CAR-T cell targeting NKG2DL and secreting IL-15 and preparation method thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a CAR-T cell targeting NKG2DL and secreting IL-15 and a preparation method thereof, the method comprises the step of infecting a T cell with a recombinant lentivirus, and the recombinant lentivirus contains a nucleic acid molecule of a chimeric antigen receptor encoding NKG2D and IL-15 or is prepared through an expression vector containing the nucleic acid molecule. The CAR-T cell expresses a chimeric antigen receptor targeting NKG2DL and can secrete a cell factor IL-15, the proliferation and effector functions of the CAR-T cell are promoted, and the killing activity on tumors is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a CAR-T cell targeting NKG2DL and secreting IL-15, and a preparation method and application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy, which belongs to a type of immunotherapy, is mainly used for the clinical treatment of malignant blood diseases and malignant tumors. This therapy expresses one or more specific CARs on T cells through genetic engineering technology, enabling them to target and eliminate tumors. Clinical results show that CAR-T cells have great advantages in the treatment of hematological malignancies, but there are still limitations such as tumor recurrence and poor treatment effects in solid tumors. One of the reasons for this limitation is that the expansion ability and self-persistence of CAR-T cells are limited, thus affecting their long-term anti-tumor activity.

[0003] NKG2D is a C-type lectin-like receptor molecule composed of homodimers, mainly expressed on the surface of natural killer (NK) cells and CD8 + T cells. NKG2D plays an important role in innate immunity and can participate in the recognition of virus-infected cells and the killing of tumor cells by NK cells. The biological function of NKG2D is closely related to its ligand, namely NKG2DL: NKG2DL can play an immune surveillance role, and the recognition of NKG2DL by NKG2D can mediate the tumor-killing effect of immune cells (Lerner EC et al, 2023). Research shows that NKG2DL is expressed in a variety of cancer cell lines and primary tumors such as cervical cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lymphoma, etc. (Jin Xin et al, 2023). Therefore, NKG2DL-CAR-T is expected to become an effective means for treating these tumors. Currently, the in-research drugs and therapies targeting NKG2DL mainly focus on the early clinical stage. Consistent with in vitro studies and animal models, the maintenance time of CAR-T cells targeting NKG2DL, namely NKG2DL-CAR-T cells, is short, which limits the tumor treatment effect.

[0004] In addition, NKG2DL-CAR-T cell therapy is expected to become an effective and selective anti-aging therapy for treating aging and age-related diseases driven by aging. It has been found that the expression of NKG2DL is upregulated in senescent cells cultured in vitro (Deng Yushuang, 2024), and also in various tissues of senescent mice and non-human primates. NKG2DL-CAR-T cell therapy can selectively target and eliminate senescent cells with high NKG2DL expression in vitro, in mice, and in non-human primates, with no significant effect on normal cells (Dong Yang et al, 2023).

[0005] CAR-T therapy has shown great potential in tumor treatment. However, the efficacy of CAR-T therapy is affected by many factors, such as antigen heterogeneity, antigen loss, limited cell persistence, poor infiltration ability, and inhibition of the tumor microenvironment. To improve the effectiveness of CAR-T therapy, a new generation of CAR-T that can autonomously express cytokines has emerged. Some cytokines have an impact on the proliferation, maintenance, and function of T cells. Preparing CAR-T cells that can autonomously secrete these cytokines can enhance the immune response of CAR-T cells and also recruit or activate other immune cells to combat tumor cells. For example, IL-15 and IL-12 can resist immunosuppressive factors in the tumor microenvironment (Tang Lin et al, 2023).

[0006] IL-15 is a potent immune-stimulating cytokine that can induce the activation, proliferation, and survival of T cells and contribute to the generation and maintenance of highly active antigen-specific memory CD8+ T cells. In addition, IL-15 is also involved in the development, maintenance, and activation of NK, NKT, and γ / δ T cells and has good application prospects in adoptive immunotherapy. In clinical trials of patients with metastatic melanoma and renal cell carcinoma, IL-15 has shown a significant ability to promote the expansion of effector T cells and NK cells (Yang Xu et al, 2014). Many studies have shown that the application of IL-15 to CAR-T cells such as CD19 and GPC3 can extend cell lifespan and enhance cell killing function, and is expected to improve the efficacy of CAR-T cell immunotherapy (Tatsuki Ueda et al, 2022), which suggests that IL-15 may be able to extend the maintenance time of NKG2DL-CAR-T cells.

