In vitro amplification method of dry memory T cells

Abstract

The invention provides an in-vitro amplification method of dry memory T cells. The method comprises the step of culturing a first cell population containing T cells in vitro in the presence of succinic acid or pharmaceutically acceptable salt or ester thereof, so as to obtain a second cell population containing dry memory T cells. The method disclosed by the invention can be used for efficiently amplifying or preparing the dry memory T cells in vitro. Besides, the anti-tumor function of T cells can be improved through pretreatment of succinic acid or salt thereof, and tumor can be synergistically inhibited and the level of IFN gamma < + > CD8 < + > T cells can be remarkably improved through combined use of succinic acid or salt thereof and immunodetection point drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an in vitro expansion method of stem memory T cells. Background Art

[0002] Tumor antigen-specific CD8 + T cells are the main immune cell subset controlling tumor development, but the tumor suppressive microenvironment induces CD8 + Functional exhaustion of T cells greatly limits their anti-tumor immune response. Continuous stimulation of tumor antigens and nutritional restriction induce mitochondrial metabolic damage and epigenetic fixation of dysfunction in infiltrating T cells. Studies have shown that the anti-tumor effects of immune checkpoint therapy and adoptive transfer of TCR-T and CAR-T cell therapy depend on antigen-specific T cell populations with memory characteristics (stemness). Therefore, it is particularly important to improve the metabolic competitiveness of T cells and the epigenetic openness of stemness-related genes during in vitro culture. However, there are potential off-target effects and unknown risks when using drugs to target T cell mitochondrial metabolism or epigenetics.

[0003] In summary, it is necessary to develop efficient methods for expanding stem memory T cells in vitro and tumor immunotherapy drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for efficiently expanding stem memory T cells in vitro and its application.

[0005] The first aspect of the present invention provides an in vitro method for preparing stem memory T cells, comprising the steps of:

[0006] (a) culturing a first cell population containing T cells in vitro in the presence of succinic acid or a pharmaceutically acceptable salt or ester thereof to obtain a second cell population containing stem memory T cells.

[0007] In another preferred embodiment, the first cell population is a primary or passaged T cell population isolated from a human or non-human mammal.

[0008] In another preferred embodiment, the first cell population is T cells sorted by CD3.

[0009] In another preferred embodiment, the T cells sorted by CD3 are CD3 + T cells.

[0010] In another preferred embodiment, in the first cell population, CD3 + The content of T cells is 80-99%, preferably 85-95%.

[0011] In another preferred embodiment, the first cell population is an activated T cell population.

[0012] In another preferred embodiment, the activation treatment includes activation treatment with CD3 antibody, CD28 antibody or a combination thereof.

[0013] In another preferred embodiment, the activation treatment comprises treatment with a predetermined concentration of CD3 antibody, CD28 antibody or a combination thereof (wherein the predetermined concentration is 0.1-1 ug / ml, preferably 0.3-0.7 ug / mL, such as about 0.5 ug / mL).

[0014] In another preferred embodiment, after activation treatment, cell survival, stemness phenotype and / or proliferation ability are significantly promoted.

[0015] In another preferred embodiment, in the in vitro culture, the concentration of succinic acid or its pharmaceutically acceptable salt or ester is about 5-200 mM, preferably about 10-100 mM, more preferably about 15-80 mM, and most preferably about 20-70 mM, calculated as succinic acid.

[0016] In another preferred embodiment, succinic acid or a pharmaceutically acceptable salt or ester thereof is added or supplemented during the in vitro culture.

[0017] In another preferred embodiment, the in vitro culture uses a culture medium suitable for T cells.

[0018] In another preferred embodiment, the culture medium comprises components selected from the group consisting of fetal bovine serum (FBS), a buffer system, amino acids, antibiotics, a reducing agent, and carbohydrates.

[0019] In another preferred embodiment, the culture medium comprises components selected from the group consisting of: RPMI culture medium containing 10% FBS, Hepes buffer, sodium pyruvate, penicillin-streptomycin, L-glutamine, MEM non-essential amino acids and β-mercaptoethanol.

