added at a ratio of 1:200. The cells were mixed with the target cells and co-cultured for 4 hours. Tumor-free NK cells were retained as controls in each group. 4) Wash twice with FACS buffer according to the flow cytometry method, stain the surface with marker antibody, add APC anti-NGFR antibody at a ratio of 1:500 in 100μL system, and stain at 4℃ for 20 minutes in the dark. 5) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution to each sample, and then perform flow cytometry analysis.

[0100] The results are as follows Figure 22 As shown, after CAR-NK cells come into contact with target cells, the CD107a degranulation response is significantly enhanced, and the degranulation of dual-secreting CAR-NK cells is slightly higher than that of single-secreting CAR-NK cells.

[0101] 3. Long-term real-time in vitro killing experiment (RTCA experiment): 1) Take out the E-plate 16 (Agilent, catalog number 300600890), preheat the culture medium to 37°C, 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; 2) Target cell plating: Collect AGS-CLDN18.2-Luc-GFP cells and adjust the cell density to 10,000 cells per well with a volume of 150 μL; 3) Add 150 μL of cell suspension to each well of the E-plate 16, 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; 4) When the OD value reaches approximately 2.0, centrifuge and wash the CAR-NK cells, resuspend them in preheated culture medium, count them, adjust the CAR% to be consistent, and adjust the CAR density to 1.25 × 10⁻⁶. 4 / mL; 5) Effector cell plating: Interrupt the RTCA experiment, remove the E-plate 16, discard the original culture medium, add 200 μL of CAR-NK cells of adjusted density to each well according to the group, and retain the negative control (only target cells); 6) Place the E-plate 16 back onto the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-NK cells against target cells.

[0102] The results are as follows Figure 23 As shown, CAR-NK can significantly kill AGS cells expressing CLDN18.2 antigen under a 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-secreting CAR-NK is significantly stronger than that of single-secreting CAR-NK.

[0103] Example 11. In vitro functional validation of BCMA dual-secreting CAR-NK cells

[0104] A cell line co-expressing luciferase CBR (Crec. beetle red luciferase, which catalyzes the excitation of red light from luciferin substrates) was constructed using human multiple myeloma cells MM.1S (purchased from the Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). CBR and GFP were simultaneously introduced into MM.1S cells. This cell line was used as target cells for short-term in vitro CAR-NK cell killing experiments.

[0105] 1. Luciferase-labeled cell killing assay: 1) Target cell plating: Collect MM.1S tumor cells in the logarithmic growth phase, centrifuge, count, and adjust the cell density to 2×10⁻⁶. 5 / mL, add 50μL / well to the 96-well U-type substrate, i.e., 1×10⁶ μL / well. 4 One target cell; 2) Adjusting CAR%: Collect BCMA-CAR-IL15 / BCMA-CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells at the CAR-NK cell concentration required for the most effective target ratio (calculate cell density at 50 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. max Using Triton-100 and a negative control (K) min (using NK medium); 4) Gently mix the cell mixture in each well, incubate in an incubator for 4 hours, preheat the Luciferin substrate, 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; read the data K using a microplate reader in chemiluminescence mode. 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0106] The results are as follows Figure 24As shown, compared with MOCK, BCMA-CAR-IL15 and BCMA-CAR-IL10-IL15 CAR-NK cells both showed significantly enhanced killing function against MM.1S cells expressing BCMA antigen under different effector-to-target ratio conditions, and the killing ability of dual-secreting CAR-NK cells was stronger than that of single-secreting CAR-NK cells.

[0107] 2. CD107a degranulation experiment: 1) Target cell plating: Collect logarithmic growth phase myeloma cells MM.1S, centrifuge, count, and plate at 1×10⁶ cells per well. 5 50 μL of cells were inoculated into a 96-well U-plate; 2) Adjusting CAR%: Collect BCMA-CAR-IL15 / BCMA-CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-NK cell by flow cytometry, resuspend the CAR-NK cells in preheated NK medium, and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells. 3) Effector cell plating: The cell density of each CAR-NK cell was adjusted to a target-effect ratio of 1:1 and the cells were inoculated into 96-well U plates. Protein transport inhibitor (BD, catalog number 554724) was added to each well at a ratio of 1:1500 of the total volume, and PE-Cy7 anti-hCD107a antibody (BioLegend, catalog number 328618) was added at a ratio of 1:200. The cells were mixed with the target cells and co-cultured for 4 hours. Tumor-free NK cells were retained as controls in each group. 4) Wash twice with FACS buffer according to the flow cytometry method, stain the surface with marker antibody, add APC anti-NGFR antibody at a ratio of 1:500 in 100μL system, and stain at 4℃ for 20 minutes in the dark. 5) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution to each sample, and then perform flow cytometry analysis.

