Application of overexpressed SLC29A1 in enhancing internal and external activities of T cells

Abstract

The invention provides application of overexpressed SLC29A1 in enhancing internal and external activities of T cells, the killing effect of the T cells on target cells is remarkably enhanced through in-vitro overexpressed SLC29A1, in-vivo feedback has stronger anti-tumor and anti-infection functions compared with wild type T cells, and the T cells can be used for preparing drugs for resisting malignant tumors and infectious diseases and have good application prospects. And the curative effect of T cell adoptive therapy is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and specifically to the application of overexpressing SLC29A1 for enhancing the internal and external activities of T cells. Background Art

[0002] Adoptive T cell therapy (ACT) is a method of immunotherapy using the patient's own or donor's T cells, which 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), tumor-infiltrating T cells (TIL), etc., which have shown great clinical applications and market potential in infections, malignant tumors, autoimmune diseases, etc. However, ACT therapy still faces many challenges and difficulties. Among them, the difficulty of T cells to survive for a long time and maintain anti-tumor function in chronic inflammatory microenvironments such as tumor microenvironments is an important reason that limits the effectiveness of ACT.

[0003] T cell metabolism is an important factor affecting its activation, proliferation, differentiation and killing of target cells. In chronic inflammatory microenvironments, including tumor microenvironments, T cells face tremendous survival pressure. First, the widespread hypoxic environment in the microenvironment inhibits the energy metabolism of T cells; second, a variety of metabolites in the chronic inflammatory environment, such as adenosine, kynurenine, lactic acid, etc., can inhibit the anti-tumor function of T cells, leading to their death or differentiation into terminally exhausted T cells; in addition, there is competition for nutrients between various cell subpopulations. For example, tumor cells take in a large amount of glutamine, which leads to the inhibition of T cell function due to insufficient glutamine intake. At present, enhancing the adaptability of T cells to the environment is an important way to enhance 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] The present invention proposes the use of overexpressing SLC29A1 to enhance the activity of T cells inside and outside. By overexpressing SLC29A1 in vitro, the killing effect of T cells on target cells is significantly enhanced, and the in vivo transfusion has stronger anti-tumor and anti-infection functions than wild-type T cells. The invention can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The first aspect of the present invention is to propose the use of enhanced T cells in the preparation of anti-tumor or infectious disease drugs, wherein the enhanced T cells are T cells that overexpress SLC29A1.

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

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

[0010] a) Survive longer;

[0011] b) increased proliferation;

[0012] c) Improved anti-tumor activity;

[0013] d) Improved 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 SLC29A1-overexpressing T cells are obtained by infecting T cells with lentivirus, adenovirus, plasmid or mRNA carrying the SLC29A1 gene.

[0017] Furthermore, the tumor is a benign tumor or a malignant tumor.

[0018] Preferably, the tumor includes but is not limited to melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, cervical squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, and mesothelioma.

[0019] Furthermore, the infectious disease is a disease caused by an infectious agent, specifically an infectious disease caused by pathogens invading the human body, and the pathogens include viruses, bacteria, fungi, parasites, etc.

[0020] The 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; said enhancing T cell function comprises at least one of the following:

[0021] a) Survive longer;

[0022] b) increased proliferation;

[0023] c) Improved anti-tumor activity;

[0024] d) Improved anti-infective activity.

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

[0026] The fourth aspect of the present invention is to provide a method for improving the ability of T cells to secrete cytokines in vitro, comprising the steps of constructing T cells that overexpress SLC29A1, and adding an activator to amplify the T cells.

[0027] The fifth aspect of the present invention is to provide a method for improving the ability of T cells to differentiate into memory cells in vitro, or reducing T cells to be exhausted in vitro, the steps comprising:

[0028] 1) Construction of T cells overexpressing SLC29A1;

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

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

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

[0032] The present invention verifies that overexpression of SLC29A1 enhances the in vivo and in vitro activity of T cells, and finds that in vitro overexpression of SLC29A1 significantly enhances the killing effect of T cells on target cells, and in vivo reinfusion has stronger anti-tumor and anti-infection functions than 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 that the killing ability of target cells in T cell adoptive therapy is inhibited due to environmental influences. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

[0036] Figure 3In the embodiment of the present invention, overexpression of SLC29A1 promotes CD8 + The expression of CD62L, a marker of T cell differentiation and memory, is shown as the result of flow cytometry detection.

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

[0038] Figure 5 CD8 overexpressing SLC29A1 in the present invention + OT1 cells were infused in vivo to treat B16-F10-OVA melanoma subcutaneous xenografts, and the tumor volume growth curve was obtained.

