Use of t cells overexpressing prdm16 in preparation of drugs for resisting tumor or treating infectious diseases

Abstract

This invention discloses the application of T cells overexpressing PRDM16 in the preparation of anti-tumor or infectious disease drugs. Through experiments, this invention verifies that overexpression of PRDM16 enhances the in vivo and in vitro activity of T cells. It was found that in vitro overexpression of PRDM16 significantly enhances the killing effect of T cells on target cells. In vivo reinfusion of PRDM16 has stronger anti-tumor and anti-infection functions compared to wild-type T cells. Therefore, it can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy and solve the defect of T cell adoptive therapy where the killing power of target cells is suppressed due to environmental influences.

Description

Technical Field

[0001] This invention relates to the field of tumor and infectious disease technology, specifically to the application of T cells overexpressing PRDM16 in the preparation of anti-tumor or infectious disease treatment drugs. Background Technology

[0002] Adoptive T cell therapy (ACT), a highly promising immunomodulatory intervention, uses autologous or allogeneic T lymphocytes as its core. These cells are modified or activated in vitro and then reinfused into the patient to precisely identify and eliminate tumor cells or pathogens. ACT has demonstrated significant efficacy in the treatment of various malignant tumors and infectious diseases. Currently, this technology has developed into several important branches, including chimeric antigen receptor-modified T cells (CAR-T), T cell receptor-engineered T cells (TCR-T), and tumor-infiltrating lymphocytes (TILs). With its high specificity and precise targeting, it has broad clinical application prospects and commercial transformation value in hematologic malignancies, solid tumors, and chronic infectious diseases. However, the widespread adoption and improvement of ACT efficacy are still limited by several key bottlenecks. One of the core issues is the insufficient survival ability and functional stability of T cells in the tumor microenvironment and long-term inflammatory environment, which severely restricts the durability and reliability of the treatment effect.

[0003] In the long-term inflammatory microenvironment induced by tumors or chronic infections, adoptive T cells are prone to exhaustion, a key factor leading to ACT treatment failure, tumor recurrence, or persistent infection. Exhausted T cells exhibit a series of functional defects, specifically: significantly reduced proliferation capacity, decreased secretion of effector cytokines (such as IFN-γ and TNF-α), and abnormal upregulation of inhibitory receptors such as PD-1, TIM-3, LAG-3, TIGIT, and CTLA4, while the expression levels of key genes regulating T cell activation, signal transduction, and effector functions, such as CCR3, RHOU, PIK3CG, IFNG, and GZMB, are significantly downregulated. More importantly, exhausted T cells undergo intracellular energy metabolism reprogramming, failing to effectively maintain normal biosynthesis and effector function requirements, further exacerbating their functional decline. Therefore, effectively inhibiting or reversing T cell exhaustion, enhancing their survival and functional durability in the pathological microenvironment, and improving their ability to recognize and eliminate target cells are crucial for improving the efficacy of adoptive immunotherapy. Summary of the Invention

[0004] The main objective of this invention is to propose the application of T cells overexpressing PRDM16 in the preparation of anti-tumor or infectious disease drugs, aiming to solve the problems of existing T cell adoptive therapy, such as T cell exhaustion, poor survival ability in the pathological microenvironment, and poor treatment effect.

[0005] To achieve the above objectives, this invention proposes the application of T cells overexpressing the PRDM16 gene in the preparation of drugs for treating tumors or infectious diseases, wherein the nucleotide sequence of the PRDM16 gene is shown in SEQ ID NO:1.

[0006] Preferably, the T cells are CD8. + T cells or CAR-T cells.

[0007] Preferably, the infection is a bacterial infection.

[0008] Preferably, the T cells overexpressing PRDM16 are obtained by infecting T cells with lentivirus, adenovirus, plasmid, LNP or mRNA carrying the PRDM16 gene.