[0108] The results are as follows Figure 25 As shown, after CAR-NK cells come into contact with target cells, the CD107a degranulation response is significantly enhanced, and the degranulation of dual-secreting CAR-NK cells is slightly higher than that of single-secreting CAR-NK cells.

[0109] Example 12. Preparation of CAR-T cells

[0110] Based on the scFv sequence of the FMC63 monoclonal antibody targeting CD19, a lentiviral vector with a CAR structure using 4-1BB as a co-stimulatory factor and the CD3zeta activation domain was designed and constructed, and IL-15 and IL-10 were co-expressed using the Furin-2A short peptide. The resulting CD19-targeting dual-secreting CAR-T was 19CAR-IL10-IL15, and the single-secreting CAR-T was 19CAR-IL15. The specific steps for CAR-T preparation are shown below: 1. Acquisition and cryopreservation of human peripheral blood mononuclear cells The specific steps for isolating mononuclear cells (PBMCs) from peripheral blood are as follows: 1) Prepare peripheral blood from healthy individuals: Draw 10-15 mL of peripheral blood from healthy individuals into a blood collection tube (BD, catalog number 365974), shake well, transport at low temperature to the laboratory, disinfect the surface, remove it from the blood collection tube with a 20 mL syringe, transfer it to a sealed 50 mL centrifuge tube, and measure the volume of peripheral blood at the same time. 2) Human peripheral blood dilution: Peripheral blood was diluted with an equal volume of PBS (Gino, catalog number GMN20012-5); 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, 800 g, 30 minutes, breakoff (increase 1, decrease 0); 4) Obtaining mononuclear cells: After centrifugation, a clear white membrane layer of cells can be seen. Discard the plasma and transfer the white membrane layer cells to a new centrifuge tube. Add sterile flow cytometry washing buffer (PBS containing 2% fetal bovine serum, i.e., 2% FBS+PBS, hereinafter referred to as FASC Buffer). 5) Wash cells with FASC Buffer: 300g, 10 minutes, wash twice, take a small amount of cells for counting; 6) Cell identification: A small number of cells from step 5) were used for flow cytometry to detect the expression of CD3 / CD45; 7) Cell cryopreservation: The obtained mononuclear cells were cryopreserved, and the number of cryopreserved cells was calculated as total cell count × CD45%; CryoStor CS5 (STEMCELL, catalog number 7933) cryopreservation solution was used, at a ratio of 2 × 10⁻⁶ cells / year. 6 Cells were resuspended at a density of 2 × 10⁶ mL. 6 One cell was cryopreserved per unit, and the obtained mononuclear cells were cryopreserved in a -80℃ / liquid nitrogen tank.

[0111] 2. Preparation of lentiviral vectors

[0112] The specific steps for preparing a lentiviral vector for infecting T cells to express CAR molecules are as follows: 1) Transfect 293T cells when the confluence is 80-90%. Change the culture medium (10% FBS Opti-MEM) for packaging the virus 2 hours before transfection. 2) Preparation of lentivirus using a four-plasmid packaging system: This system includes three helper plasmids and a target plasmid (19CAR-IL15 and 19CAR-IL10-IL15), and a transfection reagent mixture is prepared by adding transfection reagents such as Lipo2000. 3) After mixing, incubate at room temperature for 15-25 minutes, then add to 293T cells along the side wall; 4) Collect viral supernatant: Collect viral supernatant after 48 and 72 hours respectively; 5) After filtering cell debris through a 0.45 μm pore size filter membrane, the supernatant was concentrated using ultracentrifugation; 6) Lentiviral titers were detected using the NK-92MI cell line, and the functional titers were found to be between 0.5 and 1 × 10⁻⁶. 8 Within the TU / mL range, lentiviruses that can effectively transfect cells can be obtained for subsequent experiments.