[0007] Currently, there is no research on NKG2DL-IL-15-CAR-T cells in this field. It is necessary to prepare a CAR-T cell that can secrete IL-15 and target NKG2DL to provide a new strategy for the durable treatment of malignant tumors. Summary of the Invention

[0008] The object of the present invention is to provide a CAR-T cell drug targeting NKG2DL and secreting IL-15 and its applications. The main technical problems to be solved include: first, enhancing the ability of CAR-T cells to kill target cells by targeting NKG2DL; second, improving the survival and proliferation ability of terminally exhausted CAR-T cells and enhancing their tumor killing activity by co-expressing IL-15.

[0009] Therefore, in a first aspect, the present invention provides a CAR (NKG2DL-IL15-CAR) targeting NKG2DL and comprising IL-15, which comprises the following elements: 1) a signal peptide region; 2) NKG2D; 3) an IL-15R domain; 4) a hinge region; 5) a transmembrane region; 6) an intracellular signaling region; 7) a co-stimulatory region; 8) a stimulatory region; 9) an insulin signal peptide; 10) a 2A sequence; and 11) a human IL-15 sequence.

[0010] The amino acid sequence of NKG2D is as shown in SEQ ID NO:2.

[0011] In an embodiment of the present invention, the signal peptide region may be a signal peptide of a mammalian cell surface protein, such as a human albumin signal peptide, a human insulin signal peptide, a human CD8 signal peptide or a murine IgG kappa signal peptide; preferably, the signal peptide is a human albumin signal peptide, and its amino acid sequence is as shown in SEQ ID NO:1.

[0012] In an embodiment of the present invention, the amino acid sequence of the IL-15R domain is as shown in SEQ ID NO:3.

[0013] In an embodiment of the present invention, the hinge region is the hinge region of human CD8α, human CD28 or human IL-15R. Preferably, the hinge region is the hinge region of human IL-15R, and its amino acid sequence is as shown in SEQ ID NO:4.

[0014] In an embodiment of the present invention, the transmembrane region may be the transmembrane region of human CD8α, human CD28 or human IL-15R. Preferably, the transmembrane region is the transmembrane region of human CD8α, and its amino acid sequence is as shown in SEQ ID NO:5.

[0015] In an experimental protocol of the present invention, the amino acid sequence of the intracellular signaling region is as shown in SEQ ID NO:6.

[0016] In an embodiment of the present invention, the intracellular co-stimulatory region is the intracellular co-stimulatory region of human CD28, and its amino acid sequence is as shown in SEQ ID NO:7.

[0017] In an embodiment of the present invention, the intracellular stimulation region is the human CD38 zeta intracellular stimulation region, and its amino acid sequence is as shown in SEQ ID NO:8.

[0018] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is as shown in SEQ ID NO:9;

[0019] In an embodiment of the present invention, the amino acid sequence of the human insulin signal peptide is as shown in SEQ ID NO:10;

[0020] In an embodiment of the present invention, the amino acid sequence of the IL-15 sequence is as shown in SEQ ID NO:11.

[0021] In a second aspect, the present invention provides a nucleic acid molecule encoding the NKG2DL-IL15-CAR of the first aspect.

[0022] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect above.

[0023] In a fourth aspect, the present invention provides a recombinant lentivirus, which comprises the nucleic acid molecule of the second aspect of the present invention or is prepared by the expression vector of the third aspect.

[0024] In a fifth aspect, the present invention provides a method for preparing a recombinant lentivirus, comprising co-transfecting a mammalian host cell with the expression vector of the third aspect and an auxiliary plasmid to obtain a recombinant lentivirus.