[0020] In another preferred embodiment, the culture medium comprises: RPMI culture medium containing 10% FBS, Hepes buffer (10 mM), sodium pyruvate (1 mM), penicillin-streptomycin, L-glutamine (2 mM), MEM non-essential amino acids (1×) and β-mercaptoethanol (50 μM).

[0021] In another preferred embodiment, the succinic acid or a pharmaceutically acceptable salt or ester thereof is selected from the group consisting of succinic acid, sodium succinate, potassium succinate, dimethyl succinate, or a combination thereof.

[0022] In another preferred embodiment, in the in vitro culture, the concentration of succinic acid or its pharmaceutically acceptable salt or ester is about 5-200 mM, preferably about 10-100 mM, more preferably about 15-80 mM, and most preferably about 20-70 mM, calculated as succinic acid.

[0023] In another preferred embodiment, in the in vitro culture, the culture time in the presence of succinic acid or a pharmaceutically acceptable salt or ester thereof is 0.5-10 days, preferably 4-8 days, more preferably 5-7 days.

[0024] In another preferred embodiment, the number S2 of the stem memory T cells in the second cell population is about 700-950, based on 1000 raw material T cells in the first cell population.

[0025] In another preferred embodiment, the number S2 of stem memory T cells in the second cell population is significantly higher than the number S1 of stem memory T cells in the first cell population.

[0026] In another preferred embodiment, the ratio of the number S2 of stem memory T cells in the second cell population to the number S1 of stem memory T cells in the first cell population (S2 / S1) is ≥10, preferably ≥100, and more preferably ≥1000.

[0027] In another preferred embodiment, the concentration C2 of stem memory T cells in the second cell population is about 70-95%.

[0028] In another preferred embodiment, the concentration C2 of the stem memory T cells in the second cell population is significantly higher than the concentration C1 of the stem memory T cells in the first cell population.

[0029] In another preferred embodiment, the ratio of the concentration C2 of the stem memory T cells in the second cell population to the concentration C1 of the stem memory T cells in the first cell population (C2 / C1) is ≥1, preferably ≥2, and more preferably ≥3.

[0030] In another preferred embodiment, the stem memory T cells are TCF1-positive memory T cells.

[0031] In another preferred embodiment, the second cell population contains a lymphocyte subset of stem memory T cells selected from the group consisting of: CD62L + CCR7 + Labeled stem memory T cells.

[0032] In another preferred embodiment, the second cell population contains a lymphocyte subset of stem memory T cells selected from the group consisting of: CD4 + T cells, CD8 + T cells, γδ T cells, or a combination thereof.

[0033] In another preferred embodiment, the method further comprises:

[0034] (a1) culturing the first cell population containing T cells in vitro under the same culture conditions but in the absence of succinic acid or a pharmaceutically acceptable salt or ester thereof, thereby obtaining a third cell population containing T cells.

[0035] In another preferred example, the number S2 of stem memory T cells in the second cell population is significantly higher than the number S3 of stem memory T cells in the third cell population.

[0036] In another preferred embodiment, the ratio (S2 / S3) of the number S2 of the stem memory T cells in the second cell population to the number S3 of the stem memory T cells in the third cell population is ≥2, preferably ≥5, and more preferably ≥10.

[0037] In another preferred example, the concentration C2 of stem memory T cells in the second cell population is significantly higher than the concentration C3 of stem memory T cells in the third cell population.

[0038] In another preferred embodiment, the ratio of the concentration C2 of the stem memory T cells in the second cell population to the concentration C3 of the stem memory T cells in the third cell population (C2 / C3) is ≥2, preferably ≥4, and more preferably ≥5.