[0113] 3. Preparation of CAR-T cells

[0114] 1) On day 0, resuscitate and count human mononuclear cells; 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 at a 1:1 ratio in MOCKS culture medium. TM AIMV TM SFM (Gibco, catalog number 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 number 25030-081) + 1% HEPES (1 M) (Gibco, catalog number 15630080) + 1% MEM non-essential amino acid solution (100×) (Gibco, catalog number 11140050) + 1% sodium pyruvate (100 mM) (Gibco, catalog number 11360-070); 3) Lentiviral transduction on day 2: The cell count is calculated based on the total count of the previous day. The amount of lentivirus added is calculated according to the following formula: (cell count × MOI) / virus titer, where MOI is 5. At the same time, polybrene (Beyotime, catalog number C0351-50mg) is added as a transduction aid to a final concentration of 8μg / mL. 4) Add lentivirus and conversion aid to T cells as described in step 3) above, mix well, and add 1–3 × 10⁶ cells per well. 5 Add 200 μL of the system to a 96-well plate, and centrifuge the plate at 1200 g at 37 ° C for 90 minutes. 5) After centrifugation, place the cells in an incubator and incubate for 4 hours. Then change the cell culture medium, discard the virus solution, and replace it with fresh T cell culture medium to continue culturing. 6) Day 4, remove the activated antibody beads: After sterilizing the surface of the DynaMag-15 magnetic rack, place it in a clean bench for later use; transfer the CAR-T cell and antibody bead mixture to a 15mL centrifuge tube, repeatedly pipette to mix thoroughly to separate the cells from the antibodies, and place it on the DynaMag-15 magnetic rack. Let it stand for 1-3 minutes, and then gently remove the cell suspension on the magnetic rack and transfer it to a new 15mL centrifuge tube, being careful not to touch the centrifuge tube wall when removing the cell suspension; after centrifuging the cell suspension with the antibody beads removed, continue culturing, maintaining a cell density of 2×10⁻⁶ cells / mL. 6 / mL; 7) On days 12-16, proliferated CAR-T cells were obtained for subsequent functional studies.

[0115] Example 13. Identification of Cytokine Secretion in Dual-Secretory CAR-T Cells

[0116] CAR-T cells were prepared according to Example 1. CAR-T cells were cultured for 13 days and CAR expression was detected by flow cytometry. The CAR%, total number of cells and cell density were adjusted to be consistent. After three days of culture, the cell culture supernatant was collected and the secretion of IL-10 and IL-15 was detected by ELISA.

[0117] 1) CAR-T cells were prepared according to the above protocol. During the preparation of CAR-T cells, the number of CAR-T and MOCK cells was counted on days 10, 13, 15, 17 and 20 respectively. 2) Flow cytometry detection of CAR expression (a) On day 13, take 1×10 5 CAR-T cells and MOCK cells were prepared. The cells were washed twice by centrifugation with FACS Buffer and resuspended in 50 μL of FACS Buffer. (b) Surface Marker staining: APC anti-FMC63 (Bioswan, catalog number 300402) was added to a 50 μL system with antibody at a ratio of 1:500, and stained at 4°C in the dark for 20 minutes. (c) After washing twice with FACS buffer, resuspend the cells in 200 μL of FACS buffer, add 1 μL of the live / dead dye 7AAD-Solution (Biogems, catalog number 61410-00-200) to each sample, and perform flow cytometry analysis.

[0118] 3) Collect cell culture supernatant

[0119] (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 cell was adjusted to be consistent with the total number of T cells. (b) Adjusting cell density: 2 × 10⁻⁶ cells from CAR-T and T cells with consistent CAR% adjustment. 6 The cells were adjusted to a density of 1×10⁻⁶. 6 / mL, inoculated into 12-well plates and cultured for three days. (c) Collect cell culture supernatant: Take out the CAR-T cells and MOCK cells cultured for three days and transfer them to a 15mL centrifuge tube. Centrifuge at 400g for 5 minutes to collect the cell supernatant. Place it in a 1.5mL EP tube. Label each supernatant with the name, CAR%, cell density, culture time, and collection date. Store at -80℃ for later use. (d) The cell culture supernatant stored at -80℃ was used to detect the specific secretion of IL-1v and IL-15 in the cell supernatant using an ELISA kit for IL-10 (Eco-Syne, catalog number EH006-96) and IL-15 (Eco-Syne, catalog number EH057-96).