[0009] Preferably, the T cells overexpressing PRDM16 include CD8 cells overexpressing PRDM16. + T cells, CD8 cells overexpressing PRDM16 + T cells exhibit enhanced proliferative capacity, higher levels of IFN-γ and granzyme B secretion, higher levels of T cell memory differentiation, and lower expression levels of exhaustion-related molecules PD-1 and TIM-3.

[0010] Preferably, the T cells overexpressing PRDM16 include CD8 cells overexpressing PRDM16. + T cells, CD8 cells overexpressing PRDM16 + T cells can inhibit tumor growth in vivo; the PRDM16-overexpressing T cells include PRDM16-overexpressing CAR-T cells, which, after being reinfused in vivo, can inhibit tumor growth.

[0011] Preferably, the T cells overexpressing PRDM16 include CD8 cells overexpressing PRDM16. + T cells or CAR-T cells overexpressing PRDM16, wherein the CD8+ overexpressing PRDM16 + T cells or CAR-T cells overexpressing PRDM16 have a stronger tumor-killing effect.

[0012] The present invention also proposes a pharmaceutical composition in which the active ingredient comprises the T cells overexpressing the PRDM16 gene.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] Preferably, the pharmaceutical composition is in the form of an injectable preparation.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) The T cells overexpressing PRDM16 provided by this invention have significant advantages in the preparation of anti-tumor drugs, providing a highly efficient candidate direction for optimizing tumor T cell adoptive therapy. This invention has clearly verified through experiments that overexpression of PRDM16 can significantly enhance the in vitro and in vivo activity of T cells, with the in vitro experiment showing a significant increase in the killing effect of these cells on target cells—whether CD8+ or CD9+. + Both T cells and CAR-T cells, after overexpressing PRDM16, exhibited stronger tumor-killing efficacy under different effector-to-target ratios. In vivo experiments showed that, compared to wild-type T cells, infusion of these cells significantly inhibited tumor growth, especially the anti-tumor effect of PRDM16-overexpressing CAR-T cells. Furthermore, these cells can significantly enhance their own proliferation capacity and increase the proportion of cell divisions, providing a sufficient cellular basis for anti-tumor immune responses. Simultaneously, they greatly promote the secretion of effector molecules such as IFN-γ and granzyme B, further enhancing killing efficacy. They also alleviate T cell exhaustion by increasing the proportion of central memory cell subsets and reducing the expression of exhaustion-related molecules such as PD-1 and TIM-3, maintaining long-term anti-tumor activity. This effectively addresses the deficiency in traditional T-cell adoptive therapy where target cell killing ability is easily suppressed by environmental influences, providing important support for improving the effectiveness and durability of tumor immunotherapy.

[0017] (2) The PRDM16-overexpressing T cells provided by this invention also have key application value in the preparation of drugs for treating infectious diseases, and can effectively enhance the efficacy of adoptive T cell therapy for anti-infection. Experimental verification of this invention shows that after in vivo reinfusion of PRDM16-overexpressing T cells, they have stronger anti-infection function compared with wild-type T cells. Taking the lethal dose infection model of bacteria expressing OVA antigen (Lm-OVA) as an example, after infusion treatment with OT1 cells overexpressing PRDM16, all infected mice survived, while the mortality rate of the non-overexpressing OT1 cell group reached 33.3%, and the survival rate of the PBS control group was 0%. This advantage stems from the comprehensive enhancement of T cell function by PRDM16. It can not only enhance the proliferation capacity of T cells to expand the anti-infection effector cell population, but also optimize the functional state of T cells and enhance the ability to clear infectious pathogens. It effectively solves the defect that the killing ability of traditional T cell adoptive therapy is easily suppressed by the environment, and provides an efficient cell therapy strategy for treating bacterial and other pathogen infections (especially lethal dose infections), and is expected to overcome the treatment problems of some refractory infectious diseases in clinical practice. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 In Example 2 of this invention, overexpression of PRDM16 affects CD8. + Figure A shows the effect of T cell proliferation capacity; Figure A is the control CD8. + T cells, CD8 cells overexpressing PRDM16 + Figure B shows the distribution of T cell division generations; Figure B is the control CD8. + T cells, CD8 cells overexpressing PRDM16 + Bar chart of average fluorescence intensity of T cells.