[0025] In an embodiment of the fifth aspect, the auxiliary plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0026] In an embodiment of the fifth aspect, the mammalian host cell can be, but is not limited to, HEK293 cells, human PER.C6 cells, human HeLa cells, murine CHO cells, etc., and preferably HEK293 cells.

[0027] In a sixth aspect, the present invention provides CAR-T cells (NKG2DL-IL15-CAR-T cells) that target NKG2DL and secrete IL-15, and the NKG2DL-IL15-CAR-T cells express the NKG2DL-IL15-CAR of the first aspect of the present invention.

[0028] In a seventh aspect, the present invention provides a method for preparing NKG2DL-IL15-CAR-T cells, comprising infecting T cells with the recombinant lentivirus of the fifth aspect. In a specific embodiment of the seventh aspect of the present invention, the method for preparing NKG2DL-IL15-CAR-T cells comprises the following steps:

[0029] S1) Construct a recombinant lentiviral expression vector carrying a nucleotide sequence encoding NKG2DL-IL15-CAR;

[0030] S2) Transfect host cells with the above recombinant lentiviral expression vector and auxiliary plasmids to prepare recombinant lentivirus capable of infecting T cells;

[0031] S3) Isolate peripheral blood mononuclear cells from the peripheral blood provided by the donor, and use magnetic beads to isolate and activate T cells;

[0032] S4) Infect T cells with the recombinant lentivirus obtained in step S2) to generate NKG2DL-IL15-CAR-T cells expressing NKG2DL-IL15-CAR.

[0033] In a further embodiment, the auxiliary plasmids can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0034] In a further embodiment, the method may further include the following steps after step S4):

[0035] S5) In vitro culture the cells obtained in step S4);

[0036] S6) Greatly amplify the cells obtained in step S5);

[0037] S7) Collect NKG2DL-IL15-CAR-T cells.

[0038] In an eighth aspect, the present invention provides a disease treatment drug, which comprises the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant lentivirus of the fourth aspect, or the NKG2DL-IL15-CAR-T cells of the sixth aspect of the present invention.

[0039] In a ninth aspect, the present invention provides the use of the above nucleic acid molecule, the above expression vector, the above recombinant lentivirus, or the above NKG2DL-IL15-CAR-T cells in the preparation of a disease treatment drug.

[0040] In the embodiments of the present invention, the diseases include cervical cancer, ovarian cancer, colorectal cancer, relapsed / refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, multiple myeloma, osteosarcoma, and aging-related diseases, etc., which are characterized by high expression of NKG2DL on the cell surface.

[0041] In an embodiment of the present invention, CAR represents a chimeric antigen receptor, NKG2DL-CAR represents a chimeric antigen receptor targeting NKG2DL, NKG2DL-IL15-CAR represents a chimeric antigen receptor targeting NKG2DL and containing IL-15, CAR-T cell represents a chimeric antigen receptor T cell, NKG2DL-CAR-T cell represents a chimeric antigen receptor T cell targeting NKG2DL, and NKG2DL-IL15-CAR-T cell represents a chimeric antigen receptor T cell targeting NKG2DL and autonomously secreting IL-15.

[0042] In the present invention, NKG2DL-IL15-CAR-T cells specifically recognize and bind to NKG2DL on target cells through the expressed NKG2DL-CAR, and then kill the target cells. At the same time, the cytokine IL-15 secreted by the NKG2DL-IL15-CAR-T cells of the present invention can promote the expansion and maintenance of T cells, improve the tumor killing ability, and is expected to make up for the deficiencies of NKG2DL-CAR-T cells in clinical applications. Brief Description of the Drawings

[0043] Figure 1 is a schematic structural diagram of NKG2DL-IL15-CAR.

[0044] Figure 2 is a detection graph of the positive rate of NKG2DL-CAR in NKG2DL-IL15-CAR-T cells on the 3rd day after virus transfection. Among them, A is the flow cytometry graph of the detection of the positive rate of NKG2DL-CAR, and B is the statistical graph of the detection of the positive rate of NKG2DL-CAR.

