Application of SLC29A1 overexpression to enhance T cell intracellular and extracellular activity

Abstract

This invention proposes the application of overexpressing SLC29A1 to enhance the intracellular and in vitro activity of T cells. By overexpressing SLC29A1 in vitro, the killing effect of T cells on target cells is significantly enhanced. When reinfused in vivo, it has stronger anti-tumor and anti-infection functions compared with wild-type T cells. It can be used to prepare drugs for anti-malignant tumors and infectious diseases, and enhance the efficacy of T cell adoptive therapy.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy technology, specifically to the application of overexpressing SLC29A1 to enhance the intracellular and extracellular activity of T cells. Background Technology

[0002] Adoptive T cell therapy (ACT) is an immunotherapy method that utilizes the patient's own or a donor's T cells, and it has shown significant efficacy in the treatment of certain types of cancer. Currently, ACT mainly includes chimeric antigen receptor T cells (CAR-T), T cell receptor T cells (TCR-T), and tumor-infiltrating T cells (TIL), which have demonstrated enormous clinical application and market potential in infections, malignant tumors, and autoimmune diseases. However, ACT therapy still faces many challenges and difficulties. Among them, the difficulty for T cells to survive long-term and maintain anti-tumor function in chronic inflammatory microenvironments such as the tumor microenvironment is a significant factor limiting the effectiveness of ACT.

[0003] T cell metabolism is a crucial factor influencing their activation, proliferation, differentiation, and target cell killing. In the chronic inflammatory microenvironment, including the tumor microenvironment, T cells face immense survival pressure. First, the widespread hypoxic environment inhibits T cell energy metabolism. Second, various metabolites in the chronic inflammatory environment, such as adenosine, kynurenine, and lactate, can suppress T cell anti-tumor function, leading to cell death or differentiation into terminally exhausted T cells. Furthermore, competition for nutrients exists among different cell subsets; for example, tumor cells consume large amounts of glutamine, resulting in insufficient glutamine uptake by T cells and suppressed T cell function. Currently, enhancing T cell adaptability to the environment is an important pathway to improve their function.

[0004] Therefore, enhancing the killing effect of T cells on target cells is of great significance for adoptive immunotherapy. Summary of the Invention

[0005] This invention proposes the application of SLC29A1 overexpression to enhance the intracellular and extracellular activity of T cells. In vitro overexpression of SLC29A1 significantly enhances the cytotoxic effect of T cells against target cells, and in vivo reinfusion of SLC29A1 results in stronger anti-tumor and anti-infection functions compared to wild-type T cells. This invention can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy.

[0006] The technical solution of this invention is implemented as follows:

[0007] The first aspect of this invention is to propose the use of enhanced T cells in the preparation of drugs for antitumor or infectious diseases, wherein the enhanced T cells are T cells overexpressing SLC29A1.

[0008] Furthermore, the T cells overexpressing SLC29A1 can enhance T cell function and increase the killing effect on target cells; the drug is used for adoptive immunotherapy.

[0009] Furthermore, the enhancement of T cell function includes at least one of the following:

[0010] a) Longer survival time;

[0011] b) Increased proliferation;

[0012] c) Increased antitumor activity;

[0013] d) Increased anti-infective activity.

[0014] Furthermore, the T cells are selected from chimeric antigen receptor T cells, T cell receptor T cells, and tumor-infiltrating lymphocytes.

[0015] Furthermore, the T cells are CD8. + or CD4 + T cells.

[0016] Furthermore, the T cells overexpressing SLC29A1 are obtained by infecting T cells with lentivirus, adenovirus, plasmid, or mRNA carrying the SLC29A1 gene.

[0017] Furthermore, the tumor may be benign or malignant.

[0018] Preferably, the tumors include, but are not limited to, melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, stomach cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, squamous cell carcinoma of the neck, squamous cell carcinoma of the esophagus, Hodgkin's lymphoma, and mesothelioma.

[0019] Furthermore, the infectious disease is a disease caused by an infectious agent, specifically referring to an infectious disease caused by a pathogen invading the human body, including pathogens such as viruses, bacteria, fungi, and parasites.

[0020] A second aspect of the present invention is to provide a method for preparing functionally enhanced T cells in vitro, comprising the steps of infecting T cells with an expression vector carrying the SLC29A1 gene and performing proliferation culture; wherein the enhanced T cell function comprises at least one of the following:

[0021] a) Longer survival time;

[0022] b) Increased proliferation;

[0023] c) Increased antitumor activity;

[0024] d) Increased anti-infective activity.

