CAR-T cell targeting NKG2DL and secreting IL-10 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-10 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-10 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-10, the proliferation and effector functions of the CAR-T cell are promoted, and the killing activity on tumors is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a CAR-T cell that targets NKG2DL and secretes IL-10, its preparation method and application. Background Technology

[0002] Chimeric antigen receptor (CAR) T-cell therapy, a type of immunotherapy, is primarily used for the clinical treatment of hematologic malignancies and malignant tumors. This therapy utilizes genetic engineering to express one or more specific CARs onto T cells, enabling them to target and eliminate tumors. Clinical results indicate that CAR-T cells have significant advantages in treating hematologic malignancies, but limitations remain, such as tumor recurrence and poor efficacy in solid tumors. One reason for these limitations is the limited expansion capacity and persistence of CAR-T cells, which affects their long-term anti-tumor activity.

[0003] NKG2D is a C-type lectin-like receptor molecule composed of homodimers, primarily expressed on natural killer (NK) cells and CD8 cells. + NKG2D plays a crucial role in innate immunity, participating in the recognition of virus-infected cells and the killing of tumor cells by NK cells. The biological functions of NKG2D are closely related to its ligand, NKG2DL: NKG2DL can perform immune surveillance, and NKG2D recognition of NKG2DL can mediate the tumor-killing effect of immune cells (Lerner EC et al, 2023). Studies have shown that NKG2DL is expressed in various cancer cell lines and primary tumors, including cervical cancer, colorectal cancer, ovarian cancer, pancreatic cancer, and lymphoma (Jin Xin et al, 2023). Therefore, CAR-T cells targeting NKG2DL (i.e., NKG2DL-CAR-T cells) hold promise as an effective treatment for these tumors. Currently, investigational drugs and therapies targeting NKG2DL are mainly in early clinical stages. Consistent with in vitro studies and animal models, the maintenance time of CAR-T cells targeting NKG2DL is relatively short, limiting the therapeutic effect on tumors.

[0004] Furthermore, NKG2DL-CAR-T cell therapy holds promise as an effective and selective anti-aging therapy for treating aging and age-related diseases driven by aging. Studies have found that NKG2DL expression is upregulated in cultured senescent cells (Deng Yushuang, 2024), and also in various tissues of senescent mice and senescent non-human primates. NKG2DL-CAR-T cell therapy can selectively target and eliminate NKG2DL-highly expressed senescent cells in vitro, in mice, and in non-human primates, without significantly affecting normal cells (Dong Yang et al, 2023).

[0005] CAR-T therapy has shown great potential in cancer treatment; however, its efficacy is influenced by many factors, such as antigen heterogeneity, antigen loss, limited cell persistence, poor invasiveness, and suppression by the tumor microenvironment. To improve the effectiveness of CAR-T therapy, a new generation of CAR-T cells capable of autonomously expressing cytokines has emerged. Some cytokines affect the proliferation, maintenance, and function of T cells. Preparing CAR-T cells that autonomously secrete these cytokines can enhance the immune response of CAR-T cells and recruit or activate other immune cells to fight tumor cells. For example, IL-15 and IL-12 can resist immunosuppressive factors in the tumor microenvironment (Tang Lin et al., 2023).

[0006] IL-10 is a pleiotropic cytokine associated with the pathogenesis and development of autoimmune diseases and tumors. Studies have shown that the effects of IL-10 on tumors may be related to the following three biological activities: (1) IL-10 can enhance the activity and proliferation of CD8+ T cells and improve their cytolysis function; (2) it inhibits the antigen presentation process and the production of pro-inflammatory cytokines, especially IL-12 and IL-23, but whether this affects antitumor activity is unclear; (3) IL-10 reduces the tumor-promoting effects mediated by chronic inflammation (Wenjun Ouyang et al, 2019). Pegylated IL-10 (peg-IL-10) has shown good antitumor activity as a single drug and has produced antitumor effects in patients with renal cell carcinoma (Aung Naing et al, 2016). In mouse models of colon cancer, breast cancer, melanoma, and pancreatic cancer, CAR-T cells capable of autonomously secreting IL-10 exhibited more intact mitochondrial morphology and lower expression of the programmed death receptor PD-1 compared to conventional CAR-T cells, demonstrating stronger proliferative capacity. This suggests that cell exhaustion was suppressed to some extent, leading to complete tumor regression (Zhao Yang et al., 2024). Although IL-10 promotes the proliferation and killing of CD8+ T cells, seemingly playing a powerful inhibitory role in tumor progression, its ultimate effect on tumors requires further investigation due to its inhibitory effect on antigen-presenting cells, particularly tumor-infiltrating dendritic cells.