[0039] In another preferred embodiment, the method comprises:

[0040] (t1) After coating the plate with CD3 antibodies, T cells are activated and expanded using CD28 antibodies, IL-2, and other cytokines;

[0041] (t2) during T cell activation and culture, adding a sodium succinate solution having a predetermined concentration (e.g., 20-100 mM, preferably about 40±10 mM) to the T cell culture medium;

[0042] (t3) supplementing the culture medium with IL-2 and a sodium succinate solution at a predetermined concentration (e.g., 20-100 mM, preferably about 40±10 mM) every 2-3 days;

[0043] (t4) After culturing for a period of time (e.g., about 4-8 days, preferably 5-7 days, such as 6 days), a lymphocyte population mainly composed of memory stem T cells can be obtained.

[0044] In a second aspect of the present invention, there is provided the use of succinic acid or a pharmaceutically acceptable salt or ester thereof for preparing a formulation or composition for one or more uses selected from the group consisting of:

[0045] (a) Promote tumor antigen-specific CD8 + Increased number of T cells;

[0046] (b) preparing or expanding stem memory T cells in vitro;

[0047] (c) increasing T cell activity or reducing T cell apoptosis rate;

[0048] (d) improving the stemness of T cells;

[0049] (e) Increase the basal and maximal oxygen consumption of T cells;

[0050] (f) Increase mitochondrial ATP production in T cells;

[0051] (g) Promotes chromatin opening and expression of stemness-related molecules in T cells.

[0052] In another preferred embodiment, the tumor antigen-specific CD8 + T cells including IFNγ + CD8 + T cells.

[0053] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0054] In another preferred embodiment, the pharmaceutical composition is administered to a subject with a tumor.

[0055] In another preferred embodiment, the subjects include humans and non-human mammals (such as rodents and non-human primates).

[0056] In another preferred embodiment, the rodents include mice and rats.

[0057] In another preferred embodiment, the preparation includes a laboratory preparation.

[0058] A third aspect of the present invention provides an active ingredient combination, comprising:

[0059] (Z1) succinic acid or a pharmaceutically acceptable salt or ester thereof; and

[0060] (Z2) Drugs targeting immune checkpoints.

[0061] In another preferred embodiment, the drug targeting the immune checkpoint comprises an antibody.

[0062] In another preferred embodiment, the drug targeting the immune checkpoint is selected from the following group: anti-PD-L1 antibody, anti-LAG3 antibody, or a combination thereof.

[0063] A fourth aspect of the present invention provides a medicine kit, comprising:

[0064] (Y1) a first pharmaceutical composition comprising (Y1a) succinic acid or a pharmaceutically acceptable salt or ester thereof; and (Y1b) a pharmaceutically acceptable carrier;

[0065] (Y2) a second pharmaceutical composition comprising (Y2a) an immune checkpoint active ingredient; and (Y2b) a pharmaceutically acceptable carrier.

[0066] The fifth aspect of the present invention provides the use of the active ingredient combination of the third aspect of the present invention for preparing a medicament for treating tumors.

[0067] In another preferred embodiment, the tumor includes: a hematological tumor, a solid tumor or a combination thereof.

[0068] In another preferred embodiment, the tumor is selected from the following group: liver cancer, gastric cancer, intestinal cancer, melanoma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, gastrointestinal stromal cancer, pheochromocytoma, paraganglioma, renal cell carcinoma, and pituitary adenocarcinoma.

[0069] In a sixth aspect, the present invention provides the use of an immune checkpoint inhibitor for preparing an anti-tumor drug, which is administered to a subject suffering from a succinate-rich tumor.

[0070] In another preferred embodiment, the immune checkpoint inhibitor comprises: PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, or a combination thereof.

[0071] In another preferred embodiment, the succinate-enriched tumor is a tumor with an inactivated mutation of the succinate dehydrogenase complex (SDH).

[0072] In another preferred example, the succinate dehydrogenase complex (SDH) inactivation mutation tumor comprises a cancer having an SDH subunit mutation selected from the following group: SDHA, SDHB, SDHC, SDHD, SDHAF2, or a combination thereof; the cancer is selected from: liver cancer, gastric cancer, intestinal cancer, melanoma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, gastrointestinal stromal cancer, pheochromocytoma, paraganglioma, renal cell carcinoma, pituitary adenocarcinoma, or a combination thereof.

