Use of trim21 inhibitors in the preparation of a product for enhancing tumor immune effect

Abstract

The application provides use of a TRIM21 inhibitor in preparation of a product for enhancing tumor immunity effect. Specifically, the application provides use of a TRIM21 inhibitor in preparation of a product for inhibiting TRIM21 from improving application of an immune checkpoint CTLA-4 therapeutic antibody and CAR T cell therapy in tumor treatment. + The application finds that, in a mouse tumor model, whole-body knockout of Trim21 significantly reduces expression amount of an immune checkpoint PD-1 protein and activates cytotoxic CD8 T lymphocytes, thereby enhancing sensitivity of the tumor to the CTLA-4 therapeutic antibody. Moreover, the CAR T cell with the knockout of Trim21 has stronger anti-tumor capacity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of TRIM21 inhibitors in the preparation of products for enhancing tumor immune response. Background Technology

[0002] The advent of tumor immunotherapy has transformed some deadly cancers into manageable chronic diseases. Since T cells are key participants in anti-tumor immunity, most current research in tumor immunotherapy revolves around inducing T cell activation-mediated anti-tumor immune responses. PD-1 is a major inhibitory receptor expressed on the surface of activated T cells and other immune cells. PD-1 weakens the activity of cytotoxic T cells by binding to its ligand PD-L1, leading to T cell dysfunction and exhaustion, thereby suppressing tumor immunity. Therefore, by using PD-1 / PD-L1 blocking antibodies to inhibit the binding of PD-1 to PD-L1, an anti-tumor response of cytotoxic T cells can be induced, thereby killing tumor cells. Currently, several PD-1 / PD-L1 blocking antibodies have been approved clinically for treating different types of tumors. However, only a small percentage of cancer patients show sustained response to PD-1 / PD-L1 blocking antibody treatment; most patients do not respond to this therapy or develop significant resistance after a brief response.

[0003] Therefore, there is an urgent clinical need to find products that can enhance the efficacy of tumor immunity. Summary of the Invention

[0004] The purpose of this invention is to provide the use of TRIM21 inhibitors in the preparation of products for enhancing tumor immune response. Through research, this invention has found that TRIM21 inhibitors can downregulate PD-1 protein expression by inhibiting TRIM21 gene expression, protein expression, or protein activity, thereby activating the anti-tumor immune response in the tumor microenvironment and enhancing the efficacy of tumor immunotherapy.

[0005] To achieve the aforementioned objective, the present invention adopts the following technical solution:

[0006] In a first aspect of the invention, the use of a TRIM21 inhibitor in the preparation of a product for enhancing tumor immune efficacy is provided.

[0007] Furthermore, the TRIM21 inhibitor includes at least one of a product that inhibits TRIM21 gene expression, a product that inhibits TRIM21 protein, and a product that inhibits the functional activity of TRIM21 protein.

[0008] Further, the TRIM21 inhibitor includes a product that inhibits TRIM21 gene expression or the product that inhibits TRIM21 protein expression includes at least one of polynucleotides, plasmids packaged with lentiviruses or retroviruses, viruses, lipids, and TRIM21 inhibitory antibodies; the product that inhibits the functional activity of TRIM21 protein includes at least one of proteins, peptides, enzymes, and small molecule compounds that inhibit the activity of TRIM21 protein.

[0009] Furthermore, the tumors in the product for enhancing tumor immunity include at least one of lung cancer, colon cancer, ovarian cancer, breast cancer, myeloma, neuroblastoma-derived CNS tumors, monocytic leukemia, B-cell-derived leukemia, T-cell-derived leukemia, B-cell-derived lymphoma, T-cell-derived lymphoma, and mast cell-derived tumors.

[0010] In a second aspect of the invention, the use of a combination of a TRIM21 inhibitor and an anti-CTLA-4 therapeutic antibody in the preparation of a product for enhancing tumor immune response is provided.

[0011] In a third aspect of the invention, the use of a TRIM21 inhibitor in the preparation of products for enhancing the ability of CD19-CAR T cells to kill tumor cells is provided.

[0012] In a fourth aspect of the invention, a product for enhancing tumor immune response is provided, said product comprising a TRIM21 inhibitor.

[0013] In a fifth aspect of the invention, a product for enhancing tumor immune response is provided, the product comprising a composition of a TRIM21 inhibitor and an anti-CTLA-4 therapeutic antibody.