[0007] Currently, there are no studies in this field on CAR-T cells that target NKG2DL and secrete IL-10. Summary of the Invention

[0008] The purpose of this invention is to provide a CAR-T cell drug that targets NKG2DL and secretes IL-10 and its application. The main technical problems to be solved include: first, enhancing the ability of CAR-T cells to kill target cells by targeting NKG2DL; and second, improving the survival and proliferation ability of terminally exhausted CAR-T cells and enhancing their tumor-killing activity by co-expressing IL-10.

[0009] Therefore, in a first aspect, the present invention provides a CAR (NKG2DL-IL10-CAR) that targets NKG2DL and contains IL-10, comprising the following elements: 1) a signal peptide region; 2) NKG2D; 3) a hinge region; 4) a transmembrane region; 5) a co-stimulatory region; 6) a stimulatory region; 7) a 2A sequence; and 8) an IL-10 sequence.

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

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

[0012] 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 of human CD8α, whose amino acid sequence is shown in SEQ ID NO:3.

[0013] 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 shown in SEQ ID NO:4.

[0014] In an embodiment of the present invention, the intracellular co-stimulatory region is the human CD28 intracellular co-stimulatory region, the amino acid sequence of which is shown in SEQ ID NO:5.

[0015] In an embodiment of the present invention, the intracellular stimulation region is the human CD38zeta intracellular stimulation region, the amino acid sequence of which is shown in SEQ ID NO:6.

[0016] In an embodiment of the present invention, the amino acid sequence of sequence 2A is shown in SEQ ID NO:7;

[0017] In an embodiment of the present invention, the amino acid sequence of the IL-10 sequence is shown in SEQ ID NO:8.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding NKG2DL-IL10-CAR of the first aspect.

[0019] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect above.

[0020] In a fourth aspect, the present invention provides a recombinant lentivirus comprising the nucleic acid molecule of the second aspect of the present invention or prepared by the expression vector of the third aspect.

[0021] In a fifth aspect, the present invention provides a method for preparing a recombinant lentivirus, comprising co-transfecting mammalian host cells with the expression vector and helper plasmid of the third aspect to obtain a recombinant lentivirus.

[0022] In the implementation of the fifth aspect, the helper plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, or pRSV-REV-Kana.

[0023] In the fifth aspect of the implementation scheme, the mammalian host cell can be, but is not limited to, HEK293 cells, human PER.C6 cells, human HeLa cells, and mouse CHO cells, with HEK293 cells being preferred.

[0024] In a sixth aspect, the present invention provides CAR-T cells (NKG2DL-IL10-CAR-T cells) that target NKG2DL and secrete IL-10, the NKG2DL-IL10-CAR-T cells expressing the NKG2DL-IL10-CAR of the first aspect of the present invention.

[0025] In a seventh aspect, the present invention provides a method for preparing NKG2DL-IL10-CAR-T cells, comprising infecting T cells with a recombinant lentivirus as described in the fifth aspect. In a specific embodiment of the seventh aspect of the present invention, the method for preparing NKG2DL-IL10-CAR-T cells includes the following steps:

[0026] S1) Construct a recombinant lentiviral expression vector carrying a nucleotide sequence encoding NKG2DL-IL10-CAR;

[0027] S2) Use the above-mentioned recombinant lentiviral expression vector and helper plasmid to transfect host cells to prepare recombinant lentivirus that can infect T cells;

[0028] S3) Peripheral blood mononuclear cells are isolated from peripheral blood provided by the donor, and T cells are separated and activated using magnetic beads;

[0029] S4) Infect T cells with the recombinant lentivirus obtained in step S2) to generate NKG2DL-IL10-CAR-T cells expressing NKG2DL-IL10-CAR.

[0030] In a further embodiment, the helper plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, or pRSV-REV-Kana.