[0073] A seventh aspect of the present invention provides a reagent combination, comprising:

[0074] (A) detection reagents; and

[0075] (B) Immune checkpoint inhibitors.

[0076] In another preferred embodiment, the detection reagent is used to detect whether the succinate dehydrogenase complex (SDH) in tumor cells has an inactivating mutation or reduced activity.

[0077] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Succinate was shown to promote tumor antigen-specific CD8 + Increase in the number of T cells.

[0079] Figure 2 Sodium succinate was shown to promote T cell survival.

[0080] Figure 3 Sodium succinate was shown to promote T cell stemness.

[0081] Figure 4 It was shown that succinate induces mitochondrial metabolic and epigenetic changes in T cells.

[0082] Figure 5 Succinate pretreatment of T cells showed anti-tumor function. DETAILED DESCRIPTION

[0083] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that succinic acid (or its salts or esters) can significantly promote T cell survival and T cell stemness. The inventors also provided a method for preparing succinic acid-based stem memory T cells. After treatment with succinic acid (or its salts or esters), stem memory T cells can be efficiently expanded or prepared in vitro. This was the basis for the present invention.

[0084] Specifically, experiments have shown that succinic acid and its salts can significantly promote tumor antigen-specific CD8 + T cell numbers or density increase in the early stages of tumor development or in response to PD-L1 antibody therapy. Treatment with succinate and its salts significantly reduces T cell apoptosis, promotes T cell survival, and significantly enhances T cell stemness. Furthermore, succinate induces changes in T cell mitochondrial metabolism and epigenetic changes.

[0085] In terms of anti-tumor, pretreatment with succinic acid or its salts can enhance the anti-tumor function of T cells, and the combination of succinic acid or its salts with immune checkpoint drugs can synergistically inhibit tumors and significantly increase IFNγ + CD8 + T cell levels.

[0086] Abbreviations and terms

[0087] As used herein, the term "stem memory T cells" refers to TCF1-positive T cells that have a gene expression profile similar to that of memory T cells and can rapidly proliferate and differentiate into cytotoxic T cells after being reactivated by antigens.

[0088] As used herein, the term "knockout" or "KO" refers to the destruction of a gene through genetic engineering technology (such as gene editing), so that the gene is silenced, or its expression is significantly reduced, or an inactive protein is expressed.

[0089] SDH complex

[0090] The succinate dehydrogenase complex (SDH) is composed of four subunits: SDHA, SDHB, SDHC, and SDHD. Its activity requires the assistance of succinate dehydrogenase complex assembly factor 2 (SDHAF2). A functional SDH complex is located in the inner membrane of mitochondria and is the hub connecting tricarboxylic acid cycle metabolism and the mitochondrial electron transport chain, catalyzing the oxidation of succinate to fumarate.

[0091] Inactivation of any of SDHA, SDHB, SDHC, SDHD, and SDHAF2 can lead to loss of SDH complex function, damage to mitochondrial tricarboxylic acid cycle metabolism and the mitochondrial electron transport chain, accumulation of succinate in cells, and increased extracellular succinate levels. In this study, SDHB subunit knockout was used to construct MC38 and B16-OVA succinate-accumulating tumors and test their ability to respond to immune checkpoint blockade therapy.

[0092] Succinic acid and pharmaceutically acceptable salts or esters thereof

[0093] In the present invention, succinic acid and pharmaceutically acceptable salts or esters thereof can be used.

[0094] In the present invention, representative pharmaceutically acceptable salts of succinic acid include (but are not limited to): alkali metal salts (such as Na salts, K salts), alkaline earth metal salts (such as Ca salts, Mg salts, etc.), or ammonium salts of succinic acid.

[0095] In the present invention, representative pharmaceutically acceptable esters of succinic acid include (but are not limited to): esters formed between succinic acid and lower alkanols (such as C1-C6 alkanols), such as dimethyl succinate, diethyl succinate, and the like.