[0120] 4) ELISA detection of IL-10 and IL-15 secretion

[0121] (a) Remove the IL-10 and IL-15 ELISA kits from the refrigerator and allow them to equilibrate to room temperature; (b) Remove the required strips from the sealed bag that has been equilibrated to room temperature. Put the unused strips and desiccant back into the aluminum foil bag and seal it in a refrigerator at 2-8°C. (c) Preparation of standards and samples: After dissolving the cell culture supernatant stored at -80℃, prepare the original cell culture supernatant, 25-fold dilution and 50-fold dilution respectively. Prepare three replicates for each sample. Dissolve the standard protein IL-10 / IL-15 provided with the ELISA kit in Assay Diluent to a sample concentration of 1000 pg / mL. Let stand for 15 minutes, then perform a two-fold serial dilution to obtain samples of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL and 15.6 pg / mL. Prepare three replicates for each standard. (d) Add 100 μL of standard or sample of different concentrations to the corresponding wells, leaving blank wells as controls. Seal the reaction wells with sealing tape and incubate at 37°C for 90 minutes. (e) Prepare biotinylated antibody working solution: Dilute HumanIL-10 / IL-15 100X Biotin-Antibody 100 times with Assay Diluent according to the amount required for the current experiment to prepare biotinylated antibody working solution. Prepare 30 minutes before use and use only on the same day. (f) Dilute 20X Wash Buffer Concentrate with deionized water to prepare 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 well with sealing tape, and incubate at 37°C for 60 minutes. (g) Prepare enzyme conjugation working solution: Dilute 100XHRP-Streptavidin 100 times with Assay Diluent according to the amount required for the current experiment to prepare enzyme conjugation working solution. Prepare 30 minutes before use and use only on the same day. (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 at 37°C for 60 minutes. (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 incubator in the dark for 15 minutes. (j) Add Stop Solution, 100 μL / well, mix well, and immediately detect CD using a microplate reader. 450 value The results are as follows Figure 26As shown, 19CAR-IL10-IL15 secretes a large amount of IL-10 and IL-15, while 19CAR-IL15 only secretes IL-15 cytokine. The IL-10 secretion of dual-secreting CAR-T cells can reach more than 3 ng / mL.

[0122] Example 14. In vitro functional verification of dual-secreting CAR-T cells

[0123] We constructed Raji-CBR-GFP and JeKo-1-CBR-GFP cell lines co-expressing luciferase CBR and green fluorescent protein GFP using Raji lymphoma cells (purchased from ATCC) and JeKo-1 cells (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). (CBR stands for click beetle redluciferase, which catalyzes the excitation of red light from luciferin substrates; CBR and GFP were simultaneously introduced into Raji and JeKo-1 cells). These cell lines were used as target cells for in vitro CAR-T cell killing, degranulation, and IFN-γ / TNF-α cytokine secretion experiments. Simultaneously, we constructed a cell line overexpressing human CD19 antigen, 3T3-CD19, using mouse fibroblast 3T3 cells (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). This cell line was used as target cells for long-term, real-time in vitro CAR-T cell killing function monitoring.

[0124] 1. Luciferase-labeled cell killing assay

[0125] 1) Target cell plating: Collect Raji and JeKo-1 tumor cells in logarithmic growth phase, centrifuge, count, and adjust cell density to 1×10⁻⁶. 5 / mL, add 100μL / well to the 96-well U-type substrate, i.e., 1×10⁶ μL / well. 4 One target cell; 2) Adjusting CAR%: Collect 19CAR-IL15 / 19CAR-IL10-IL15 / MOCK cells, detect the CAR% of each CAR-T cell by flow cytometry, resuspend the CAR-T cells in the preheated T cell culture medium, and adjust the CAR% of each CAR-T cell 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 at the concentration required for the most effective target ratio (calculate cell density at 100 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. max Using Triton-100 and a negative control (K) min (using T cell culture medium) 4) Gently mix the cell mixture in each well and incubate in an incubator for 4-6 hours. Preheat the Luciferin substrate. After washing 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. Use a microplate reader in chemiluminescence mode to read the data. 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0126] The results are as follows Figure 27 As shown, compared to MOCK, CAR-T 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-to-target ratios. At the same time, the dual-secreting CAR-T cells showed stronger killing function in vitro than the single-secreting CAR-T cells, with significant differences. 2. Long-term real-time in vitro killing experiment (RTCA experiment)

