Methods for inducing t cell memory differentiation in vitro and uses thereof

Abstract

The application discloses a method for inducing T cell immune memory differentiation in vitro and application thereof. + The application can be used in malignant tumors and infectious diseases to enhance the curative effect of T cell adoptive therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy, in particular to a method for inducing T cell immune memory differentiation in vitro and application thereof. BACKGROUND

[0002] Adoptive cell immunotherapy based on T cells such as CAR-T, TCR-T, TIL cells, etc. has shown great clinical application and market potential in infectious diseases, malignant tumors and other diseases. However, T cells are prone to exhaustion or death after being reinfused in vivo, resulting in poor efficacy. Studies have shown that inducing T cells to differentiate into memory cells in vitro can significantly improve their immune function after reinfusion in vivo.

[0003] Itaconic acid is an intermediate metabolite produced by the decarboxylation of the key metabolite cis-aconitate in the tricarboxylic acid cycle, which is catalyzed by the immune response gene 1 (Immune-Responsive Gene 1, IRG1) encoded cis-aconitate decarboxylase ACOD1. It is mainly derived from myeloid immune cells such as macrophages and neutrophils. Genetic analysis of Irg1 gene-deficient mice has confirmed that itaconic acid plays an anti-inflammatory role in various infectious disease models or sterile inflammation models, including sepsis, viral infection, psoriasis, gout, ischemia / reperfusion injury and pulmonary fibrosis. New evidence shows that itaconic acid can also inhibit anti-tumor immune responses, the main mechanisms of which include inhibiting CD8 + T aspartate, serine and glycine biosynthesis, and up-regulating CD8 + T cell PD-1 and TIM-3 expression, leading to CD8 + T cell exhaustion.

[0004] Current studies have shown that the continuous presence of itaconic acid in vivo can inhibit the anti-tumor function of effector CD8 + T cells. However, there is no report on the in vitro induction of itaconic acid. SUMMARY

[0005] The present application provides a method for inducing T cell immune memory differentiation in vitro and application thereof, which enhances CD8 + T memory differentiation by supplementing itaconic acid or its derivatives in vitro, thereby enhancing anti-tumor and anti-infectious immune responses after reinfusion in vivo. It can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy.

[0006] In view of this, the solution of the present application is as follows:

[0007] The first aspect of the present application is to propose the application of a culture medium in T cell in vitro culture, which promotes the differentiation of T cells into memory cells, and the culture medium comprises a basic culture medium and an additive, and the additive is itaconic acid or its derivatives.

[0008] Further, the in-vitro culture promotes CD8 + T cells to differentiate into memory cells.

[0009] Further, the in-vitro culture promotes the expression of memory T cell related genes Tcf7, Sell, Id3, Bcl6, Ccr7.

[0010] Further, the basic medium is 1640 medium or DMEM, and 10% fetal bovine serum, 20 ng / mL IL-2 are added.

[0011] Further, the concentration of itaconic acid is 1 mM; and the itaconic acid derivative is 4-OI with a concentration of 125 μM.

[0012] The second aspect of the present application is to provide a method for inducing T cell immune memory differentiation in vitro, which promotes CD8 + After the T cells are activated, the T cells are cultured in a medium, and immune memory differentiated T cells are obtained by collecting the T cells; the medium comprises a basic medium and an additive, and the additive is itaconic acid or a derivative thereof.

[0013] The third aspect of the present application is to provide the use of the immune memory differentiated T cells obtained by the method of the second aspect in the preparation of an immunotherapy drug.

[0014] Further, the drug is used for anti-tumor or anti-infectious diseases.

[0015] Preferably, the tumor includes melanoma, lung cancer, lymphoma, hemangioma, lymphangioma, renal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, neck squamous cell carcinoma, esophageal squamous cell carcinoma, lymphoma, mesothelioma, leukemia, and other solid tumors and hematological tumors.

[0016] Preferably, the infectious disease includes hepatitis B, AIDS, tuberculosis, HPV infection, EBV infection, and other infectious diseases.

[0017] Further, the drug is used for adoptive immunotherapy; the adoptive immunotherapy includes but is not limited to adoptive cell therapy selected from chimeric antigen receptor T cells (CAR-T), T cell receptor T cells (TCR-T), tumor infiltrating lymphocytes (TIL), and the like.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application verifies that the in-vitro itaconic acid or its derivative supplement for T cell in-vitro culture can enhance CD8 +T memory differentiation, and then enhance the anti-tumor and anti-infection immune response after in vivo reinfusion. The application can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy, and provides a new option for the preparation of T cell adoptive therapy drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 In the embodiments of the application, itaconic acid and itaconic acid derivative 4-OI promote CD8 + RT-PCR detection results of T cell memory differentiation.