[0025] A third aspect of the present invention is to provide a method for improving the in vitro proliferation capacity of T cells, comprising the steps of constructing T cells overexpressing SLC29A1 and culturing the T cells for proliferation.

[0026] A fourth aspect of the present invention is to provide a method for enhancing the ability of T cells to secrete cytokines in vitro, comprising the steps of constructing T cells overexpressing SLC29A1 and expanding the T cells by adding an activator.

[0027] A fifth aspect of this invention is to provide a method for improving the in vitro memory cell differentiation capacity of T cells or reducing the in vitro exhaustion of T cells, the steps of which include:

[0028] 1) Construct T cells overexpressing SLC29A1;

[0029] 2) Add activators to expand T cells;

[0030] 3) Add tumor cells and T cells for co-culture.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] This invention verifies the effect of SLC29A1 overexpression on enhancing the in vivo and in vitro activity of T cells. It finds that in vitro overexpression of SLC29A1 significantly enhances the killing effect of T cells on target cells, and in vivo reinfusion of SLC29A1 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. Attached Figure Description

[0033] 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 drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 In this embodiment of the invention, overexpression of SLC29A1 promotes CD8. + T cell proliferation.

[0035] Figure 2 In this embodiment of the invention, overexpression of SLC29A1 promotes CD8. + T cells secrete more IFN-γ and granzyme B.

[0036] Figure 3In this embodiment of the invention, overexpression of SLC29A1 promotes CD8. + The expression of CD62L, a marker of T cell memory T cell differentiation, is shown in the flow cytometry results.

[0037] Figure 4 In this embodiment of the invention, overexpression of SLC29A1 reduces CD8. + The expression of T cell exhaustion-related molecules PD-1 and TIM-3 is shown in the flow cytometry results.

[0038] Figure 5 In this embodiment of the invention, CD8 overexpressing SLC29A1 + In vivo infusion of OT1 cells for the treatment of B16-F10-OVA melanoma subcutaneous xenografts, tumor volume growth curve.

[0039] Figure 6 In this embodiment of the invention, CD8 overexpressing SLC29A1 + Survival curve of mice after in vivo reinfusion of OT1 cells for the treatment of Listeria infection. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] In one embodiment, it was verified that in vitro overexpression of SLC29A1 can promote CD8. + T cell proliferation, cytokine secretion, and memory cell differentiation were enhanced, promoting the expression of the memory cell-related marker CD62L. In a mouse B16-F10-OVA subcutaneous melanoma model, intravenous injections of physiological saline and 5×10⁻⁶ mol / L serum were administered. 6 Control wild-type OT1 cells (OVA antigen-specific CD8) + T cells), 5×10 6 OT1 cells overexpressing SLC29A1 were treated, and tumor volume curves were recorded. The results showed that OT1 cells overexpressing SLC29A1 exhibited stronger anti-tumor function. These results indicate that SLC29A1 overexpression can promote CD8... + T cell proliferation, cytokine secretion, and memory cell differentiation were enhanced, and the in vivo reinfusion resulted in a stronger anti-tumor therapeutic effect. This validated the efficacy of CD8 cells overexpressing SLC29A1 in vitro. + T cells, when reinfused into the body, enhance the anti-tumor therapeutic effect of T cells.

[0042] In another embodiment, it was verified that in vitro overexpression of SLC29A1 can enhance CD8. + The therapeutic effect of T cells on Listeria infection was demonstrated by the following: Listeria monocytogenes overexpressing OVA antigen was intravenously infused into C57BL / 6 mice, infecting them with a lethal dose of Listeria. The mice were then administered PBS via the tail vein. 5 1 OT1 cell, 1×10 5 In vitro treatment with OT1 cells overexpressing SLC29A1 resulted in mouse survival time recording and survival curve plotting. The results showed that all mice in the OT1 cell treatment group (overexpressing SLC29A1) survived (71.4% survival rate), while the survival rate in the PBS group was 0%. These results indicate that CD8+ overexpressing SLC29A1... + T-cell in vivo reinfusion resulted in stronger anti-infection efficacy, validating the efficacy of CD8 cells overexpressing SLC29A1 in vitro. + T cells, when reinfused into the body, enhance the anti-infection therapeutic effect of T cells.