[0039] Figure 6 CD8 overexpressing SLC29A1 in the present invention + Survival time curve of mice after in vivo reinfusion of OT1 cells for the treatment of Listeria infection. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In one example, it was verified that overexpression of SLC29A1 in vitro can promote CD8 + T cell proliferation, cytokine secretion and memory cell differentiation, and promoted the expression of memory cell-related marker CD62L; in the mouse B16-F10-OVA melanoma subcutaneous tumor model, intravenous injection of normal saline, 5×10 6 control wild-type OT1 cells (OVA antigen-specific CD8 + T cells), 5×10 6 The OT1 cells overexpressing SLC29A1 were treated and the tumor volume curve was recorded. It was found that the OT1 cells overexpressing SLC29A1 had stronger anti-tumor function. The above results indicate that SLC29A1 overexpression can promote CD8 + T cell proliferation, cytokine secretion and memory cell differentiation, and has a stronger anti-tumor effect after in vivo infusion. + T cells, in vivo infusion enhances the effect of T cell anti-tumor therapy.

[0042] In another embodiment, it was verified that overexpression of SLC29A1 in vitro can enhance CD8 + The therapeutic effect of T cells on Listeria infection was investigated by transfusing Listeria monocytogenes overexpressing OVA antigen into C57BL / 6 mice through the tail vein to infect the mice with a lethal dose of Listeria monocytogenes. The mice were then given PBS and 1×10 5 OT1 cells, 1×10 5 The mice were treated with OT1 cells overexpressing SLC29A1 in vitro, and the survival time of mice was recorded and the survival curve of mice was drawn. It was found that all mice in the OT1 cell treatment group overexpressing SLC29A1 survived, with a survival rate of 71.4% in the OT1 cell group and 0% in the PBS group. The above results indicate that CD8 + T cells have stronger anti-infection efficacy after infusion in vivo, which verifies the CD8 + T cells, in vivo infusion enhances the effect of T cells in anti-infection therapy.

[0043] The following is a specific example for constructing a CD8 + T cells are infused in vivo for anti-tumor and anti-infection treatment. Unless otherwise specified, the materials or reagents used are commonly used in the art, and the experimental methods used are well-known to those skilled in the art.

[0044] Example

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

[0046] Peripheral blood was collected, red blood cells were lysed, and purified CD8 + CD8 T cells were infected by lentivirus or adenovirus carrying SLC29A1 gene + T cells were overexpressed with SLC29A1, and the overexpression was confirmed by RT-PCR.

[0047] 2. CD8 overexpression of SLC29A1 + T cell proliferation ability assay

[0048] CD8 + After T cells successfully overexpressed SLC29A1, the CFDA-SE fluorescent labeling method was used to detect T cell proliferation. The specific method is:

[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 + T, incubate on ice for 10 min;

[0051] ③ Add 5 mL of 1640 medium containing 10% fetal bovine serum to terminate the incubation, centrifuge at 500 g for 5 min, and wash away the residual CSDA-SE with 4°C precooled PBS buffer to obtain CD8 + T;

[0052] ④ Continue to culture the cells for 48 hours, collect the cells, detect the cell fluorescence intensity by flow cytometry, and analyze the cell division by the fluorescence intensity.

[0053] The proliferation of T cells was detected by CFDA-SE fluorescent probe labeling. Figure 1 As shown, Figure A is the control CD8 + Distribution diagram of T cell division generations. Figure B shows CD8 overexpressing SLC29A1. + Distribution diagram of T cell division generations. Figure 1 CD8 overexpression of SLC29A1 can be seen + The above results indicate that CD8 + The proliferation ability of T cells was significantly stronger than that of the control group.

[0054] 3. CD8 overexpression of SLC29A1 + Detection of IFN-γ and granzyme B secretion by T cells

[0055] CD8 + After successful overexpression of SLC29A1 in T cells, CD8 + T cells, and the cell culture supernatant was collected 24 hours later, and the expression of IFN-γ and granzyme B in the supernatant was detected by ELISA. Figure 2 As shown, CD8 + The expression of IFN-γ and granzyme B in T cells (SLC29A1-OE) was significantly higher than that in the control group. *** indicates statistical P < 0.001.

[0056] 4. CD8 overexpression of SLC29A1 + Expression of CD62L, a marker of T cell differentiation and memory T cells

[0057] Using mouse OT1 cells (OVA antigen-specific CD8 + T cells) overexpressed mouse SLC29A1, and CD3 / CD28 magnetic beads were added to promote CD8 +T cells were expanded for 3 days, and then B16-F10-OVA cells were added to OT1 cells for mixed culture (cell number, 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 the expression of CD62L on T cells to detect the level of T cell memory differentiation. 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 P < 0.001.