[0020] Figure 2 In Example 3 of this invention, overexpression of PRDM16 affects CD8. + Figure A shows the effects of T cell secretion of IFN-γ and granzyme B; Figure A shows the CD8+ groups of WT, WT+ tumor cells, Prdm16 overexpression, and Prdm16 overexpression+ tumor cells. + Figure B shows the expression levels of IFN-γ and granzyme B in T cells; Figure B shows the expression levels of CD8 in different treatment groups. + Bar chart showing the ratio of IFN-γ to granzyme B-positive cells in T cells.

[0021] Figure 3 In Example 4 of this invention, overexpression of PRDM16 affects CD8. + The effect of T cell central memory T differentiation on T cell differentiation; Figure A is a flow cytometry scatter plot of the WT group and the Prdm16.oe group; Figure B is the CD8+ central memory T cell differentiation of the WT group and the Prdm16.oe group. + A chart showing the percentage of different T cell subsets.

[0022] Figure 4 In Example 5 of this invention, overexpression of PRDM16 affects CD8. + Figure A shows the effects of PD-1 and TIM-3 expression on T cell exhaustion-related molecules; Figure A shows the effects of WT group and CD8 overexpression of PRDM16. + Contour scatter plots of PD-1 and TIM-3 in the T cell group; Figure B shows the WT group and CD8 cells overexpressing PRDM16. + CD8 in the T cell group + Bar chart showing the percentage of T cells.

[0023] Figure 5In Example 6 of this invention, overexpression of PRDM16 affects CD8. + The effect of T cells on tumor cell killing in vitro.

[0024] Figure 6 In Example 7 of this invention, overexpression of PRDM16 affects CD8. + Figure A shows the effect of T cells on anti-tumor function in vivo; Figure A shows the WT group and CD8 cells overexpressing PRDM16. + Figure B shows the tumor volume growth curve of the T cell group; Figure B shows the WT group and the CD8 group overexpressing PRDM16. + T-cell group tumor weight bar chart.

[0025] Figure 7 Figure 8 shows the effect of PRDM16 overexpression on the killing effect of CAR-T cells on tumor cells in vitro. Figure A shows the survival / apoptosis of tumor cells in different treatment groups WT, WT-CAR, Prdm16.oe, and Prdm16.oe-CAR. Figure B is a bar chart showing the proportion of exhausted cells in different treatment groups WT, WT-CAR, Prdm16.oe, and Prdm16.oe-CAR.

[0026] Figure 8 Figure 9 shows the effect of PRDM16 overexpression on inhibiting tumor growth of CAR-T cells in Example 9 of the present invention; Figure A shows the trend of tumor volume changes at different time points after tumor inoculation in different treatment groups: Blank control group, WT CAR group, and Prdm16.oe-CAR group; Figure B shows the tumor volume bars of WT, WT CAR group, and Prdm16.oe-CAR group.

[0027] Figure 9 CD8 overexpressing PRDM16 in Example 10 of this invention + Survival time curve of mice after in vivo reinfusion of OT1 cells for the treatment of bacterial infection.

[0028] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1: CD8 overexpressing PRDM16 + Preparation of T cells

[0032] 1. CD8 + Isolation and purification of T cells

[0033] (1) Collect 10 mL of peripheral blood sample;

[0034] (2) Take the above peripheral blood sample, add 10 mL of PBS buffer to dilute it, and follow the instructions of the red blood cell lysis buffer to add 2 times the volume of red blood cell lysis buffer to the diluted peripheral blood. Incubate at room temperature for 10 min. After lysis, centrifuge to collect the precipitate, wash and centrifuge again to obtain white blood cell suspension.

[0035] (3) CD8 antibody-conjugated magnetic beads were used to separate and purify CD8 using magnetic cell sorting (MACS) technology. + T cells were used to obtain purified CD8. + T cells.