[0014] Furthermore, the product for enhancing tumor immune efficacy also includes pharmaceutically additive excipients.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0016] 1. This invention provides the use of TRIM21 inhibitors in the preparation of products for enhancing tumor immune response. By using TRIM21 inhibitors to reduce TRIM21 expression or inhibit TRIM21 catalytic activity, the expression of PD-1 protein is further downregulated, thereby activating the anti-tumor immune response in the tumor microenvironment and enhancing the effect of tumor immunotherapy.

[0017] 2. This invention has found through research that when TRIM21 inhibitors are used in combination with CTLA-4 therapeutic antibodies, they have a synergistic effect and enhance the therapeutic effect on tumors.

[0018] 3. Trim21 gene knockout significantly enhances the ability of CD19-CAR T cells to kill tumor cells, indicating that TRIM21 inhibitors can be used in the preparation of products that enhance the ability of CD19-CAR T cells to kill tumor cells. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention.

[0020] Figure 1 To knock down / knock out the Trim21 gene in cells or mice to downregulate the expression of PD-1 protein; Figure 1 In the diagram, Figure A shows the protein expression of TRIM21 and PD-1 after infecting Jurkat cells with lentivirus and knocking down Trim21 in Jurkat cells with shRNA; Figure B shows the protein expression of TRIM21 and PD-1 after infecting MOLT-4 cells with lentivirus and knocking down Trim21 in MOLT-4 cells with shRNA; Figure C shows the isolation of CD8 from the spleens of WT / Trim21 KO mice. + T cells were used to detect the protein expression of TRIM21 and PD-1; Figure D shows the protein expression of TRIM21 and PD-1 in spleen tissues from WT / Trim21 KO mice; Figure E shows the protein expression of TRIM21 and PD-1 in thymus tissues from WT / Trim21 KO mice; Figure F shows the protein expression of TRIM21 and PD-1 in lymph node (LN) tissues from WT / Trim21 KO mice.

[0021] Figure 2 In an LLC tumor-bearing mouse model, knocking out Trim21 followed by treatment with the CTLA-4 therapeutic antibody enhances the efficacy of tumor immunotherapy by activating the immune response. Figure 2 Figure A shows the effect of subcutaneous transplantation of LLC cells in mice, WT mice, mice with systemic Trim21 gene knockout, and treatment with CTLA-4 therapeutic antibody alone or after systemic Trim21 knockout followed by CTLA-4 therapeutic antibody on tumor growth in mice. Figure B shows a comparison of LLC tumor size after subcutaneous transplantation of LLC cells in mice, WT mice, mice with systemic Trim21 knockout, and treatment with CTLA-4 therapeutic antibody alone or after systemic Trim21 knockout followed by CTLA-4 therapeutic antibody. Figure C shows the analysis of immune cells in mouse LLC tumor tissue, including infiltrating CD8 cells. +The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes; Figure D shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + T lymphocyte infiltration; Figure E shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of interferon-γ (IFNγ) in T lymphocytes; Figure F shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of tumor necrosis factor (TNF) in T lymphocytes; Figure G shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + Expression of granzyme B (GzmB) in T lymphocytes.

[0022] Figure 3 In the MC38 tumor-bearing mouse model, knocking out Trim21 followed by treatment with the CTLA-4 therapeutic antibody can enhance the efficacy of tumor immunotherapy by activating the immune response. Figure 3 Figure A shows the effect of subcutaneous transplantation of MC38 cells into mice, WT mice, mice with systemic Trim21 knockout, and mice treated with CTLA-4 therapeutic antibody alone or after systemic Trim21 knockout. Figure B shows a comparison of MC38 tumor size after subcutaneous transplantation of MC38 cells into mice, WT mice, mice with systemic Trim21 knockout, and mice treated with CTLA-4 therapeutic antibody alone or after systemic Trim21 knockout. Figure C shows the analysis of immune cells in mouse MC38 tumor tissue, including infiltrating CD8+ cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes; Figure D shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + T lymphocyte infiltration; Figure E shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of IFNγ in T lymphocytes; Figure F shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of TNF in T lymphocytes; Figure G shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + Expression of GzmB in T lymphocytes.