[0031] In a further embodiment, the method may also include the following steps after step S4):

[0032] S5) Culture the cells obtained in step S4) in vitro;

[0033] S6) Expand the cells obtained in step S5) in large quantities;

[0034] S7) Collect NKG2DL-IL10-CAR-T cells.

[0035] In an eighth aspect, the present invention provides a disease treatment drug comprising 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-IL10-CAR-T cell of the sixth aspect.

[0036] In a ninth aspect, the present invention provides the use of the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant lentivirus, or the above-mentioned NKG2DL-IL10-CAR-T cell in the preparation of a disease treatment drug.

[0037] In 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, which are characterized by high expression of NKG2DL on the cell surface.

[0038] In embodiments of the present invention, CAR represents a chimeric antigen receptor, NKG2DL-CAR represents a chimeric antigen receptor targeting NKG2D ligand (NKG2DL), NKG2DL-IL10-CAR represents a chimeric antigen receptor targeting NKG2DL and containing IL-10, 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-IL10-CAR-T cell represents a chimeric antigen receptor T cell targeting NKG2DL and autonomously secreting IL-10.

[0039] The NKG2DL-IL10-CAR-T cells of this invention specifically recognize and bind to NKG2DL on target cells through the expression of NKG2DL-CAR, thereby achieving targeted killing of tumor cells. Simultaneously, the cytokine IL-10 secreted by the NKG2DL-IL10-CAR-T cells of this invention can significantly enhance the proliferation and maintenance of T cells, thereby improving tumor-killing ability. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of NKG2DL-IL10-CAR.

[0041] Figure 2 This is a graph showing the NKG2DL-CAR positivity rate of NKG2DL-IL10-CAR-T cells on day 3 after viral transfection.

[0042] Figure 3 This is a comparison chart of the proliferation trends of NKG2DL-IL10-CAR-T cells and untransduced T cells.

[0043] Figure 4This is a comparison of the killing results of NKG2DL-IL10-CAR-T cells and untransduced T cells. The horizontal axis represents the effector-to-target ratio, i.e., the ratio of the number of T cells or CAR-T cells to the number of target HeLa cells, and the vertical axis represents the killing rate. A and B represent the killing results on day 10 and day 20 of culture, respectively. Detailed Implementation

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 2012), or as recommended by the manufacturer's instructions.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] Example 1. Design and construction of plasmid encoding NKG2DL-IL10-CAR

[0047] The plasmid encoding NKG2DL-IL10-CAR was synthesized by Nanjing Baode Biotechnology Co., Ltd. From the 5' to 3' direction of the CAR structure, the plasmid contains nucleic acid sequences encoding the following elements: signal peptide region (SEQ ID NO:1), NKG2D (SEQ ID NO:2), hinge region (SEQ ID NO:3), transmembrane region (SEQ ID NO:4), co-stimulatory region (SEQ ID NO:5), stimulatory region (SEQ ID NO:6), 2A sequence (SEQ ID NO:7), and IL-10 sequence (SEQ ID NO:8). This plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newp Biotechnology Co., Ltd., catalog number: V000092) to obtain the plasmid encoding NKG2DL-IL10-CAR, named pMSGV-NKG2DL-IL10, which serves as a recombinant lentiviral expression vector in lentiviral packaging. The structure of NKG2DL-IL10-CAR is shown below. Figure 1 As shown.

[0048] Example 2. Preparation of recombinant lentivirus

[0049] Recombinant lentivirus NKG2DL-IL10, capable of infecting T cells, can be prepared by co-transfecting the WayneLVPro HEK293 cell line (Suspension Adapted) (Zhongshan Kangcheng Biotechnology Co., Ltd., catalog number A23109, hereinafter referred to as 293T cells) with the master plasmid pMSGV-NKG2DL-IL10 and three helper plasmids pMDLG-pRRE-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0374), pMD2G-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0648), and pRSV-REV-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0370). The specific steps for preparing the recombinant lentivirus are as follows:

[0050] 2.1 Cell Culture: 293T cells were cultured in DMEM (Stenofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30243.01) complete medium containing 10% FBS (Stenofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30256.01) and cultured in a 37℃, 5% CO2 cell culture incubator until the confluence reached about 80%.