[0036] 2. Construction and packaging of viral vectors for the PRDM16 gene

[0037] (1) The PRDM16 gene (gene sequence as shown in SEQ ID NO:1) was cloned into a lentiviral vector (such as pLVX-Puro) to construct a recombinant viral expression vector;

[0038] SEQ ID NO:1:

[0039]

[0040] (2) The recombinant vector and helper plasmid were co-transfected into the packaging cell line (293T cells), and cultured for 56 h after transfection;

[0041] (3) Collect the culture supernatant of lentivirus containing the PRDM16 gene, filter and concentrate it, and then determine the viral titer to ensure that the titer reaches 1×10⁻⁶. 7 TU / ml or higher.

[0042] 3. CD8 + Viral transduction of T cells

[0043] (1) The purified CD8 + T cells were seeded in culture plates at a density of 1×10⁶. 6 cells / ml;

[0044] (2) Add lentivirus containing the PRDM16 gene, with a multiplicity of infection (MOI) of 20;

[0045] (3) Polybrene (final concentration 8 μg / ml) was added to improve transduction efficiency;

[0046] (4) After culturing at 37℃ and 5% CO2 for 48 h, candidate CD8 overexpressing PRDM16 were obtained. + T cells.

[0047] 4. Validation of PRDM16 overexpression

[0048] (1) Total RNA was extracted from the above-mentioned candidate cells using an RNA extraction kit;

[0049] (2) Using the extracted total RNA as a template, reverse transcription was performed according to the instructions of the reverse transcription kit to synthesize cDNA; specific primers were designed based on the PRDM16 gene sequence (upstream primer sequence: 5'-CAGCCAATCTCACCAGACACCT-3', SEQ ID NO:2; downstream primer sequence: 5'-GTGGCACTTGAAAGGCTTCTCC-3', SEQ ID NO:3), and internal control primers were designed using the GAPDH gene as the internal control gene; the PCR reaction system included: cDNA template, upstream and downstream primers, PCR Mix, and enzyme-free water. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; and 72℃ final extension for 10 min; the PCR amplification products were subjected to agarose gel electrophoresis, and the electrophoretic bands were observed using a gel imaging system. The results showed that the candidate cell group showed a specific band of the PRDM16 gene, and the band brightness was significantly higher than that of the negative control group. The brightness of the internal control gene band was consistent, proving that CD8+ PRDM16 is overexpressed in T cells.

[0050] Example 2: CD8 overexpressing PRDM16 + T cell proliferation assay

[0051] 1. Experimental Materials

[0052] Cell samples: CD8 cells confirmed by the above RT-PCR to be successfully overexpressing PRDM16 were selected. + T cells were used as the experimental group (PRDM16-OE group), with untransduced CD8 cells. + T cells served as the control group, and the proliferation capacity of the two groups of cells was detected by CFDA-SE fluorescent labeling method.

[0053] 2. Experimental Methods

[0054] (1) Add 1 μL of 2 mM CFDA-SE solution to 1 mL of PBS buffer, mix well, and prepare a 2 μM CFDA-SE PBS solution;

[0055] (2) Resuspend CD8 in the 2 μM CFDA-SE solution prepared above. + T cells (cell density 1×10⁻⁶) 6 (number / mL), incubate on ice in the dark for 10 min;

[0056] (3) Add 5 mL of RPMI 1640 containing 10% fetal bovine serum to terminate the incubation, centrifuge at 500g for 5 min, and wash away residual CSDA-SE with PBS buffer pre-cooled at 4℃ to obtain CD8 labeled with CFDA-SE. + T;

[0057] (4) Place the marked CD8 + T cells were resuspended in complete culture medium, seeded in 24-well plates, and cultured at 37°C in a 5% CO2 incubator for 48 hours. Cells were collected, washed with PBS, resuspended in PBS, and their fluorescence intensity was detected by flow cytometry. Cell division was analyzed by fluorescence intensity.