[0127] 1) Take out the E-plate 16 (Agilent, catalog number 300600890), preheat the culture medium to 37°C, 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; 2) Target cell plating: Collect 3T3-CD19 cells and adjust the cell density to 10,000 cells per well with a volume of 150 μL; 3) Add 150 μL of cell suspension to each well of the E-plate 16, 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; 4) When the OD value reaches approximately 1.0, centrifuge and wash the CAR-T cells, resuspend them in preheated T cell culture medium, count them, adjust the CAR% to be consistent, and adjust the CAR density to 1.667 × 10⁻⁶. 4 / mL; 5) Effector cell plating: Interrupt the RTCA experiment, remove the E-plate 16, discard the original culture medium, add 200 μL of CAR-T cells of adjusted density to each well according to the group, and retain the negative control (only target cells); 6) Place the E-plate 16 back onto the RTCA instrument and continue to detect the OD value to evaluate the specific killing function of CAR-T cells against target cells.

[0128] The results are as follows Figure 28As shown, CAR-T cells can significantly kill 3T3 cells expressing CD19 antigen under a 1:3 effector-to-target ratio. At the same time, it can be seen that in long-term real-time killing, the killing function of dual-secreting CAR-T cells is stronger than that of single-secreting CAR-T cells, which can rapidly kill target cells in a short time and the killing advantage persists for a long time.

[0129] Example 15. Validation of the in vivo antitumor function of dual-secreting CAR-T cells using a mouse xenograft model.

[0130] JeKo-1-CBR-GFP cells, which were constructed with luciferase, were injected via the tail vein into severely immunodeficient mice (NSG) to prepare CAR-T cells, which were then used to treat tumor-burdened mice (NSG) intravenously.

[0131] 1) Venous tumorigenesis of lymphoma cells

[0132] (a) Cell collection: Collect JeKo-1-CBR-GFP cells in the logarithmic growth phase, 400g, 5 minutes, centrifuge and discard the supernatant, wash twice with serum-free PBS to ensure no serum residue; (b) Adjusting cell density: Resuspend cells in pre-cooled PBS and count them, administering 1 × 10⁶ cells per NSG mouse. 4 One tumor cell, 300 μL / cell, was used to adjust the tumor cell density to 3.34 × 10⁻⁶. 4 / mL, ready for tail vein injection; (c) Tumor cells were injected into each mouse via the tail vein at a volume of 300 μL to establish the model. Two mice were reserved as blank controls without any treatment. 2) Animal in vivo imaging grouping: Small animals were in vivo imaged 48 hours after venous tumor formation. The tumor formation was assessed based on the imaging results, and the animals were randomly grouped as follows: (a) MOCK group: injected with untransduced CAR plain T cells (6 animals); (b) 19CAR-IL15 group: injected with single secretory CAR-T cells (6 animals); (c) 19CAR-IL10-IL15 group: injected with dual-secreting CAR-T cells (6 animals); (d) Blank group: blank control group, no treatment was given (2 animals); 3) CAR-T cell tail vein therapy 72 hours after venous tumor formation. (a) Collect CAR-T cells in the logarithmic growth phase and detect CAR% by flow cytometry. Adjust the CAR% of 19CAR-IL15, 19CAR-IL10-IL15 and MOCK cells to be consistent, and ensure that the total number of T cells is consistent. (b) Wash twice with pre-cooled PBS without serum to ensure no serum residue remains; (c) Resuspend CAR-T cells in pre-chilled PBS without serum and count them, adjusting the CAR density to 3.34 × 10⁻⁶. 5 / mL, prepare for tail vein treatment; (d) In accordance with the grouping in step 2), each mouse was injected with the corresponding MOCK and CAR-T cells via the tail vein at a volume of 300 μL. (e) Animal in vivo imaging was performed twice a week during the first week of intravenous treatment, followed by weekly animal in vivo imaging to assess tumor progression. 4) Analyze fluorescence curves and survival curves based on tumor burden and mouse survival status.

[0133] The results are as follows Figure 29 As shown, the tumor burden in the MOCK group increased sharply with the increase of tumor burden time; in the early stage, the tumor burden in the 19CAR-IL15 group and 19CAR-IL10-IL15 group increased for a short period of time and was almost completely cleared, with no significant increase in tumor burden; after one week of treatment, the tumor burden in the single-secreting CAR-T cell group was significantly enhanced, while the tumor burden in the dual-secreting CAR-T group did not increase, and both groups showed the same degree of animal mortality; the fluorescence curve of NSG mice showed that the therapeutic effect of dual-secreting CAR-T was significantly stronger than that of single-secreting CAR-T cells.