[0045] Figure 3 is a comparison graph of the proliferation trends of NKG2DL-IL15-CAR-T cells and untransduced T cells.

[0046] Figure 4 is a comparison graph of the killing results of NKG2DL-IL15-CAR-T cells and untransduced T cells. The horizontal axis is the effector-to-target ratio, that is, the number ratio of T cells or CAR-T cells to target cells HeLa, and the vertical axis is the killing rate. Among them, A and B are the killing results on the 10th day and the 20th day of culture, respectively. Detailed Embodiments

[0047] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as in the Molecular Cloning Experimental Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2012), or according to the conditions recommended by the manufacturer's instructions.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained through commercial channels.

[0049] Example 1. Design and construction of plasmid encoding NKG2DL-IL15-CAR

[0050] The plasmid encoding NKG2DL-IL15-CAR was synthesized by Nanjing Biaode Biotechnology Co., Ltd. This plasmid contains nucleic acid sequences encoding the following elements in the 5' to 3' direction of the CAR structure: signal peptide region (SEQ ID NO:1), NKG2D (SEQ ID NO:2), IL-15R domain (SEQ ID NO:3), hinge region (NO:4), transmembrane region (SEQ ID NO:5), intracellular signaling region (SEQ ID NO:6), co-stimulatory region (SEQ ID NO:7), stimulatory region (SEQ ID NO:8), human insulin signal peptide (SEQ ID NO:9), 2A sequence (SEQ ID NO:10), and IL-15 sequence (SEQ ID NO:11). This plasmid was cloned into the lentiviral backbone plasmid pMSGV-IRES (Shanghai New Bio-Tech Co., Ltd., product number: V000092) to obtain the plasmid encoding NKG2DL-IL15-CAR, which is called pMSGV-NKG2DL-IL15 and is a recombinant lentiviral expression vector in lentiviral packaging. The NKG2DL-IL15-CAR structure is as Figure 1 shown.

[0051] Example 2. Preparation of recombinant lentivirus

[0052] Co-transfect the backbone plasmid pMSGV-NKG2DL-IL15 with three helper plasmids pMDLG-pRRE-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0374), pMD2G-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0648), and pRSV-REV-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0370) into the WayneLVPro HEK293 cell line (Suspension Adapted) (Zhongshan Kangsheng Biotechnology Co., Ltd., product number A23109, hereinafter referred to as 293T cells), and the NKG2DL-IL15-CAR recombinant lentivirus that can infect T cells can be prepared. The specific operation steps for the preparation of the recombinant lentivirus are as follows:

[0053] 2.1 Cell culture: 293T cells were cultured in DMEM (Thermo Fisher Scientific (Hangzhou) Co., Ltd., product number: SH30243.01) complete medium containing 10% FBS (GE Healthcare Life Sciences (Hangzhou) Co., Ltd., product number: SH30256.01) and cultured in a cell incubator at 37°C and 5% CO2 until the confluence reached about 80%.

[0054] 2.2 Take 144 μL of PEI (Merck KGaA, product number: 937762) with a concentration of 1 mg / mL, add it to 1 mL of OPTI-MEM (Thermo Fisher Scientific, product number: 11058021), mix well, and let it stand at room temperature for 20 min.

[0055] 2.3 Add 16 μg of the main plasmid pMSGV-NKG2DL-IL15, 12 μg of pMDLG-pRRE-Kana, 4 μg of pMD2G-Kana, and 4 μg of pRSV-REV-Kana helper plasmids to 1 mL of OPTI-MEM and mix well.

[0056] 2.4 Add the PEI-OPTI-MEM mixed solution in step 2.2 to the diluted plasmids in step 2.3, mix well, and let it stand at room temperature for 20 min.

[0057] 2.5 Take 8 mL of DMEM complete medium containing 10% FBS, add it to the PEI-plasmid mixture in step 2.4, and mix well.

[0058] 2.6 Discard the medium in the 293T cell culture flask, add the medium in step 2.5, and continue to culture the cells in the incubator.