[0058] 3. Results Analysis

[0059] T cell proliferation was detected using CFDA-SE fluorescent probe labeling, and the results are as follows: Figure 1 As shown, Figure A is a comparison of CD8. + T cells, CD8 cells overexpressing PRDM16 + Figure B shows the distribution of T cell division generations; Figure B is the control CD8. + T cells, CD8 cells overexpressing PRDM16 +A histogram of mean fluorescence intensity of T cells. Figure 1 CD8 cells that overexpress PRDM16 can be observed. + The proportion of T cells reaching generation 3 is higher. These results indicate that CD8 cells overexpressing PRDM16... + T cell proliferation capacity was significantly stronger than that of the control group.

[0060] Example 3: Overexpression of PRDM16 on CD8 + Effects of T cell secretion of IFN-γ and granzyme B

[0061] (1) The CD8 overexpressing PRDM16 prepared in Example 1 was used to... + T cells and control cells were seeded in 24-well plates at a cell density of 1 × 10⁶ cells / well. 6 cells / mL;

[0062] (2) Add CD3 / CD28 to activate magnetic beads at a ratio of 1:1 to cells. After culturing in a 37°C, 5% CO2 incubator for 24 hours, collect the cell culture supernatant, centrifuge at 12,000×g for 5 minutes to remove cell debris, and detect the expression of IFN-γ and granzyme B in the supernatant by ELISA.

[0063] The results are as follows Figure 2 As shown: CD8 overexpressing PRDM16 + The expression of IFN-γ and granzyme B in T cells (PRDM16-OE) was significantly higher than that in the control group. This indicates a statistically significant p < 0.01. The statistical significance is P < 0.001. This indicates that overexpression of PRDM16 can significantly enhance the effector function of CD8+ T cells and promote the secretion of IFN-γ and granzyme B.

[0064] Example 4: Overexpression of PRDM16 on CD8 + The Influence of T Cell Memory Differentiation

[0065] 1. Experimental Materials

[0066] Animals: Wild-type (WT) C57BL / 6 mice, 6-8 weeks old; CD8 + Transgenic mice that specifically overexpress PRDM16 for T cells (PRDM16-OE), 6-8 weeks old;

[0067] Tumor cells: MC38 colon cancer cell line (mouse-derived tumor cells)

[0068] Major antibodies (for flow cytometry): PE-labeled anti-mouse CD62L antibody; APC-labeled anti-mouse CD44 antibody.

[0069] 2. Experimental Methods

[0070] (1) 5×10 5 One MC38 tumor cell was suspended in 100 μL PBS and subcutaneously injected into the right groin of WT and PRDM16-OE mice. The tumors were allowed to grow to approximately 500 mm in volume. 3 (Approximately 14 days after vaccination);

[0071] (2) Under aseptic conditions, mouse spleens were removed, ground into a single-cell suspension, treated with erythrocyte lysis buffer for 5 minutes, washed with PBS, and CD8α magnetic beads were used to sort CD8α cells. + T cells;

[0072] (3) Separate CD8 + After T cells were activated with CD3 / CD28 magnetic beads (magnetic beads: cells = 1:1) for 48 hours, the magnetic beads were removed, and the cells were co-cultured with tumor cells MC38 at a ratio of T cells: tumor cells = 10:1.

[0073] (4) After co-culturing for 48 hours, T cells were collected, washed with PBS, and anti-CD62L and anti-CD44 antibodies were added. The cells were incubated at 4°C in the dark for 30 minutes, washed twice with PBS, and resuspended in 300 μL PBS for flow cytometry analysis.

[0074] 3. Experimental Results

[0075] The results are as follows Figure 3 As shown, CD8 overexpressing PRDM16 + T cells (PRDM16-OE) and its central memory cell subset (CD62L) + CD44 + The expression of ) was significantly higher than that of the control group. This indicates a statistically significant p < 0.05. This indicates a statistically significant p-value < 0.01. The statistical significance is P < 0.001. This indicates that overexpression of PRDM16 can promote CD8. + T cells differentiating into the central memory phenotype are beneficial for maintaining long-term anti-tumor immune responses.