[0021] Figure 2 In the embodiments of the application, itaconic acid and itaconic acid derivative 4-OI induce memory CD8 + T cell in vivo reinfusion for malignant tumor treatment, tumor growth volume curve.

[0022] Figure 3 In the embodiments of the application, itaconic acid and itaconic acid derivative 4-OI induce memory CD8 + T cell in vivo reinfusion for Listeria infection treatment, survival time curve of mice. DETAILED DESCRIPTION

[0023] The technical solutions of the application will be described below in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0024] A large number of studies have shown that the sustained presence of itaconic acid in vivo can inhibit the anti-tumor function of effector CD8 + T cells. However, there is no report on the in vitro induction effect of itaconic acid. However, our previous studies have shown that short-term stimulation of itaconic acid can induce CD8 + T cells to differentiate into memory cells, which have stronger anti-tumor and anti-infection abilities after in vivo reinfusion. Therefore, the in vitro induction of CD8 + T cells to differentiate into memory cells by itaconic acid in the application is an original finding.

[0025] In one embodiment, it is verified that in vitro treatment of itaconic acid or itaconic acid derivative 4-OI can promote CD8 + T cells to differentiate into memory cells, promote the expression of memory cell-related genes Tcf7, Sell, Id3, Bcl6 and Ccr7 genes; in a mouse B16-F10-OVA melanoma subcutaneous tumor model, 5×10 6 OT1 cells (OVA antigen-specific CD8+ T cells), 5 x 10 6 The OT1 cells treated by in vitro itaconic acid or itaconic acid derivative 4-OI for 6 days were used for treatment, the tumor volume curve was recorded, and it was found that the OT1 cells treated by in vitro itaconic acid or 4-OI had stronger anti-tumor function. The above results show that the CD8 + T cells treated by in vitro itaconic acid or 4-OI differentiate into memory cells, have stronger anti-tumor effect after in vivo reinfusion, and verify the feasibility of enhancing T cell anti-tumor therapy after in vitro treatment of CD8 + T cells by in vitro itaconic acid or 4-OI.

[0026] In the above embodiments, in addition to melanoma, the tumor can also be any one of lung cancer, lymphoma, hemangioma, lymphangioma, renal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, colorectal cancer, bladder cancer, neck squamous cell carcinoma, esophageal squamous cell carcinoma, Hodgkin's lymphoma, mesothelioma, and the like.

[0027] In another embodiment, the therapeutic effect of CD8 + T cells treated by in vitro itaconic acid or 4-OI for the treatment of Listeria infection: C57BL / 6 mice were reinfused with Listeria expressing OVA antigen in the tail vein, so that the mice were infected with a lethal dose of Listeria, and then PBS, 1 x 10 5 OT1 cells, 1 x 10 5 The OT1 cells treated by in vitro itaconic acid or itaconic acid derivative 4-OI for 6 days were used for treatment, the tumor volume curve was recorded, and it was found that the OT1 cells treated by in vitro itaconic acid or 4-OI had stronger anti-tumor function. The above results show that the CD8 + T cells treated by in vitro itaconic acid or 4-OI differentiate into memory cells, have stronger anti-tumor effect after in vivo reinfusion, and verify the feasibility of enhancing T cell anti-tumor therapy after in vitro treatment of CD8 + T cells by in vitro itaconic acid or 4-OI.

[0028] In the above embodiments, in addition to Listeria infection, it can also be infectious diseases such as hepatitis B, AIDS, tuberculosis, HPV infection, and EBV infection.

[0029] The following is a specific embodiment for inducing CD8 + T cells to differentiate into memory cells by in vitro itaconic acid or itaconic acid derivative 4-OI, and then enhancing CD8 +T cell adoptive immunotherapy for infectious diseases and malignant tumors. Unless otherwise specified, the materials or reagents used are common in the art, and the experimental methods used are well known to those skilled in the art.

[0030] Examples

[0031] 1. Preparation of T cell culture medium containing 1 mM itaconic acid and 125 μM 4-OI

[0032] First, prepare the T cell culture medium: add 10% fetal bovine serum and 20 ng / mL IL-2 to 1640 medium or DMEM medium.

[0033] Add itaconic acid to the T cell culture medium according to the 130.10 ug / mL standard, dissolve in a 37°C water bath, and then filter using a sterile filtration device to obtain T cell culture medium containing 1 mM itaconic acid.