[0023] Figure 4 Knocking out Trim21 can enhance the anti-tumor effect of CD19-CAR T cells in LLC tumors. Figure 4Figure A shows the growth curve of LLC tumors after subcutaneous transplantation of human CD19-expressing LLC cells (LLC-hCD19) in mice, followed by tail vein injection of engineered WT and Trim21 KO anti-CD19 CAR T cells; Figure B shows the weight statistics of LLC tumors after subcutaneous transplantation of LLC-hCD19 cells in mice, followed by tail vein injection of engineered WT and Trim21 KO anti-CD19 CAR T cells; Figure C shows the analysis of immune cells in mouse LLC tumor tissue, including infiltrating CD8+ cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes; Figure D shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of IFNγ on T lymphocytes; Figure E shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + TNF expression in T lymphocytes; Figure F shows the analysis of immune cells in mouse LLC tumor tissue, CD8 + Expression of GzmB in T lymphocytes.

[0024] Figure 5 Knocking out Trim21 can enhance the anti-tumor effect of CD19-CAR T cells in MC38 tumors. Figure 5 Figure A shows the growth curve of MC38 tumors in mice after subcutaneous transplantation of MC38 cells expressing human CD19 (MC38-hCD19) followed by tail vein injection of engineered WT and Trim21 KO anti-CD19-CAR T cells; Figure B shows the tumor weight of MC38 tumors in mice after subcutaneous transplantation of MC38-hCD19 cells followed by tail vein injection of engineered WT and Trim21 KO anti-CD19-CAR T cells; Figure C shows the analysis of immune cells in mouse MC38 tumor tissue, including infiltrating CD8+ cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes; Figure D shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of IFNγ in T lymphocytes; Figure E shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + TNF expression in T lymphocytes; Figure F shows the analysis of immune cells in mouse MC38 tumor tissue, CD8 + Expression of GzmB in T lymphocytes. Detailed Implementation

[0025] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0026] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0027] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0028] The materials used in the embodiments of this invention are as follows:

[0029] 1. Cell lines

[0030] The MOLT-4 lymphocyte cell line and LLC mouse lung cancer cell line were purchased from the ATCC cell bank. The Jurkat lymphocyte cell line was kindly provided by Professor Liu Hudan of Wuhan University, and the MC38 mouse colon cancer cell line was kindly provided by the laboratory of Dr. Arlene H. Sharpe of Harvard Medical School. The LLC and MC38 cell lines were cultured in complete medium (DMEM, Hyclone) with 10% fetal bovine serum (Cellmax), 100U penicillin, and 100mg / ml streptomycin (Hyclone). The Jurkat and MOLT-4 cells were cultured in complete medium (1640, Hyclone) with 10% fetal bovine serum (Cellmax), 100U penicillin, and 100mg / ml streptomycin (Hyclone).

[0031] 2. Mice

[0032] Trim21 KO C57BL / 6 mice were purchased from Cyagen Pharmaceuticals and bred into homozygous knockout mice after validation at the DNA, RNA, and protein levels. C57BL / 6 mice were purchased from Jicui Pharmaceutical Co., Ltd. All protocols and experiments for the mouse model were conducted in accordance with relevant ethical guidelines and approved by the Institutional Animal Care and Use Committee of Wuhan University (MRI2021-LAC213, MRI2021-LAC217, and MRI2022-LAC015). All mice used in the experiments were housed in standard housing conditions within a pathogen-free facility at the Institute of Medical Sciences, Wuhan University.

[0033] 3. Antibodies

[0034] Flow cytometry antibodies Anti-Mouse PD-1PE, Anti-Mouse CD8αBV510, Anti-Mouse IFNγAPC, and Anti-Mouse TNF PE were purchased from BD Biosciences; Anti-Mouse GzmB was purchased from eBioscience; and Anti-Human PD-1PE was purchased from BioLegend. Western blot antibodies Anti-PD-1(D4W2J) rabbit mAb, Anti-PD-1(Intracellular Domain)(D7D5W) rabbit mAb, and Anti-Tubulin rabbit pAb were purchased from CST; Anti-TRIM21 mouse mAb and Anti-TRIM21 rabbit pAb were purchased from Proteintech; and Anti-Vinculin(VIN-11-5) mouse mAb was purchased from Sigma-Aldrich. The therapeutic antibody In vivoMAb anti-mouse CTLA-4 (clone 9H10) was purchased from BioX Cell.

[0035] The following will describe in detail the use of the TRIM21 inhibitor of this application in the preparation of products for enhancing tumor immune response, in conjunction with examples and experimental data.