[0076] Example 5: Effects of PRDM16 overexpression on the expression of CD8+ T cell exhaustion-related molecules PD-1 and TIM-3

[0077] 1. Experimental Materials

[0078] Animals: Wild-type (WT) C57BL / 6 mice, 6-8 weeks old; CD8 + Transgenic mice that specifically overexpress PRDM16 for T cells (PRDM16-OE), 6-8 weeks old;

[0079] Tumor cells: MC38 colon cancer cell line (mouse homologous tumor cells);

[0080] Major antibodies: BV421-labeled anti-mouse PD-1 antibody; BV711-labeled anti-mouse TIM-3 antibody.

[0081] 2. Experimental Methods

[0082] (1) A tumor-bearing mouse model was established according to the method in Example 4, and CD8 cells of the spleen were isolated. + T cells, activated by CD3 / CD28, were co-cultured with MC38 tumor cells for 48 hours.

[0083] (2) Collect co-cultured T cells, wash with PBS, add anti-PD-1 and anti-TIM-3 antibodies, incubate at 4°C in the dark for 30 minutes, wash with PBS twice, and use flow cytometry to detect the expression of PD-1 and TIM-3 in T cells to detect the level of T cell exhaustion.

[0084] 3. Experimental Results

[0085] Test results as follows Figure 4 As shown in Figures A and B, CD8 cells overexpressing PRDM16 are respectively displayed. + The expression of PD-1 and TIM-3 in T cells was significantly lower than that in the WT control group. This indicates a statistically significant p < 0.01. The statistical significance is P < 0.001. This indicates that overexpression of PRDM16 can reduce CD8. + The expression of T cell exhaustion markers can slow down T cell exhaustion and maintain its anti-tumor activity.

[0086] Example 6: CD8 overexpressing PRDM16 + Assessment of T-cell in vitro tumor-killing function

[0087] 1. Experimental Materials

[0088] Effector cells: CD8 cells derived from WT and PRDM16-OE mice prepared using the same method as in Example 4. + T cells;

[0089] Target cells: MC38-Luc tumor cells (MC38 cell line that stably expresses luciferase);

[0090] Main reagents:

[0091] CellTiter-Glo luminescence detection kit.

[0092] 2. Experimental Methods

[0093] (1) CD8 was isolated from the spleen of tumor-bearing WT and PRDM16-OE mice according to the method in Example 4. + T cells were activated with CD3 / CD28 magnetic beads for 48 hours, the beads were removed, and the cells were counted for later use.

[0094] (2) MC38-Luc cells were digested with trypsin, and the cell concentration was adjusted to 1×10⁻⁶ after counting. 5 Units / mL available for use;

[0095] (3) Add 1×10 to each well of the 96-well plate 4 MC38-Luc target cells were added to effector cells at effector-to-target ratios (E:T ratios) of 0.1:1, 0.25:1, 0.5:1, 1:1, 2.5:1, 5:1, and 10:1, respectively, with 3 replicates per group, and co-cultured in a 37°C, 5% CO2 incubator.

[0096] (4) After co-culturing for 24 hours, add 100 μL of CellTiter-Glo reagent to each well and incubate at room temperature in the dark for 10 minutes. Use a multi-functional microplate reader to detect the luminescence signal and calculate the killing rate (killing rate = (fluorescence value of control group - fluorescence value of experimental group) / fluorescence value of control group).

[0097] 3. Experimental Results

[0098] The results are as follows Figure 5 As shown, PRDM16 overexpresses CD8 + T cells exhibit stronger tumor-killing effects at different effector-to-target ratios, and the killing effect is enhanced as the effector-to-target ratio increases. This indicates a statistically significant p < 0.01. The statistical significance is P < 0.001. This indicates that overexpression of PRDM16 can significantly enhance CD8. + The ability of T cells to kill tumor cells in vitro.