[0043] The following are specific examples for constructing CD8 overexpressing SLC29A1 in vitro. + T cells, in vivo reinfusion for anti-tumor and anti-infective therapy. Unless otherwise specified, all materials and reagents used are commonly used in the field, and all experimental techniques used are well-practiced by those skilled in the art.

[0044] Example

[0045] 1. Preparation of CD8 cells overexpressing SLC29A1 + T cells

[0046] Peripheral blood was collected, and after lysing red blood cells, purified CD8 was obtained by magnetic bead sorting. + T cells. Infecting CD8 cells with lentiviruses or adenoviruses carrying the SLC29A1 gene. + T cells were induced to overexpress SLC29A1, and the overexpression was confirmed by RT-PCR.

[0047] 2. Overexpression of SLC29A1 in CD8 + T cell proliferation assay

[0048] CD8 was identified by RT-PCR + After successful overexpression of SLC29A1 by T cells, T cell proliferation was detected using the CFDA-SE fluorescent labeling method. The specific method was as follows:

[0049] ① Add 1 μL of 2 mM CFDA-SE solution to 1 mL of PBS buffer to prepare a 2 μM CFDA-SE PBS solution;

[0050] ② Resuspend CD8 in the prepared CFDA-SE PBS solution. + T, incubate on ice for 10 minutes;

[0051] ③ Add 5 mL of 1640 medium 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;

[0052] ④ Continue culturing the cells for 48 hours, collect the cells, and detect the cell fluorescence intensity by flow cytometry. Analyze the cell division status by analyzing the fluorescence intensity.

[0053] 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 cell division generation distribution diagram. Figure B shows CD8 cells overexpressing SLC29A1. + T cell division generation distribution diagram. (From...) Figure 1 CD8 overexpression of SLC29A1 can be observed. + The proportion of T cells reaching generation 3 is higher. These results indicate that CD8+ overexpressing SLC29A1... + T cell proliferation capacity was significantly stronger than that of the control group.

[0054] 3. Overexpression of SLC29A1 in CD8 + Detection of T cell IFN-γ and granzyme B secretion

[0055] CD8 was identified by RT-PCR + After T cells successfully overexpressed SLC29A1, CD8 cells were stimulated with CD3 / CD28 magnetic beads. + T cells were cultured, and the supernatant was collected 24 hours later. The expression of IFN-γ and granzyme B in the supernatant was detected by ELISA. Figure 2 As shown, CD8 overexpression of SLC29A1 + The expression of IFN-γ and granzyme B in T cells (SLC29A1-OE) was significantly higher than that in the control group. *** indicates statistical significance P < 0.001.

[0056] 4. Overexpression of SLC29A1 in CD8 + Expression of CD62L, a marker of T cell memory T cell differentiation

[0057] Using mouse OT1 cells (OVA antigen-specific CD8) + T cells overexpressing murine SLC29A1, with the addition of CD3 / CD28 magnetic beads promoting CD8 +T cells were expanded for 3 days, and then B16-F10-OVA cells were added to OT1 cells for co-culture (cell ratio: OT1 cells:B16-F10-OVA = 10:1) for 3 days. CD62L is one of the markers of memory cells. Flow cytometry was used to detect CD62L expression in T cells to detect the level of T cell memory differentiation. Detection... Figure 3 As shown, CD62L expression in CD8+ T cells overexpressing SLC29A1 (SLC29A1-OE) was significantly higher than that in the control group. *** indicates statistical significance (P < 0.001).

[0058] 5. Overexpression of SLC29A1 in CD8 + Detection of the expression of T cell exhaustion-related molecules PD-1 and TIM-3

[0059] Using mouse OT1 cells (OVA antigen-specific CD8) + T cells overexpressing murine SLC29A1, with the addition of CD3 / CD28 magnetic beads promoting CD8 + T cells were expanded for 3 days, and then B16-F10-OVA cells were added to OT1 cells for co-culture (cell ratio: OT1 cells:B16-F10-OVA = 10:1) for 3 days. PD-1 and TIM-3 are important markers of exhausted T cells. Flow cytometry was used to detect the expression of PD-1 and TIM-3 in T cells to detect the level of T cell exhaustion. The detection results are as follows. Figure 4 As shown in Figures A and B, CD8+ overexpressing SLC29A1 is displayed. + The expression of PD-1 and TIM-3 in T cells (SLC29A1-OE) was significantly lower than that in the control group. *** indicates statistical significance P < 0.001.