[0134] Part of the sequence in this article

[0135] The amino acid sequence of the 19CAR-IL10-IL15 CAR molecule (SEQ ID NO.1):

[0136] in, Signal peptide sequence (SEQ ID NO.2): MALPVTALLLPLALLLHAARP Anti-CD19 scFv sequence (SEQ ID NO.3):

[0137] 4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO.4):

[0138] IL-10 sequence (SEQ ID NO.5):

[0139] IL-15 sequence (SEQ ID NO.6):

[0140] DNA sequence of the 19CAR-IL10-IL15 CAR molecule (SEQ ID NO.7):

[0141] in, Signal peptide sequence (SEQ ID NO.8):

[0142] Anti-CD19 scFv sequence (SEQ ID NO.9):

[0143] 4-1BB-CD3z intracellular activation domain sequence (SEQ ID NO.10):

[0144] IL-10 sequence (SEQ ID NO.11):

[0145] IL-15 sequence (SEQ ID NO.12):

[0146] Targeting the CLDN18.2 CAR-binding domain amino acid sequence (SEQ ID NO.13):

[0147] DNA sequence corresponding to the amino acid sequence of the CLDN18.2 CAR-binding domain (SEQ ID NO.14):

[0148] Targeting the BCMACAR binding domain amino acid sequence (SEQ ID NO.15):

[0149] DNA sequence corresponding to the amino acid sequence targeting the BCMACAR binding domain (SEQ ID NO.16):

[0150] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Although the present invention 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 substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant construct, characterized in that, Encoding IL-10, IL-15, and CAR, fragments or variants thereof, wherein the antigen of said CAR includes 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. Preferably, The amino acid sequence of IL-10 is shown in SEQ ID NO.5, or a fragment thereof or a variant thereof; The amino acid sequence of IL-15 is shown in SEQ ID NO.6, or a fragment thereof or a variant thereof; The amino acid sequence targeting CD19 in CAR is the monoclonal antibody scFv sequence shown in Genebank: HM852952.1; The amino acid sequence targeting the CLDN18.2 region in the CAR is shown in SEQ ID NO.13; The amino acid sequence targeting the BCMA region in the CAR is shown in SEQ ID NO.

15.

2. The 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 as shown in SEQ ID NO.7, its fragment or variant, or 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, its fragment or variant.

3. The recombinant construct according to claim 1, characterized in that, The CAR antigen is CLDN18.

2. The CLDN18.2 targeting nucleic acid sequence in the recombinant construct is shown in SEQ ID NO.14, or a fragment or variant thereof. The IL-10 nucleic acid sequence in the recombinant construct is shown in SEQ ID NO.11, or a fragment or variant thereof. The IL-15 nucleic acid sequence in the recombinant construct is shown in SEQ ID NO.12, or a fragment or variant thereof. The CAR antigen is CLDN18.

2. The amino acid sequence targeting the CLDN18.2 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.

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

5. A CAR-modified cell, characterized in that, Includes the recombinant constructs as described in any one of claims 1 to 4.

6. The CAR-modified cells according to claim 5, characterized in that, The CAR-modified cells can be either CAR-NK cells or CAR-T cells.

7. The CAR-modified cells according to claim 5, characterized in that, The CAR-modified cells are autologous, allogeneic, or xenologous.

8. A pharmaceutical composition, characterized in that, include: The recombinant construct according to any one of claims 1 to 4 or the CAR-modified cell according to any one of claims 5 to 7, and At least one pharmaceutically acceptable carrier.

9. Use of a recombinant construct according to any one of claims 1 to 4, or a CAR-modified cell according to any one of claims 5 to 7, or a pharmaceutical composition according to claim 8, in the preparation of a kit for the diagnosis / treatment of malignant tumors or autoimmune diseases. Preferably, the malignant tumor is any one of gastric adenocarcinoma, B-cell malignant tumor, or myeloma.

10. The use of a recombinant construct according to any one of claims 1 to 4, or a CAR-modified cell according to any one of claims 5 to 7, or a pharmaceutical composition according to claim 8, in the preparation of a drug for malignant tumors or autoimmune diseases. Preferably, the malignant tumor is any one of gastric adenocarcinoma, B-cell malignant tumor, or myeloma.

Images