[0099] Example 7: CD8 overexpressing PRDM16 + Assessment of T cell anti-tumor function in vivo

[0100] 1. Experimental Materials

[0101] animal:

[0102] WT: Wild-type C57BL / 6 mice, 6-8 weeks old, female;

[0103] Prdm16.oe: 16CD8 + Transgenic mice, 6-8 weeks old, female, that specifically overexpress PRDM16 for T cells;

[0104] Tumor cells: MC38 colon cancer cell line.

[0105] 2. Experimental Methods

[0106] (1) Collect MC38 cells in the logarithmic growth phase, digest with trypsin, count and adjust the cell concentration to 1×10⁻⁶. 5 The tumor cells were resuspended in 100 μL PBS and injected subcutaneously into the right groin of mice in the wild-type control group and the experimental group.

[0107] (2) Starting from the 7th day after inoculation, the long diameter (L) and short diameter of the tumor were measured every 2 days using vernier calipers, and the tumor growth curve was plotted.

[0108] 3. Experimental Results

[0109] The results are as follows Figure 6 As shown, the tumor volume growth in the PRDM16 overexpression group (Prdm16.oe) was significantly slower than that in the WT control group. These data indicate that PRDM16 overexpression can significantly enhance the anti-tumor function of T cells. This indicates a statistically significant p < 0.01. This indicates a statistical p < 0.001.

[0110] Example 8: Evaluation of the in vitro tumor-killing function of CAR-T cells overexpressing PRDM16

[0111] 1. Experimental Materials

[0112] Effector cells: PRDM16-overexpressing CAR-T cells in the experimental group; control group T cells, control group PRDM16-overexpressing T cells, and control group CAR-T cells;

[0113] Target cells: MC38-Luc tumor cells (MC38 cell line that stably expresses luciferase);

[0114] 2. Experimental Methods

[0115] (1) Constructing chimeric antigen receptor T cells (CAR-T cells) overexpressing PRDM16.

[0116] (2) Control group T cells, control group T cells overexpressing PRDM16, and control group CAR-T cells were set up as controls. The four types of cells were co-cultured with tumor cells in vitro at an effector-target ratio of 0.5:1.

[0117] (3) Flow cytometry was used to detect the killing activity of each group of cells against tumor cells.

[0118] 3. Experimental Results

[0119] The results are as follows Figure 7As shown, CAR-T cells overexpressing PRDM16 had the strongest tumor-killing effect compared to the control group. This indicates a statistically significant p < 0.01. The statistical significance is P < 0.001. This indicates that PRDM16 overexpression can significantly enhance the in vitro tumor-killing ability of CAR-T cells.

[0120] Example 9: Evaluation of the in vivo antitumor function of CAR-T cells overexpressing PRDM16

[0121] 1. Experimental Materials

[0122] Animals: NOD-SCID-γ (NSG) immunodeficient mice, 6-8 weeks old

[0123] cell:

[0124] Target cells: Human tumor cells expressing the corresponding antigens (such as Raji lymphoma cells)

[0125] Effector cells: Control group T cells, control group CAR-T cells, and experimental group PRDM16-overexpressing CAR-T cells prepared in Example 8.

[0126] 2. Experimental Methods

[0127] (1) 5×10 5 One tumor cell was resuspended in 100 μL PBS and subcutaneously injected into the right groin of NSG mice;

[0128] (2) On the 7th day after inoculation, 1 million control T cells, control CAR-T cells and PRDM16 overexpressing CAR-T cells were infused respectively, and tumor volume was monitored regularly and growth curves were plotted.

[0129] 3. Experimental Results

[0130] The results are as follows Figure 8 As shown, the tumor volume growth in the PRDM16-overexpressing CAR-T group was significantly slower than that in the control group. These data indicate that PRDM16 overexpression can significantly enhance the anti-tumor function of CAR-T cells and improve treatment efficacy. This indicates a statistically significant p < 0.01. This indicates a statistical p < 0.001.