[0036] Example 1: Experiment on the effect of inhibiting TRIM21 expression to enhance anti-tumor immunotherapy

[0037] 1. Packaging lentiviruses and infecting Jurkat and MOLT-4 cell lines

[0038] (1) Construction of the target plasmid: The shRNA of the Trim21 gene was annealed and inserted into the pLKO plasmid vector (the restriction sites are Age I and EcoRI). The shTRIM21 sequence used is as follows:

[0039] Sequence (5'-3') shTRIM21_#1 5'-TGGAAGTGGAAATTGCAATAA-3'(SEQ ID NO.1) shTRIM21_#2 5'-GAAGAGAGATTTGATAGTTAT-3'(SEQ ID NO.2) shTRIM21_#3 5'-GATCCCAGCAAGCGAGCTTTA-3'(SEQ ID NO.3)

[0040] (2) Cell preparation: 1-2 days before cell transfection, 293T cells are seeded in a 6cm dish. Transfection begins when the cell density reaches 70-80%.

[0041] (3) Virus packaging: Based on the ratio of 1 μg shTRIM21 plasmid, 1 μg pVSVG, and 1 μg pD8.9, with a PEI volume (μL) / plasmid mass (μg) ratio of 3:1, the plasmid was transfected with PEI to package the virus in 293T cells. Viral supernatant was collected twice, at 36 h and 60 h after transfection. The collected viral supernatants were mixed and filtered through a 0.45 μm filter to collect the viral solution.

[0042] (4) Prepare Jurkat or MOLT-4 cell lines at a density of approximately 40% using 6-well plates. Add 1 mL of virus solution and 1 mL of fresh complete culture medium to each well, and centrifuge at 2300 rpm for 90 min at room temperature to improve infection efficiency. After culturing in an incubator for 12 h, centrifuge at 1000 rpm for 3 min to remove the supernatant, and add virus solution and complete culture medium to the cell lines again. Continue culturing for another 12 h, then replace with fresh complete culture medium and culture for 24 h.

[0043] (5) After infection, cells were screened with 1 μg / μL of puromycin.

[0044] 2. The knockdown effect of Trim21 and changes in PD-1 expression were detected using Western blotting. The specific steps for Western blotting (WB) are as follows:

[0045] ① Collect protein samples: Lyse cells at 4°C for 15 min using cell lysis buffer (1M Tris-HCl, pH=7.5, 5M NaCl, 0.5% NP40); then centrifuge the lysed cells at 12000 rpm for 10 min at 4°C, collect the supernatant and determine the protein concentration using a BCA kit. Add 3× Loading Buffer to the supernatant, boil in a metal bath for 5 min, load the samples and perform SDS-PAGE.

[0046] ② SDS-PAGE and transfer: Perform protein electrophoresis in protein electrophoresis buffer at a constant voltage of 120V (about 70 min). After electrophoresis, transfer the protein to a membrane and perform protein transfer in protein transfer buffer at a constant current of 300mA for 1.5-2 h.

[0047] ③ Blocking and antibody application: After the transfer, place the PVDF membrane in 5% skim milk at room temperature for 1 hour. After blocking, place the blocked PVDF membrane in the antibody dilution solution required for the experiment and incubate overnight at 4°C.

[0048] ④ ECL Development: After primary antibody incubation, wash the PVDF membrane 5 times with TBST, incubate with secondary antibody at room temperature for 1 hour, and after secondary antibody incubation, continue washing the PVDF membrane 5 times with TBST. Then, develop with ECL developing solution.

[0049] 3. Experimental Results

[0050] (1) Jurkat cells were infected with lentivirus. After knocking down Trim21 in Jurkat cells with shRNA, the protein expression of TRIM21 and PD-1 was detected as follows: Figure 1 As shown in Figure A, knocking down TRIM21 in Jurkat cells can significantly reduce the expression of PD-1 protein.

[0051] (2) MOLT-4 cells were infected with lentivirus. After knocking down Trim21 in MOLT-4 cells with shRNA, the protein expression of TRIM21 and PD-1 was detected as follows: Figure 1 As shown in Figure B, knocking down TRIM21 in MOLT-4 cells can significantly reduce the expression of PD-1 protein.

[0052] (3) CD8 groups were isolated from the spleens of WT / Trim21 KO mice. + T cells were used to detect the protein expression of TRIM21 and PD-1. Figure 1 As shown in Figure C, compared with WT mice, the CD82 content isolated from the spleen of Trim21 KO mice is significantly higher. + The expression of PD-1 protein in T cells is significantly reduced.

[0053] (4) Spleen tissues were collected from WT / Trim21 KO mice to detect the protein expression of TRIM21 and PD-1. Figure 1 As shown in Figure D, compared with WT mice, the expression of PD-1 protein in the spleen tissue of Trim21 KO mice was significantly reduced.