[0034] Add 4-OI to the T cell culture medium according to the 30.28 ug / mL standard, dissolve in a 37°C water bath, and then filter using a sterile filtration device to obtain T cell culture medium containing 125 μM 4-OI.

[0035] 2. In vitro promotion of CD8 + T cell memory cell differentiation

[0036] Prepare C57BL / 6 mouse spleen sorted CD8 + T cells, expanded in vitro with IL-2 (20 ng / mL), and induced with CD3 / CD28 monoclonal antibodies to induce CD8 + T cell activation, cultured in medium supplemented with 1 mM itaconic acid or 125 μM 4-OI, and collected after 6 days. RT-PCR was used to detect T cell Tcf7, Sell, Id3, Bcl6, and Ccr7 expression to identify the level of T cell memory differentiation. The results are shown in Figure 1 Fig. 1, where *** indicates statistical P<0.001. In vitro treatment of CD8 + T cells with 125 μM 4-OI and 1 mM itaconic acid significantly increased the expression levels of T cell Tcf7, Sell, Id3, Bcl6, and Ccr7, indicating that itaconic acid or 4-OI can induce CD8 + T cells to differentiate into memory cells in vitro.

[0037] 3. T cell reinfusion for treatment of subcutaneous tumor transplantation models

[0038] 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 At that time, the mice were divided into four groups according to the following treatment:

[0039] ① Intravenous injection of PBS (solvent);

[0040] ② Intravenous injection of 5×10 6 OT1 cells (OVA antigen-specific CD8) + T cells);

[0041] ③5×10 6 Individual OT1 cell therapy involving in vitro treatment with itaconic acid or 4-OI for 6 days.

[0042] After treatment began, the long and short axes of the mouse tumors were measured every other day, and tumor growth curves were plotted. Figure 2 As shown, compared with the control group, OT1 cells treated with itaconic acid or 4-OI had a stronger inhibitory effect on tumor growth. *** indicates statistical significance (P < 0.001). This means that OT1 cells treated with itaconic acid or 4-OI in vitro have a stronger anti-tumor function.

[0043] 4. T-cell reinfusion for the treatment of infection models

[0044] Wild-type C57BL / 6 mice aged 6–8 weeks were injected via tail vein with a lethal dose of Listeria monocytogenes expressing the OVA antigen (Lm-OVA) to establish a mouse model of Listeria infection. Treatment groups:

[0045] ① Inject PBS via tail vein;

[0046] ② Tail vein injection of 1×10 5 One OT1 cell;

[0047] ③ Tail vein injection of 1×10 5 Individual OT1 cell therapy involving in vitro treatment with itaconic acid or 4-OI for 6 days.

[0048] Record the survival time of the mice and plot the mouse survival curve.

[0049] Itaconic acid and its derivatives 4-OI-induced memory CD8 + The survival curve of mice treated with in vivo T-cell infusion for Listeria infection is as follows: Figure 3*** indicates statistical P<0.001). The results of this study found that all OT1 cell treatment group mice survived after 6 days of 4-OI treatment, 12.5% of OT1 cell treatment group mice died after 6 days of itaconic acid treatment, 50% of OT1 cell treatment group mice died, and all PBS group mice died. It is shown that the survival rate of OT1 cell treatment group mice treated with itaconic acid or 4-OI for 6 days is significantly higher than that of control OT1 cell treatment group and PBS group. It is shown that itaconic acid or its derivatives can induce CD8 + T cells to differentiate into memory cells, which have stronger anti-tumor and anti-infection ability after in vivo reinfusion.

[0050] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. The application of culture medium in the in vitro culture of T cells, characterized in that, The in vitro culture promotes the differentiation of T cells into memory cells. The culture medium contains a basal medium and an additive, the additive being itaconic acid or 4-OI, and the T cells being CD8+ T cells.

2. The application according to claim 1, characterized in that, The in vitro culture promoted the increase in expression levels of memory T cell-related genes Tcf7, Sell, Id3, Bcl6, and Ccr7.

3. The application according to claim 1, characterized in that, The basal culture medium is 1640 medium or DMEM, supplemented with 10% fetal bovine serum and 20 ng / mL IL-2.

4. The application according to claim 1, characterized in that, The concentration of itaconic acid is 1 mM; the concentration of 4-OI is 125 μM.

5. A method for inducing T-cell immune memory differentiation in vitro, characterized in that, T cells are activated and cultured in a culture medium to collect T cells differentiated into immune memory; the culture medium includes a basal culture medium and an additive, the additive being itaconic acid or 4-OI, and the T cells being CD8+ T cells.

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