[0051] 2.2 Take 144 μL of PEI (Merck Biotech Ltd., catalog number: 937762) with a concentration of 1 mg / mL, add it to 1 mL of OPTI-MEM (Thermo Fisher Scientific Ltd., catalog number: 11058021), mix well, and let stand at room temperature for 20 min.

[0052] 2.3 Add 16 μg of main plasmid pMSGV-NKG2DL-IL10, 12 μg of pMDLG-pRRE-Kana, 4 μg of pMD2G-Kana and 4 μg of pRSV-REV-Kana auxiliary plasmid to 1 mL of OPTI-MEM and mix well.

[0053] 2.4 Add the PEI-OPTI-MEM mixed solution from step 2.2 to the diluted plasmid from step 2.3, mix well, and let stand at room temperature for 20 min.

[0054] 2.5 Take 8 mL of DMEM complete medium containing 10% FBS and add it to the PEI-plasmid mixture from step 2.4, and mix well.

[0055] 2.6 Discard the culture medium in the 293T cell culture flask, add the culture medium from step 2.5, and place the cells in an incubator to continue culturing.

[0056] 2.748h later, the cell culture supernatant was collected and filtered through a vacuum filter with a pore size of 0.45μm (Corning (Shanghai) Co., Ltd., catalog number: 430770) to obtain a supernatant containing recombinant lentivirus, which was then placed in a refrigerator at 2-8℃ for 2h.

[0057] 2.8 Centrifuge the supernatant from step 2.7 at 20000×g and 4℃ for 2h. After centrifugation, discard the supernatant and resuspend the recombinant lentivirus at the bottom using Lymphocyte Serum-Free Medium (Corning Life Sciences (Wujiang) Co., Ltd., catalog number: 88-581-CM). The volume ratio of the culture medium used to resuspend the virus to the original supernatant is 1:25, i.e., 25 times concentrated.

[0058] Example 3. Detection of Recombinant Lentiviral Infection Titer

[0059] 3.1 Take Jurkat cells (American Center for Biostandard Collection (ATCC), catalog number: TIB-152) that have been revived and cultured for 2 generations, 300×g, and centrifuge at room temperature for 10 min.

[0060] 3.2 Discard the culture supernatant, resuspend the cells in RPMI 1640 medium (Stenofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30809-01) and adjust the cell density to 2×10⁶ cells / year. 6 Add Protamine (Sigma-Aldrich, catalog number: 53597-25-4, stock solution concentration: 10 mg / mL) to the cell suspension at a ratio of 2 μL / mL and mix well.

[0061] 3.3 Take a 48-well plate and seed 100 μL of Jurkat cells (2 × 10⁻⁷) into wells 2-7 of the second, third, and fourth rows. 5 The cells were seeded in a suspension of 100 cells / well in 3 rows, and 3 sets of replicate experiments were performed. 1 mL of PBS was added to the wells immediately surrounding the seeded cells.

[0062] 3.4 Take a 96-well plate and add 133 μL, 120 μL, 120 μL, 120 μL, 120 μL, 120 μL, and 120 μL of serum-free RPMI 1640 medium sequentially to wells 1 through 6 of the first row. Take 7 μL of the virus sample collected in Method 2 and add it to well 1, mix well, and obtain virus dilution 1. Take 120 μL of virus dilution 1 and add it to well 2, mix well, and obtain virus dilution 2. Take 120 μL of virus dilution 2 and add it to well 3, mix well, and obtain virus dilution 3. Take 120 μL of virus dilution 3 and add it to well 4, mix well, and obtain virus dilution 4. Take 120 μL of virus dilution 4 and add it to well 5, mix well, and obtain virus dilution 5. Take 120 μL of virus dilution 5 and add it to well 6, mix well, and obtain virus dilution 6. Thus, the first set of gradient dilutions containing different amounts of virus was obtained.

[0063] 3.5 Following the procedure in step 3.4 above, the virus solution was diluted in wells 1 to 6 of the second row and wells 1 to 6 of the third row of the 96-well plate to obtain the second and third groups of virus gradient dilution solutions.

[0064] 3.6 Take 100 μL each of virus dilutions 2, 3, 4, 5, and 6 from the first group of virus gradient dilutions and add them to wells 3 through 7 of 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 well 2 as control 1. Mix thoroughly in each well.