[0059] 2.7 After 48 h, collect the supernatant of the cell culture medium, filter it with a vacuum filter (Corning (Shanghai) Co., Ltd., product number: 430770) with a pore size of 0.45 μm to obtain the supernatant containing recombinant lentivirus, and place it in a 2 - 8°C refrigerator for 2 h.

[0060] 2.8 Centrifuge the supernatant in step 2.7 at 20000×g and 4°C for 2 h. After centrifugation, discard the supernatant, and resuspend the recombinant lentivirus at the bottom with Lymphocyte Serum-Free Medium (Corning Life Sciences (Wujiang) Co., Ltd., product number: 88-581-CM). The volume ratio of the medium used to resuspend the virus to the original supernatant is 1:25, that is, it is concentrated 25 times.

[0061] Example 3. Detection of the infection titer of recombinant lentivirus

[0062] 3.1 Take Jurkat cells (American Type Culture Collection (ATCC), catalog number: TIB-152) after 2 passages of resuscitation culture, centrifuge at 300×g for 10 min at room temperature.

[0063] 3.2 Discard the culture medium supernatant, resuspend the cells with RPMI 1640 medium (Cytiva Life Sciences (Hangzhou) Co., Ltd., catalog number: SH30809-01), and adjust the cell density to 2×10 6 cells / mL, and add the transfection reagent Protamine (Sigma-Aldrich, catalog number: 53597-25-4, prepared stock solution concentration: 10 mg / mL) to the cell suspension at a ratio of 2 μL / mL, and mix well.

[0064] 3.3 Take a 48-well plate, and inoculate 100 μL of Jurkat cell (2×10 5 cells / well) suspension into each well of the 2nd - 7th wells in the second, third, and fourth rows, inoculating a total of 3 rows, and perform 3 sets of repeated experiments. For the wells in a circle adjacent to the inoculated cells, add 1 mL of PBS.

[0065] 3.4 Take a 96-well plate, and sequentially add 133 μL, 120 μL, 120 μL, 120 μL, 120 μL, and 120 μL of serum-free RPMI 1640 medium into the 1st - 6th wells in the first row of the 96-well plate. Take 7 μL of the virus solution collected and sampled in Method 2, add it into the 1st well, and mix well to obtain Virus Dilution 1. Take 120 μL of Virus Dilution 1, add it into the 2nd well, and mix well to obtain Virus Dilution 2. Take 120 μL of Virus Dilution 2, add it into the 3rd well, and mix well to obtain Virus Dilution 3. Take 120 μL of Virus Dilution 3, add it into the 4th well, and mix well to obtain Virus Dilution 4. Take 120 μL of Virus Dilution 4, add it into the 5th well, and mix well to obtain Virus Dilution 5. Take 120 μL of Virus Dilution 5, add it into the 6th well, and mix well to obtain Virus Dilution 6. Thus, the first group of gradient dilutions containing different virus amounts is obtained.

[0066] 3.5 According to the operation method in Step 3.4 above, perform virus solution dilution in the 1st - 6th wells in the second row and the 1st - 6th wells in the third row of the 96-well plate respectively, so as to obtain the second group and the third group of virus gradient dilutions.

[0067] 3.6 Take 100 μL each of Virus Dilutions 2, 3, 4, 5, and 6 in the first group of virus gradient dilutions, and add them into the corresponding 3rd - 7th wells in the second row of the 48-well plate inoculated with Jurkat cells in Step 3.3. Add 100 μL of serum-free RPMI 1640 medium to the 2nd well as Control 1. Mix each well thoroughly.

[0068] 3.7 According to the operation method in step 3.6 above, add the second and third groups of virus gradient dilutions to the third and fourth rows, wells 3 to 7 corresponding to the 48-well plate inoculated with Jurkat cells in step 3.3. Add 100 μL of serum-free RPMI 1640 medium to wells 2 in the third and fourth rows as control 2 and control 3 respectively. Mix each well thoroughly and culture in a 37 °C, 5% CO2 cell incubator.

[0069] 3.8 After 6 h, supplement 800 μL of RPMI 1640 complete medium containing 10% FBS and culture in a 37 °C, 5% CO2 cell incubator.