[0058] 5. CD8 overexpression of SLC29A1 + 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) overexpressed mouse SLC29A1, and CD3 / CD28 magnetic beads were added to promote CD8 + T cells were expanded for 3 days, and then B16-F10-OVA cells were added to OT1 cells for mixed culture (cell number, 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 on T cells to detect the level of T cell exhaustion. The test results are as follows Figure 4 As shown in Figure A and B, CD8 + The expression of PD-1 and TIM-3 in T cells (SLC29A1-OE) was significantly lower than that in the control group. *** indicates statistical P < 0.001.

[0060] 6. CD8 overexpression of SLC29A1 + OT1 cells were infused in vivo for the treatment of B16-F10-OVA melanoma subcutaneous xenografts.

[0061] Wild-type C57BL / 6 mice aged 6 to 8 weeks were prepared and subcutaneously inoculated with 100 μL of tumor cell suspension (3×10 5 B16-F10-OVA melanoma cells) were used to construct a subcutaneous tumor model of B16-F10-OVA melanoma in C57BL / 6 mice. 3 The 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 OT1 cells (OT1 group), ③5×10 6The OT1 cells overexpressing SLC29A1 were treated (SLC29A1 group). After the treatment started, the long and short diameters of the mouse tumors were measured every other day and the tumor growth curve was drawn. Figure 5 As shown in the figure, the tumor volume growth of the OT1 cell infusion group overexpressing SLC29A1 was significantly slower than that of the OT1 cell infusion group and the control group. The above data show that SLC29A1 overexpression can significantly enhance the anti-tumor function of OT1 cells. *** indicates statistical P < 0.001.

[0062] 7. In vivo transfusion of CD8+OT1 cells overexpressing SLC29A1 is used to treat Listeria infection.

[0063] Wild-type C57BL / 6 mice aged 6 to 8 weeks were prepared and a lethal dose of Listeria monocytogenes expressing OVA antigen (Lm-OVA) was injected into the tail vein to prepare a mouse model of Listeria monocytogenes infection. Treatment groups: ① Tail vein injection of PBS (Ctrl group), ② Tail vein injection of 1×10 5 OT1 cells (OT1 group), ③ tail vein injection of 1×10 5 The survival time of mice was recorded and the survival curve of mice was drawn. Figure 6 As shown, all mice in the OT1 cell treatment group overexpressing SLC29A1 survived, the mortality rate of mice in the OT1 cell group was 71.4%, and the survival rate of mice in the PBS group was 0%. The above data show that overexpression of SLC29A1 can significantly enhance the anti-infection ability of OT1 cells. Among them, *** indicates statistical 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of enhanced T cells in the preparation of anti-tumor or infectious disease drugs, characterized in that: The enhanced T cells are T cells that overexpress SLC29A1.

2. The use according to claim 1, characterized in that The T cells overexpressing SLC29A1 can enhance the killing effect on target cells; and the drug is used for adoptive immunotherapy.

3. The use according to claim 1, characterized in that 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, characterized in that The T cells are CD8 + or CD4 + T cells.

5. The use according to claim 1, characterized in that The SLC29A1-overexpressing T cells are obtained by infecting T cells with lentivirus, adenovirus, plasmid or mRNA carrying the SLC29A1 gene.

6. The use according to claim 1, characterized in that The tumors include, but are not limited to, melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, kidney cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, cervical squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, and mesothelioma.

7. A method for preparing functionally enhanced T cells in vitro, characterized in that: The method comprises the steps of infecting T cells with an expression vector carrying the SLC29A1 gene and performing proliferation culture; wherein the method of enhancing the function of T cells comprises at least one selected from the following: a) Survive longer; b) increased proliferation; c) Improved anti-tumor activity; d) Improved anti-infective activity.

8. A method for improving the proliferation ability of T cells in vitro, characterized in that: The method comprises the steps of constructing T cells overexpressing SLC29A1 and culturing the T cells for proliferation.

9. A method for improving the ability of T cells to secrete cytokines in vitro, characterized in that: The method includes the steps of constructing T cells that overexpress SLC29A1 and adding activators to expand the T cells.

10. A method for improving the ability of T cells to differentiate into memory cells in vitro, or reducing T cells exhaustion in vitro, characterized in that the steps include: 1) Construction of T cells overexpressing SLC29A1; 2) Adding activators to expand T cells; 3) Add tumor cells and T cells for mixed culture.

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