[0065] 3.7 Following the procedure in step 3.6 above, add the second and third group of viral gradient dilutions to wells 3 through 7 of the third and fourth rows of the 48-well plate inoculated with Jurkat cells in step 3.3, respectively. Add 100 μL of serum-free RPMI 1640 medium to well 2 of the third and fourth rows as controls 2 and 3. Mix thoroughly in each well and incubate at 37°C in a 5% CO2 cell culture incubator.

[0066] 3.8 After 6 hours, 800 μL of RPMI 1640 containing 10% FBS was added to complete the culture, which was then cultured in a 37°C, 5% CO2 cell culture incubator.

[0067] 3. After 24 hours, carefully aspirate 500 μL of culture medium (Jurkat cells have adhered to the bottom of the well plate) from the liquid surface of each well, add 500 μL of RPMI 1640 complete culture medium containing 10% FBS to each well, and continue culturing in a 37°C, 5% CO2 cell culture incubator.

[0068] 3.10 Sampling: After 48 hours, take 300 μL from each well into a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min at room temperature, and discard the supernatant.

[0069] 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.

[0070] 3.12 Flow cytometry staining: Add 5 μL of NKG2D antibody (Thermo Fisher Scientific, catalog number: 17-5878-42) to each sample, mix gently by pipetting, and incubate in the dark for 30 min. A blank control group was set up at the same time.

[0071] 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.

[0072] 3.14 Add 200 μL of PBS to each tube of cell sample for resuspending, and then detect the expression rate of NKG2D by flow cytometry to calculate the recombinant lentivirus titer.

[0073] Example 4. T cell isolation and activation

[0074] 4.1 Under aseptic conditions, take 15 mL of peripheral blood from a healthy 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 later use.

[0075] 4.2 In the centrifugal gap, follow Dynabeads TM According to the instructions for Mouse T-Activator CD3 / CD28 for T-CellExpansion and Activation (Thermo Fisher Scientific, catalog number: 11452D), prepare RetroNectin coating solution and add 1.5 mL / well to a 6-well plate, incubate at room temperature for 5 hours.

[0076] 4.3 Add an equal volume of PBS to the blood cells from step 4.1 and mix well. Slowly add the diluted blood to an equal volume of lymphocyte separation medium (Tianjin Haoyang Biological Products Technology Co., Ltd., catalog number: LTS1077), centrifuge at 700×g, room temperature for 20 min.

[0077] 4.4 Transfer the intermediate white membrane layer to a new centrifuge tube, add 2 volumes of PBS to wash the white membrane layer cells, centrifuge at 500×g at room temperature for 10 min, discard the supernatant, and obtain PBMC (Peripheral Blood Mononuclear Cell).

[0078] 4.5 T lymphocytes in PBMCs were sorted and counted according to the instructions of CD3 Microbeads (Human) (Medtronic Biotechnology Co., Ltd., catalog number: 130-050-101).

[0079] 4.6 Centrifuge the T cells obtained in step 4.5 at 500×g for 10 min at room temperature to wash away the separation solution and discard the supernatant. Use X-VIVO containing 40 IU / mL human interleukin-2 (IL-2) (Suzhou Nearshore Protein Technology Co., Ltd., catalog number: GMP-CD66) to wash away the separation solution. TM Resuspend cells in serum-free medium (Lonza Investment Co., Ltd., China, catalog number: DL-102) and adjust the cell concentration to 1×10⁻⁵. 6 Cells / mL.

[0080] 4.7 Discard the RetroNectin coating solution from the 6-well plate in step 4.2, and add the T cells from step 4.6 at a ratio of 3 × 10⁻⁶. 6 Cells were seeded per well in 6-well cell culture plates for the preparation of NKG2DL-IL10-CAR-T cells in Example 5. Cells were cultured at 37°C in a 5% CO2 cell culture incubator for 24-48 hours to obtain activated T cells.

[0081] Example 5. Preparation of NKG2DL-IL10-CAR-T cells

[0082] 5.1 The isolated T cells were cultured for 24-48 hours. The state of the activated T cells in Example 4 was observed. The T cells adhered to the wall, grew larger, and reached a fusion rate of 20-50%, and could be used to infect the T cells with viruses.