[0070] 3.9 After 24 h, carefully aspirate and discard 500 μL of the medium from the liquid surface of each well (Jurkat cells have adhered to the bottom of the well plate), and supplement each well with 500 μL of RPMI 1640 complete medium containing 10% FBS, and continue to culture in a 37 °C, 5% CO2 cell incubator.

[0071] 3.10 Sampling: After 48 h, take 300 μL from each well into a 1.5 mL centrifuge tube, centrifuge at 300 × g at room temperature for 5 min, and discard the supernatant.

[0072] 3.11 Washing: Add 1 mL of PBS to each tube to resuspend the cells, centrifuge at 300 × g at room temperature for 5 min, and discard the supernatant.

[0073] 3.12 Flow cytometry staining: Add 5 μL of NKG2D antibody (Thermo Fisher Scientific, catalog number: 17-5878-42) to each sample, gently pipette and mix well, and incubate in the dark for 30 min. At the same time, set up a blank control group.

[0074] 3.13 Washing: After incubation, add 1 mL of PBS to the sample to resuspend the cells, centrifuge at 300 × g at room temperature for 5 min, and discard the supernatant.

[0075] 3.14 Add 200 μL of PBS to resuspend each tube of cell sample, and then detect the expression rate of NKG2D by flow cytometry and calculate the titer of the recombinant lentivirus.

[0076] Example 4. T cell isolation and activation

[0077] 4.1 Under sterile conditions, take 15 mL of peripheral blood from a healthy human donor, let it stand at room temperature for 30 min, centrifuge at 700 × g for 20 min, and aspirate the plasma into another 15 mL centrifuge tube for standby.

[0078] 4.2 During the centrifugation interval, according to Dynabeads TMAs described in the instruction manual of Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific, catalog number: 11452D), prepare RetroNectin coating solution and add 1.5 mL / well to a 6-well plate. Place it at room temperature for 5 h.

[0079] 4.3 Add an equal volume of PBS to the blood cells in step 4.1 and mix well. Slowly add the diluted blood to an equal volume of lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., catalog number: LTS1077). Centrifuge at 700×g at room temperature for 20 min.

[0080] 4.4 Aspirate the middle white film layer into a new centrifuge tube. Add 2 volumes of PBS to wash the white film layer cells. Centrifuge at 500×g at room temperature for 10 min. Discard the supernatant to obtain PBMC (Peripheral Blood Mononuclear Cell).

[0081] 4.5 Sort T lymphocytes in PBMC according to the magnetic bead instruction manual of CD3 Microbeads (Human) (Miltenyi Biotec GmbH, catalog number: 130-050-101) and perform cell counting.

[0082] 4.6 The T cells sorted in step 4.5 are centrifuged at 500×g at room temperature for 10 min to wash away the separation solution. Discard the supernatant. Resuspend the cells with X-VIVO TM 15 serum-free medium (Lonza Investment (China) Co., Ltd., catalog number: DL-102) and adjust the cell concentration to 1×10 6 cells / mL.

[0083] 4.7 Discard the RetroNectin coating solution in the 6-well plate in step 4.2. Inoculate the T cells in step 4.6 at 3×10 6 cells / well in a 6-well cell culture plate for the preparation of NKG2DL-IL15-CAR-T cells in method 5. Place the cells in a 37°C, 5% CO2 cell culture incubator and culture for 24 - 48 h to obtain activated T cells.

[0084] Example 5. Preparation of NKG2DL-IL15-CAR-T cells

[0085] 5.1 The isolated T cells were cultured for 24-48 hours, and the activated T cells in Example 4 were observed. The T cells adhered to the wall, grew larger, and had a fusion degree of 20-50%, which allowed the virus to infect the T cells.

[0086] 5.2 Carefully aspirate and discard 1 mL of culture medium from the top of each well, and add X-VIVO 500 containing the transfection reagent Protamine (20 μg / mL). TM 15. Add 1 mL of serum-free medium to each well and mix thoroughly by pipetting gently.