[0131] Example 10 CD8 overexpressing PRDM16 + OT1 cell therapy for bacterial infections

[0132] 1. Experimental Materials

[0133] Animals: Wild-type C57BL / 6 mice, 6-8 weeks old; OT1 transgenic mice;

[0134] Bacteria: Listeria monocytogenes-OVA (Lm-OVA) expressing OVA antigen

[0135] 2. Experimental Methods

[0136] (1) Single-cell suspensions were isolated from the spleen and lymph nodes of OT1 transgenic mice. After erythrocyte lysis, CD8α was purified by sorting with CD8α magnetic beads. + T cells;

[0137] (2) OT1 cells were activated with CD3 / CD28 magnetic beads for 48 hours. Following the method in Example 1, OT1 cells were transduced with lentivirus containing the PRDM16 gene. RT-PCR was used to verify the successful overexpression of PRDM16. The cells were then amplified and cultured for later use.

[0138] (3) Culture Lm-OVA bacteria to the logarithmic growth phase and adjust the bacterial concentration to 1×10⁻⁶. 5 CFU / mL (lethal dose), 200 μL of bacterial solution was injected into wild-type C57BL / 6 mice via tail vein;

[0139] (4) Adoptive cell therapy grouping: ① Tail vein injection of PBS (Ctrl group), ② Tail vein injection of 1×10 5 10 OT1 cells (OT1 group), ③ 1×10 cells injected via tail vein 5 OT1 cell therapy with PRDM16 overexpression (PRDM16 group).

[0140] (5) Record the survival time of the mice and plot the survival curve of the mice.

[0141] 3. Experimental Results

[0142] The results are as follows Figure 9 As shown, all mice in the OT1 cell therapy group overexpressing PRDM16 survived, with a mortality rate of 33.3% in the OT1 cell group and 0% survival in the PBS group. These data indicate that PRDM16 overexpression significantly enhances the anti-infection ability of OT1 cells and improves the host's resistance to lethal bacterial infections, suggesting that PRDM16 has broad application prospects in enhancing T cell function.

[0143] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of T cells overexpressing the PRDM16 gene in the preparation of antitumor drugs, characterized in that, The nucleotide sequence of the PRDM16 gene is shown in SEQ ID NO:1; The T cells are CD8. + T cells or CAR-T cells.

2. The application of T cells overexpressing the PRDM16 gene in the preparation of drugs for treating infectious diseases, characterized in that, The nucleotide sequence of the PRDM16 gene is shown in SEQ ID NO:1; The T cells are CD8. + T cells; The infection is a bacterial infection, specifically a Listeria infection.

3. The application according to claim 1, characterized in that, The T cells overexpressing PRDM16 were obtained by infecting T cells with lentivirus, adenovirus, plasmid, LNP, or mRNA carrying the PRDM16 gene.

4. The application according to claim 1, characterized in that, The PRDM16-overexpressing T cells include CD8+ cells that overexpress PRDM16. + T cells, CD8 cells overexpressing PRDM16 + T cells exhibit enhanced proliferative capacity, higher levels of IFN-γ and granzyme B secretion, higher levels of T cell memory differentiation, and lower expression levels of exhaustion-related molecules PD-1 and TIM-3.

5. The application according to claim 1, characterized in that, The PRDM16-overexpressing T cells include CD8+ cells that overexpress PRDM16. + T cells, CD8 cells overexpressing PRDM16 + T cells can inhibit tumor growth in vivo; the PRDM16-overexpressing T cells include PRDM16-overexpressing CAR-T cells, which, after being reinfused in vivo, can inhibit tumor growth.

6. The application according to claim 1, characterized in that, The PRDM16-overexpressing T cells include CD8+ cells that overexpress PRDM16. + T cells or CAR-T cells overexpressing PRDM16, wherein the CD8+ overexpressing PRDM16 + T cells or CAR-T cells overexpressing PRDM16 have a stronger tumor-killing effect.

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