[0083] 5.2 Carefully aspirate 1 mL of culture medium from the top of each well and add X-VIVO containing the transfection aid Protamine (20 μg / mL). TM 15. Mix 1 mL of serum-free culture medium per well by gently pipetting.

[0084] 5.3 Calculate the amount of NKG2DL-IL10-CAR virus to be added per well based on MOI=3 (virus addition per well = MOI × 3 × 10). 6 (Virus titer), add virus, and mix by pipetting. The control group consists of cells supplemented with only Protamine.

[0085] 5.4 Centrifuge at 1000×g, room temperature, with both acceleration and deceleration rates set to 3, for 30 min to promote cell adhesion. Then, place the cells in a 37℃, 5% CO2 cell culture incubator.

[0086] 5.5 After adding the virus for 24 hours, add X-VIVO at a concentration of 40 IU / mL IL-12 to each well. TM 15. Serum-free culture medium.

[0087] 5.6 Samples were taken on day 3 post-infection, and the expression of NKG2DL-CAR in control T cells and NKG2DL-IL10-CAR-T cells was detected by flow cytometry. Results are as follows: Figure 2 As shown, the positivity rate of NKG2DL-CAR in NKG2DL-IL10-CAR-T cells is around 70%.

[0088] Example 6. In vitro proliferation experiment of NKG2DL-IL10-CAR-T cells

[0089] On day 3 post-infection, NKG2DL-IL10-CAR-T cells from Example 5 and control T cells were collected, centrifuged at 500×g at room temperature for 10 min, and then treated with X-VIVO containing 40 IU / mL IL-12. TM Cells were resuspended in serum-free medium, samples were taken for cell counting, and the total number of cells in each group was recorded. The cell density of each group was then adjusted to 1 × 10⁻⁵. 6 Cells / mL. Cells were packed at a ratio of 1×10⁻⁶. 6 Seeds were placed at 1 cell per well in a 6-well plate, and culture medium was added to a final volume of 3 mL. Cell counts were taken periodically. Figure 3 As shown, NKG2DL-IL10-CAR-T cells have a stronger proliferative capacity and a longer duration of action compared to untransduced T cells.

[0090] Example 7. In vitro T cell killing experiment

[0091] 7.1 Target Cell Culture: HeLa cells (Chinese Academy of Sciences Type Culture Collection Committee Cell Bank, catalog number: TCHU187) were selected as target cells. DMEM complete medium containing 10% FBS was prepared for cell culture. When cell confluence reached 80%, the medium was discarded, and 8 mL of trypsin (Thermo Fisher Scientific, catalog number: 25200072) was added for digestion at 37°C for 5 min. Digestion was then terminated by adding 20 mL of DMEM complete medium. The cells were centrifuged at 1000 rpm at room temperature for 5 min, and the supernatant was discarded. Cells were resuspended in DMEM complete medium and sampled for counting. The cell density was adjusted to 1 × 10⁶ cells / year. 6 Cells / mL, at 0.8 × 10⁻⁶ 6 1 cell / well was seeded into a 6-well plate, and culture medium was added to a final volume of 2 mL.

[0092] 7.2 Calculation of the number of cells required for different effector-to-target ratios: Untransduced T cells and NKG2DL-IL10-CAR-T cells were sampled, and the expression rate of NKG2DL-CAR was detected by flow cytometry. Actual effector cell number = T cell number × CAR expression rate (%); Number of T cells required for different effector-to-target ratios = E / T × 0.8 × 10⁻⁶ 6 / CAR positivity rate.

[0093] 7.3 Using X-VIVO TM 15. Serum-free culture medium was used to adjust the cell density of both NKG2DL-IL10-CAR-T cells and untransduced T cells to 1×10⁻⁵. 6 Cells / mL.

[0094] 7.4 Co-incubation of T cells and target cells: The effector-to-target ratio (E:T) of effector cells and HeLa target cells was set at 1:5 and 2:5, respectively. A blank control group was set up: HeLa cells were added only; a negative control group was set up: uninfected T cells were co-incubated with HeLa cells; experimental groups were set up: NKG2DL-IL10-CAR-T cells with different effector-to-target ratios were co-incubated with HeLa cells. Culture medium was added to each group to a final volume of 2 mL.