[0087] 5.3 Calculate the amount of NKG2DL-IL15-CAR virus to be added to each well according to MOI=3 (amount of virus added per well = MOI×3×10 6 / virus titer), add virus and mix thoroughly by pipetting. The control group is cells treated with only protamine.

[0088] Centrifuge at 5.41000×g for 30 min at room temperature with both the acceleration and deceleration speeds set to 3 to promote cell adhesion. Then, culture the cells in a 37°C, 5% CO2 cell culture incubator.

[0089] 5.5 24 hours after adding the virus, add X-VIVO containing 40 IU / mL IL-12 to each well TM 15 serum-free medium.

[0090] 5.6 Samples were taken on the third day after viral infection, and the expression of NKG2DL-CAR in the control group T cells and NKG2DL-IL15-CAR-T cells was detected by flow cytometry. Figure 2 As shown in Figure 3, the positive rate of NKG2DL-CAR in NKG2DL-IL15-CAR-T cells was approximately 60%.

[0091] Example 6. NKG2DL-IL15-CAR-T cell proliferation experiment in vitro

[0092] On the third day after virus infection, the NKG2DL-IL15-CAR-T cells and control group T cells in Example 5 were collected, centrifuged at 500×g for 10 min at room temperature, and washed with X-VIVO 500 μg containing 40 IU / mL IL-12. TM Resuspend the cells in serum-free medium at 15 °C, take samples for cell counting and record the total number of cells in each group, and then adjust the cell density of each group to 1 × 10 6 cells / mL. The cells were divided into 1×10 6 Cells were seeded into 6-well plates, culture medium was added to 3 mL, and samples were taken regularly to check the cell number. Figure 3 As shown in the figure, NKG2DL-IL15-CAR-T cells have stronger expansion ability and longer maintenance time than untransduced T cells.

[0093] Example 7. In vitro killing experiment of T cells

[0094] 7.1 Culture of target cells: The human cervical cancer cell line HeLa cells (Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number: TCHu187) were selected as target cells. DMEM complete medium containing 10% FBS was prepared for culturing these cells. When the cell confluence reached 80%, the medium was discarded, and 8 mL of trypsin (Thermo Fisher Scientific, catalog number: 25200072) was added and digested at 37 °C for 5 min. Subsequently, 20 mL of fresh medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded. The cells were resuspended with DMEM complete medium containing 10% FBS and sampled for counting, and the cell density was adjusted to 1×10 6 cells / mL, and inoculated into a 6-well plate at 0.8×10 6 cells / well, and the medium was supplemented to 2 mL.

[0095] 7.2 Calculate the number of cells required for different effector-to-target ratios: Samples were taken from the untransduced group cells and NKG2DL-IL15-CAR-T cells, and the NKG2DL-CAR expression rate was detected by flow cytometry. The actual number of effector cells = the number of T cells × the CAR expression rate (%), and the number of T cells required for different effector-to-target ratios = E / T × 0.8×10 6 / CAR positive rate.

[0096] 7.3 Use X-VIVO TM 15 serum-free medium to adjust the cell density of both NKG2DL-IL15-CAR-T cells and untransduced group T cells to 1×10 6 cells / mL.

[0097] 7.4 Co-incubation of T cells and target cells: Set the effector-to-target ratios (E:T) of effector cells and HeLa target cells to 1:5 and 2:5, respectively. Set a blank control group: only HeLa cells were added; a negative control group: untransduced virus T cells were co-incubated with HeLa cells; an experimental group: NKG2DL-IL15-CAR-T cells with different effector-to-target ratios were co-incubated with HeLa cells. The medium was supplemented to 2 mL for each group.

[0098] 7.5 After 24 h, measure the remaining medium volume in each well, mix well, sample the cells in each group for counting and calculate the total number of cells. At the same time, sample and detect the proportion of HeLa cells by flow cytometry. HeLa cells were labeled with MICA / B antibody (Biolegend, catalog number: 320907), and calculate the remaining number of HeLa cells and cell killing efficiency in each group.