[0054] (5) Thymus tissue was taken from WT / Trim21 KO mice, and the protein expression of TRIM21 and PD-1 was detected as follows: Figure 1 As shown in E, compared with WT mice, the expression of PD-1 protein in the thymus tissue of Trim21 KO mice was significantly reduced.

[0055] (6) Figure F shows the expression of TRIM21 and PD-1 proteins in lymph node (LN) tissues taken from WT / Trim21 KO mice. Figure 1 As shown in Figure F, the expression of PD-1 protein in the lymph node tissue of Trim21 KO mice was significantly reduced compared with WT mice.

[0056] The above experimental results indicate that the inventors of this application discovered that knocking out Trim21 downregulates the expression of the immune checkpoint protein PD-1 in various lymphocytes, including human T-lymphoblastic leukemia cells (Jurkat and MOLT-4) and mouse primary T lymphocytes. Furthermore, they found that TRIM21 can stabilize PD-1 by enhancing its interaction with PD-1 and inhibiting polyubiquitination at the PD-1 K63 position, thereby suppressing polyubiquitination at the PD-1 K48 position and proteasome degradation. In the tumor immune microenvironment, high expression of PD-1 leads to dysfunction of cytotoxic T cells, resulting in immune escape and tumor development. Therefore, inhibiting TRIM21 expression can significantly reduce PD-1 expression, further activating the anti-tumor immune response of T cells in tumor tissue, thereby enhancing the efficacy of anti-tumor immunotherapy.

[0057] Example 2: Trim21 gene knockout combined with CTLA-4 antibody therapy trial

[0058] 1. LLC and MC38 cell preparation: Digest cells with 0.05% trypsin, wash twice with PBS, then terminate digestion with DMEM and centrifuge to remove the supernatant. Resuspend cells in DMEM, thoroughly pipette the cells, and perform cell counting to adjust the cell density to 5 × 10⁶ cells / year. 6 / mL. During cell preparation, to prevent excessively long cell digestion time and excessively high cell growth density, the maximum cell density should be controlled below 80% to maintain optimal cell growth.

[0059] 2. Cell transplantation: 100 μL of LLC or MC38 cell suspension (resuspended in serum-free MDEM) was injected subcutaneously into the axilla of 6-8 week old C57BL / 6WT and Trim21 KO C57BL / 6 mice. After injection, tumor formation was closely observed.

[0060] 3. Medication Strategy: Mice injected with LLC or MC38 cells were divided into four groups: WT group, Trim21 KO group, WT mouse CTLA-4 antibody treatment group, and Trim21 KO mouse CTLA-4 antibody treatment group. ① 100 μL of LLC or MC38 cell suspension (resuspended in serum-free MDEM) was injected subcutaneously into the axilla of 6-8 week old C57BL / 6WT mice to form the WT group; ② 100 μL of LLC or MC38 cell suspension (resuspended in serum-free MDEM) was injected subcutaneously into the axilla of 6-8 week old Trim21KO mice to form the Trim21KO group; ③ WT mice were injected with CTLA-4 antibody to form the WT mouse CTLA-4 antibody treatment group; ④ Trim21 KO mice were injected with CTLA-4 antibody to form the Trim21 KO mouse CTLA-4 antibody treatment group. The dosage of the above CTLA-4 monoclonal antibody was 100 μg / mouse, administered intraperitoneally every three days.

[0061] 4. Tumor Measurement and Mouse Sacrifice: Tumor growth was observed and recorded starting on day 5 after tumor cell injection. The length and width of the mouse's tumor were measured every two days using calipers, using the formula: length × width. 2 The tumor volume in mice was calculated by multiplying the value by 0.5. According to laboratory animal ethics, a tumor volume of 2000 mm² in mice was considered inappropriate. 3 Mice were euthanized if the tumor ulceration diameter exceeded 1 cm. Tumor growth curves were recorded and plotted after cell injection, as follows:

[0062] (1) The effects of subcutaneous transplantation of LLC cells into mice, systemic knockout of the Trim21 gene in mice, and treatment with either CTLA-4 therapeutic antibody alone or systemic knockout of Trim21 followed by CTLA-4 therapeutic antibody on tumor growth in mice were as follows: Figure 2 As shown in Figure A, it can be seen that in the LLC tumor transplantation model, Trim21 KO mice showed the most effective tumor control when receiving anti-CTLA-4 treatment, which was manifested as a significant delay in tumor growth.