[0095] 7.5 After 24 hours, the remaining culture medium volume in each well was measured. After mixing, cells from each group were sampled, counted, and the total cell count was calculated. Simultaneously, the proportion of HeLa cells was detected by flow cytometry. HeLa cells were labeled with MICA / B antibody (Biolegend, catalog number: 320907), and the remaining HeLa cell count and cell killing efficiency in each group were calculated. Results are as follows... Figure 4 As shown, compared with untransduced T cells, NKG2DL-IL10-CAR-T cells significantly killed HeLa cells on days 10 and 20 after transduction.

[0096] References

[0097] [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. NatCancer. 2023; 4(9):1258-1272.doi:10.1038 / s43018-023-00600-4.

[0098] [2]Jin X,Xie D,Sun R,et al.CAR-T cells dual-target CD123 and NKG2DLsto eradicate AML cells and selectively target immunosuppressivecells.Oncoimmunology.2023;12(1):2248826.Published 2023 Aug 26.doi:10.1080 / 2162402X.2023.2248826.

[0099] [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 May4.doi:10.1038 / s41420-024-01976-7.

[0100] [4]Dong Yang et al.NKG2D-CAR T cells eliminate senescent cells inaged mice and nonhuman primates.Sci.Transl.Med.15,eadd1951(2023).

[0101] [5]Tang L,Pan S,Wei X,Xu X,Wei Q.Arming CAR-T cells with cytokinesand more:Innovations in the fourth-generation CAR-T development.MolTher.2023;31(11):3146-3162.doi:10.1016 / j.ymthe.2023.09.021.

[0102] [6]Ouyang W,O'GarraA.IL-10 Family Cytokines IL-10 and IL-22:fromBasic Science to Clinical Translation.Immunity.2019;50(4):871-891.doi:10.1016 / j.immuni.2019.03.020.

[0103] [7]Naing A,Papadopoulos KP,Autio KA,et al.Safety,Antitumor Activity,andImmune Activation ofPegylated Recombinant Human Interleukin-10(AM0010)inPatients WithAdvanced Solid Tumors.J Clin Oncol.2016;34(29):3562-3569.doi:10.1200 / JCO.2016.68.1106.

[0104] [8]Zhao Y,Chen J,Andreatta M,et al.IL-10-expressing CAR T cellsresist dysfunction and mediate durable clearance of solid tumors andmetastases.Nat Biotechnol.2024;42(11):1693-1704.doi:10.1038 / s41587-023-02060-8.

Claims

1. A chimeric antigen receptor targeting NKG2DL and containing IL-10, characterized in that, The chimeric antigen receptor comprises the following elements in sequence: 1) Signal peptide; 2) NKG2D; 3) Hinge area; 4) Transmembrane region; 5) Co-stimulatory areas; 6) Stimulation zone; 7) 2A sequence; and 8) IL-10 sequence.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor of claim 1 that targets NKG2DL and contains IL-10.

3. An expression carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2.

4. A recombinant lentivirus, characterized in that, The recombinant lentivirus comprises the nucleic acid molecule of claim 2 or is prepared using the expression vector of claim 3.

5. A method for preparing recombinant lentivirus, characterized in that, The method includes co-transfecting mammalian host cells with the expression vector and helper plasmid as described in claim 3.

6. A method for preparing CAR-T cells that target NKG2DL and secrete IL-10, characterized in that, The method includes infecting T cells with the recombinant lentivirus of claim 4.

7. CAR-T cells that target NKG2DL and secrete IL-10, characterized in that, The cells are prepared by the method of claim 6.

8. A disease treatment drug, characterized in that, The drug comprises the nucleic acid molecule of claim 2, the expression vector of claim 3, the recombinant lentivirus of claim 4, or the CAR-T cells that target NKG2DL and secrete IL-10 of claim 7.

9. The disease treatment drug according to claim 8, characterized in that, The diseases mentioned include cervical cancer, ovarian cancer, colorectal cancer, relapsed / refractory acute myeloid leukemia, high-risk myelodysplastic syndrome, multiple myeloma and osteosarcoma, as well as age-related diseases.

10. The use of the nucleic acid molecule of claim 2, the expression vector of claim 3, the recombinant lentivirus of claim 4, or the CAR-T cells that target NKG2DL and secrete IL-10 of claim 7 in the preparation of a disease treatment drug.

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