[0060] 6. Overexpression of SLC29A1 in CD8 + In vivo infusion of OT1 cells was used for the treatment of B16-F10-OVA melanoma subcutaneous xenografts.

[0061] Prepare 6-8 week old wild-type C57BL / 6 mice and subcutaneously inoculate 100 μL of tumor cell suspension (3 × 10⁻⁶ mcg) into the right groin. 5 A C57BL / 6 mouse subcutaneous melanoma model was constructed using B16-F10-OVA melanoma cells. 3 Mice were divided into three groups according to the following treatments: ① Intravenous injection of PBS (solvent) (Ctrl group); ② Intravenous injection of 5×10 6 10 OT1 cells (OT1 group), ③ 5 × 10 6OT1 cells overexpressing SLC29A1 were used for treatment (SLC29A1 group). After treatment began, the major and minor axes of mouse tumors were measured every other day, and tumor growth curves were plotted. Figure 5 As shown, tumor volume growth in the OT1 cell infusion group overexpressing SLC29A1 was significantly slower than that in the OT1 cell infusion group and the control group. These data indicate that SLC29A1 overexpression can significantly enhance the anti-tumor function of OT1 cells. *** indicates statistical significance (P < 0.001).

[0062] 7. In vivo reinfusion of CD8+OT1 cells overexpressing SLC29A1 for the treatment of Listeria infection.

[0063] Wild-type C57BL / 6 mice aged 6–8 weeks were used to establish a Listeria infection mouse model by tail vein injection of a lethal dose of Listeria monocytogenes expressing OVA antigen (Lm-OVA). Treatment groups: ① Tail vein injection of PBS (Ctrl group), ② Tail vein injection of 1×10 5 10 OT1 cells (OT1 group), ③ Tail vein injection of 1×10 5 OT1 cells overexpressing SLC29A1 were used for treatment (SLC29A1 group). Mouse survival time was recorded, and mouse survival curves were plotted. Figure 6 As shown, all mice in the OT1 cell therapy group overexpressing SLC29A1 survived, with a mortality rate of 71.4% in the OT1 cell group and 0% survival in the PBS group. These data indicate that SLC29A1 overexpression significantly enhances the anti-infection ability of OT1 cells. *** indicates statistical significance (P < 0.001).

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of an enhanced T cell in the manufacture of a medicament for the treatment of a neoplastic or infectious disease, characterized in that, The enhanced T cell is a T cell overexpressing SLC29A1; the infectious disease is a Listeria infection.

2. Use according to claim 1, wherein The T cell overexpressing SLC29A1 can enhance the killing effect on target cells; the drug is used for adoptive immunotherapy.

3. The use according to claim 1, wherein The T cell is selected from one of a chimeric antigen receptor T cell, a T cell receptor T cell, and a tumor infiltrating lymphocyte.

4. The use according to claim 1, wherein The T cells are CD8 + or CD4 + T cells.

5. The use according to claim 1, wherein The T cell overexpressing SLC29A1 is obtained by infecting the T cell with a lentivirus, adenovirus, plasmid or mRNA carrying the SLC29A1 gene.

6. The use according to claim 1, wherein The tumor is melanoma, lung cancer, liver cancer, cervical cancer, colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, or esophageal squamous cell carcinoma.

7. A method of in vitro production of functionally enhanced T cells, characterized in that, The method comprises the steps of infecting the T cell with an expression vector carrying the SLC29A1 gene and proliferating the T cell; the enhanced T cell function comprises at least one selected from the following: a) longer survival time; b) enhanced proliferation; c) improved anti-tumor activity; d) improved anti-Listeria infection activity.

8. A method of increasing the in vitro proliferative capacity of T cells, characterized in that, The method comprises the steps of constructing a T cell overexpressing SLC29A1 and proliferating the T cell.

9. A method of increasing the ability of T cells to secrete cytokines in vitro, characterized in that, The method comprises the steps of constructing a T cell overexpressing SLC29A1 and adding an activator to expand the T cell.

10. A method of increasing the ability of T cells to differentiate into memory cells in vitro, or decreasing the exhaustion of T cells in vitro, characterized by the steps of The method comprises: 1) constructing a T cell overexpressing SLC29A1; 2) adding an activator to expand the T cell; 3) adding tumor cells to mix and culture with the T cell.

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