[0063] (2) LLC cells were subcutaneously transplanted into mice, and Trim21 was knocked out systemically in mice. Treatment with either CTLA-4 therapeutic antibody alone or CTLA-4 therapeutic antibody after systemic Trim21 knockout in mice was performed. The comparison of LLC tumor growth size is shown in the figure below. Figure 2 As shown in B, in the LLC tumor transplantation model, compared with WT mice, Trim21 KO mice showed the most effective tumor control when receiving anti-CTLA-4 treatment, as evidenced by a significant decrease in tumor size.

[0064] 5. Tumor-infiltrating lymphocyte analysis: Intact tumors were harvested from euthanized mice, grouped, and photographed. Fresh tumors were minced into pieces approximately 1-3 mm in size and digested in tumor digestion buffer (1 mg / mL collagenase IV, 0.5 mg / mL collagenase I, and 0.002 mg / mL hyaluronidase dissolved in HBSS) at 37°C for 30 min. After passing through a 70 μM filter, erythrocytes in the single-cell suspension were lysed with ACK lysis buffer (A1049201, Thermo Fisher). To analyze T-cell effector function, cells were incubated with appropriate antibodies at 4°C in the dark for 20 min. After antibody staining, the cells were analyzed by flow cytometry. The specific results are as follows:

[0065] (1) In the LLC tumor-bearing mouse model,

[0066] ① Analysis of immune cells in mouse LLC tumor tissue revealed infiltrating CD8 cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes is as follows: Figure 2 As shown in Figure C, in the LLC tumor transplantation model, compared with WT mice, Trim21KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The expression level of PD-1 on the surface of T cells is reduced;

[0067] ②Analysis of immune cells in mouse LLC tumor tissue, CD8 + T lymphocyte infiltration status, such as Figure 2 As shown in Figure D, in the LLC tumor transplantation model, compared with WT mice, Trim21 KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of T cells increased significantly;

[0068] ③ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of IFNγ in T lymphocytes is as follows: Figure 2 As shown in Figure E, in the LLC tumor transplantation model, compared with WT mice, Trim21 KO mice exhibited tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of IFNγ secreted by T cells increased significantly;

[0069] ④ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of TNF in T lymphocytes is as follows: Figure 2 As shown in Figure F, in the LLC tumor transplantation model, compared with WT mice, Trim21 KO mice exhibited tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. +The percentage of TNF secreted by T cells increased significantly;

[0070] ⑤ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of GzmB in T lymphocytes is as follows: Figure 2 As shown in G, in the LLC tumor transplantation model, compared with WT mice, Trim21 KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of GzmB secreted by T cells increased significantly;

[0071] (2) In the MC38 tumor-bearing mouse model,

[0072] ① The effects of subcutaneous transplantation of MC38 cells into mice, systemic knockout of Trim21 in mice, and treatment with either CTLA-4 therapeutic antibody alone or systemic knockout of Trim21 followed by treatment with CTLA-4 therapeutic antibody on tumor growth in mice are as follows: Figure 3 As shown in A, it can be seen that in the MC38 tumor transplantation model, Trim21 KO mice showed the most effective tumor control when receiving anti-CTLA-4 treatment, which was manifested as a significant delay in tumor growth.

[0073] ② MC38 cells were subcutaneously transplanted into mice, and Trim21 was knocked out systemically in mice. Treatment with either CTLA-4 therapeutic antibody alone or CTLA-4 therapeutic antibody after systemic Trim21 knockout in mice was performed. The comparison of LLC tumor growth size is shown in the following figure. Figure 3 As shown in B, in the MC38 tumor transplantation model, compared with WT mice, Trim21 KO mice showed the most effective tumor control when receiving anti-CTLA-4 treatment, as evidenced by a significant decrease in tumor size.

[0074] ③ Analysis of immune cells in mouse MC38 tumor tissue revealed infiltrating CD8 cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes is as follows: Figure 3 As shown in Figure C, in the LLC tumor transplantation model, compared with WT mice, Trim21KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The expression level of PD-1 on the surface of T cells is reduced;

[0075] ④ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + T lymphocyte infiltration status, such as Figure 3 As shown in Figure D, in the MC38 tumor transplantation model, compared with WT mice, Trim21 KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. +The percentage of T cells increased significantly;

[0076] ⑤ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of IFNγ in T lymphocytes is as follows: Figure 3 As shown in E, in the MC38 tumor transplantation model, compared with WT mice, Trim21 KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of IFNγ secreted by T cells increased significantly;

[0077] ⑥ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of TNF in T lymphocytes is as follows: Figure 3 As shown in Figure F, in the MC38 tumor transplantation model, compared with WT mice, Trim21 KO mice exhibited tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of TNF secreted by T cells increased significantly.