[0099] The results are as Figure 4As shown, compared with untransduced T cells, NKG2DL-IL15-CAR-T cells could significantly kill HeLa cells at both 10 days and 20 days after transduction.

[0100] References

[0101] [1] Lerner EC, Woroniecka KI, D'Anniballe VM, et al. CD8+ T cells maintain killing of MHC-I-negative tumor cells through the NKG2D-NKG2DL axis. Nat Cancer. 2023;4(9):1258-1272. doi:10.1038 / s43018-023-00600-4

[0102] [2] Jin X, Xie D, Sun R, et al. CAR-T cells dual-target CD123 and NKG2DLs to eradicate AML cells and selectively target immunosuppressive cells. Oncoimmunology. 2023;12(1):2248826. Published 2023 Aug 26. doi:10.1080 / 2162402X.2023.2248826

[0103] [3] Deng Y, Kumar A, Xie K, et al. Targeting senescent cells with NKG2D-CAR T cells. Cell Death Discov. 2024;10(1):217. Published 2024 May 4. doi:10.1038 / s41420-024-01976-7

[0104] [4] Dong Yang et al. NKG2D-CAR T cells eliminate senescent cells in aged mice and nonhuman primates. Sci. Transl. Med. 15, eadd1951(2023).

[0105] [5]Tang L, Pan S, Wei X, Xu X, Wei Q. Arming CAR-T cells with cytokines and more: Innovations in the fourth-generation CAR-T development. Mol Ther. 2023;31(11):3146 - 3162. doi:10.1016 / j.ymthe.2023.09.021

[0106] [6]Ueda T, Shiina S, Iriguchi S, et al. Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells. Nat Biomed Eng. 2023;7(1):24 - 37.

[0107] [7]Xu Y, Zhang M, Ramos CA, et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood. 2014;123(24):3750 - 3759. doi:10.1182 / blood-2014-01-552174

Claims

1. A chimeric antigen receptor targeting NKG2DL and comprising IL-15, characterized in that, The chimeric antigen receptor sequentially comprises the following elements: 1) A signal peptide; 2) NKG2D; 3) An IL-15R domain; 4) A hinge region; 5) A transmembrane region; 6) An intracellular signaling region; 7) A co-stimulatory region; 8) A stimulatory region; 9) An insulin signal peptide; 10) A 2A sequence; and 11) An IL-15 sequence.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor targeting NKG2DL and comprising IL-15 as claimed in claim 1.

3. Expression vector, characterized in that, The vector comprises the nucleic acid molecule as claimed in claim 2.

4. Recombinant lentivirus, characterized in that, The recombinant lentivirus comprises the nucleic acid molecule as claimed in claim 2 or is prepared by the expression vector as claimed in claim 3.

5. A method for preparing recombinant lentivirus, characterized in that, The method comprises co-transfecting a mammalian host cell with the expression vector as claimed in claim 3 and an auxiliary plasmid.

6. A method for preparing CAR-T cells targeting NKG2DL and secreting IL-15, characterized in that, The method comprises infecting T cells with the recombinant lentivirus as claimed in claim 4.

7. CAR-T cells targeting NKG2DL and secreting IL-15, characterized in that, The cell is prepared by the method as claimed in claim 6.

8. A drug for treating diseases, characterized in that, The drug comprises the nucleic acid molecule as claimed in claim 2, the expression vector as claimed in claim 3, the recombinant lentivirus as claimed in claim 4, or the CAR-T cell targeting NKG2DL and secreting IL-15 as claimed in claim 7.

9. The therapeutic drug for diseases according to claim 8, characterized in that, The diseases are cervical cancer, ovarian cancer, colorectal cancer, relapsed / refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, multiple myeloma, and osteosarcoma, as well as aging-related diseases, etc.

10. Use of the nucleic acid molecule as claimed in claim 2, the expression vector as claimed in claim 3, the recombinant lentivirus as claimed in claim 4, or the CAR-T cell targeting NKG2DL and secreting IL-15 as claimed in claim 7 in the preparation of a medicament for treating diseases.

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