[0078] ⑦ To analyze immune cells in mouse MC38 tumor tissue, CD8 + The expression of GzmB in T lymphocytes is as follows: Figure 3 As shown in G, in the MC38 tumor transplantation model, compared with WT mice, Trim21 KO mice showed tumor-infiltrating CD8+ when receiving anti-CTLA-4 treatment. + The percentage of GzmB secreted by T cells increased significantly;

[0079] Example 3: Trim21 gene knockout CD19-CAR T therapy experiment for mouse tumors

[0080] 1. Construction of anti-CD19 CAR

[0081] The anti-CD19 single-stranded fragment variable (scFv) nucleotide was derived from a mouse antibody (clone FMC63) (Ying et al., 2019). The CD19 scFv was cloned into the PLVX-puro lentiviral vector (with EcoRI and MulI restriction sites), which was fused with the mouse CD8α hinge, CD28 transmembrane and intracellular domains, and CD3ζ-derived cytoplasmic domains. The lentivirus was generated using a polyethyleneimine-based DNA transfection system described in a previously published article (Zhang et al., 2022b).

[0082] 2. Isolation of mouse T cells and transduction of CAR.

[0083] Mouse T cells were isolated from spleen cells of Trim21 WT and KO mice using the mouse CD3ε MicroBead Kit, and then used with Dynabeads TM Mouse T-activator CD3 / CD28 was activated for 24 hours. Then, cells were infected with lentiviruses (MOI range 5 to 10) in a culture containing... Activated T cells cultured in SFM with GMP recombinant human IL-2 (1000 IU / ml) and L-glutamine (2 mM) in CTSTMOpTmizer™ T cell expansion.

[0084] 3. Experimental Results

[0085] (1) Subcutaneous transplantation of LLC cells expressing human CD19 (LLC-hCD19) into mice.

[0086] ① After subcutaneous transplantation of LLC-hCD19 cells into mice, and subsequent treatment with engineered WT and Trim21 KO anti-CD19 CAR-T cells via tail vein injection, the growth curve of LLC tumors is as follows: Figure 4 As shown in Figure A, anti-CD19-CAR T cells showed better efficacy in inhibiting LLC-hCD19 tumor growth than control PBS treatment, indicating that engineered CAR T cells worked well; moreover, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells significantly delayed tumor growth.

[0087] ② After subcutaneous transplantation of LLC-hCD19 cells into mice, and treatment with engineered WT and Trim21 KO anti-CD19-CAR T cells via tail vein injection, the statistical results of LLC tumor weight are as follows: Figure 4 As shown in Figure B, anti-CD19-CAR T cells showed better efficacy than control PBS treatment in inhibiting LLC-hCD19 tumor growth, with significantly delayed tumor growth and reduced tumor weight. Moreover, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells showed a significant decrease in tumor weight.

[0088] ③ Analysis of immune cells in mouse LLC tumor tissue revealed infiltrating CD8 cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes is as follows: Figure 4 As shown in Figure C, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21KO anti-CD19-CAR T cells showed tumor-infiltrating CD8+. + The expression level of PD-1 on the surface of T cells is reduced;

[0089] ④ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of IFNγ in T lymphocytes is as follows: Figure 4 As shown in Figure D, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells showed tumor-infiltrating CD8+. + The percentage of IFNγ secreted by T cells increased significantly;

[0090] ⑤ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of TNF in T lymphocytes is as follows: Figure 4 As shown in Figure E, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells exhibited tumor-infiltrating CD8+. + The percentage of TNF secreted by T cells increased significantly;

[0091] ⑥ Analysis of immune cells in mouse LLC tumor tissue, CD8 + The expression of GzmB in T lymphocytes is as follows: Figure 4 As shown in Figure F, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells exhibited tumor-infiltrating CD8+. + The percentage of GzmB secreted by T cells increased significantly;

[0092] (2) Subcutaneous transplantation of MC38 cells expressing human CD19 (MC38-hCD19) into mice.

[0093] ① After subcutaneous transplantation of MC38-hCD19 cells into mice, and subsequent treatment with engineered WT and Trim21 KO anti-CD19 CAR-T cells via tail vein injection, the growth curve of MC38 tumors is as follows: Figure 5 As shown in Figure A, anti-CD19-CAR T cells showed better efficacy in inhibiting LLC-hCD19 tumor growth than control PBS treatment, indicating that engineered CAR T cells worked well; moreover, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells significantly delayed tumor growth.

[0094] ② After subcutaneous transplantation of MC38-hCD19 cells into mice, and treatment with engineered WT and Trim21 KO anti-CD19-CAR T cells via tail vein injection, the MC38 tumor weight was statistically analyzed as follows: Figure 5As shown in Figure B, anti-CD19-CAR T cells showed better efficacy than control PBS treatment in inhibiting MC38-hCD19 tumor growth, as evidenced by significantly delayed tumor growth and reduced tumor weight. Moreover, compared with mice treated with WT anti-CD19 CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells showed a significant decrease in tumor weight.

[0095] ③ Analysis of immune cells in mouse MC38 tumor tissue revealed infiltrating CD8 cells. + The mean fluorescence intensity (MFI) of PD-1 in T lymphocytes is as follows: Figure 5 As shown in Figure C, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21KO anti-CD19-CAR T cells showed tumor-infiltrating CD8+. + The expression level of PD-1 on the surface of T cells is reduced;

[0096] ④ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of IFNγ in T lymphocytes is as follows: Figure 5 As shown in Figure D, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells showed tumor-infiltrating CD8+. + The percentage of IFNγ secreted by T cells increased significantly;

[0097] ⑤ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of TNF in T lymphocytes is as follows: Figure 5 As shown in Figure E, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells exhibited tumor-infiltrating CD8+. + The percentage of TNF secreted by T cells increased significantly;

[0098] ⑥ Analysis of immune cells in mouse MC38 tumor tissue, CD8 + The expression of GzmB in T lymphocytes is as follows: Figure 5 As shown in Figure F, compared with mice treated with WT anti-CD19-CAR T cells, mice treated with Trim21 KO anti-CD19-CAR T cells exhibited tumor-infiltrating CD8+. + The percentage of GzmB secreted by T cells increased significantly;

[0099] The above experimental results indicate that Trim21 gene knockout significantly enhances the ability of CD19-CAR T cells to kill tumor cells.

[0100] In summary, in a mouse tumor-bearing model, compared to using the CLTA-4 therapeutic antibody alone, we found that treatment with the CLTA-4 therapeutic antibody after knocking out the Trim21 gene in the host mouse significantly inhibited tumor growth and prolonged the survival of mice. Simultaneously, we also found that knocking out the Trim21 gene significantly enhanced the ability of CD19-CAR T cells to kill tumor cells. More importantly, we found that whether it was knocking out the Trim21 gene alone, combining it with the CLTA-4 therapeutic antibody, or integrating Trim21 gene knockout with CD19-CAR T cell therapy, all of these significantly increased the number of CD8 cells infiltrating tumor tissue. + The number and activity of T cells.

[0101] Finally, it should be noted that 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 process, method, article, or apparatus.

[0102] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0103] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a TRIM21 inhibitor for the manufacture of a medicament for enhancing the immune effect on a tumor, characterized in that, The TRIM21 inhibitor is selected from shRNA sequences that inhibit TRIM21 gene expression, such as those shown in SEQ ID NO.1-3, and the tumor is lung cancer or colon cancer.

2. The application of a combination of a TRIM21 inhibitor and an anti-CTLA-4 therapeutic antibody in the preparation of a drug for enhancing tumor immune response, wherein the TRIM21 inhibitor is selected from shRNA sequences that inhibit TRIM21 gene expression, such as those shown in SEQ ID NO.1-3, and the tumor is lung cancer or colon cancer.

3. The use of a TRIM21 inhibitor in the preparation of a drug for enhancing the ability of CD19-CAR T cells to kill tumor cells, wherein the TRIM21 inhibitor is selected from shRNA sequences that inhibit TRIM21 gene expression as shown in SEQ ID NO.1-3, and the tumor is lung cancer or colon cancer.

4. A drug for enhancing tumor immune response, characterized in that, The drug comprises a composition of a TRIM21 inhibitor and an anti-CTLA-4 therapeutic antibody, wherein the TRIM21 inhibitor is selected from shRNA sequences that inhibit TRIM21 gene expression, such as those shown in SEQ ID NO.1-3, and the tumor is lung cancer or colon cancer.

5. The drug for enhancing tumor immunity according to claim 4, characterized in that, The drugs used to enhance tumor immunity also include pharmaceutically additive excipients.

Images