[0096] In the present invention, during in vitro expansion or culture, the concentration of succinic acid and its pharmaceutically acceptable salts or esters that can be generally used (calculated as the concentration of succinic acid) is generally about 5-200 mM, preferably about 10-100 mM, more preferably about 15-80 mM, and most preferably about 20-70 mM.

[0097] Taking succinate as an example, its concentration can generally be about 30-80 mM, such as about 40±5 mM, 50±10 mM.

[0098] Taking dimethyl succinate as an example, its concentration can generally be about 15-50 mM, such as about 20±10 mM.

[0099] Therapeutics targeting immune checkpoints

[0100] As used herein, the term "immune checkpoint-targeted therapeutic agent" or "immune checkpoint-targeted drug" refers to a therapeutic agent or active ingredient, particularly an antibody, that specifically targets immune checkpoints such as PD-L1 and LAG3. It should be understood that the term also includes small molecule compounds that target immune checkpoints.

[0101] In the present invention, representative therapeutic agents targeting immune checkpoints include (but are not limited to): PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, or a combination thereof.

[0102] In vitro expansion method of stem memory T cells

[0103] The present invention also provides an in vitro expansion method of stem memory T cells based on succinic acid pretreatment.

[0104] In a preferred embodiment, the in vitro expansion method of the present invention comprises the following steps:

[0105] (1) After coating the plate with CD3 antibodies, CD28 antibodies, IL-2, and other cytokines are used to activate and expand T cells.

[0106] (2) During the T cell activation and culture process, a sodium succinate solution having a predetermined concentration (eg, 20-100 mM, preferably about 40±10 mM) is added to the T cell culture medium.

[0107] (3) Supplementing the culture medium containing IL-2 and a sodium succinate solution at a predetermined concentration (e.g., 20-100 mM, preferably about 40±10 mM) every 2-3 days.

[0108] (4) After culturing for a period of time (e.g., about 4-8 days, preferably 5-7 days, such as 6 days), a lymphocyte population mainly composed of memory stem T cells can be obtained.

[0109] Active ingredient combinations, pharmaceutical compositions

[0110] The present invention provides an active ingredient combination, which includes (Z1) succinic acid or a pharmaceutically acceptable salt or ester thereof; and (Z2) a drug targeting an immune checkpoint.

[0111] Based on the above combination, the present invention provides a pharmaceutical composition, which can be used to prepare drugs for treating tumors.

[0112] The present invention also provides a pharmaceutical composition comprising a combination of active ingredients within a safe and effective amount range and a pharmaceutically acceptable carrier.

[0113] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, saline, buffer, glucose, water, glycerol, polysorbate, ethanol, etc.

[0114] There is no particular limitation on the administration of the active ingredient combination or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0115] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and / or cells and can be accepted by humans and / or animals.

[0116] When pharmaceutical formulations are used, a safe and effective amount of a combination of active ingredients is administered to a mammal.

[0117] It should be understood that the effective amount of the active ingredients of the present invention may vary depending on the mode of administration and the severity of the disease. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, pharmacokinetic parameters such as bioavailability, metabolism, and half-life; disease severity, patient weight, patient immune status, and route of administration.

[0118] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0119] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and spices.

[0120] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0121] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0122] The main advantages of the present invention include:

[0123] (a) The method of the present invention can efficiently prepare high-quality stem memory T cells.

[0124] (b) The method of the present invention is simple and convenient to operate.

[0125] (c) The method of the present invention can significantly improve the activity and stemness of T cells.

[0126] (d) T cells prepared by the method of the present invention have significantly improved anti-tumor ability.

[0127] (e) In the present invention, succinic acid and its pharmaceutically acceptable salts or esters, when used in combination with immune checkpoint drugs (such as antibody drugs), can synergistically inhibit tumors and synergistically increase IFNγ + CD8 + The number of T cells.

[0128] (f) The present invention discovered that succinate dehydrogenase mutations, including SDHA, SDHB, SDHC, SDHD, and SDHAF2, serve as markers for response to immune checkpoints such as PD1 and PD-L1 antibodies.

[0129] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0130] Material:

[0131] All cells used in the experiment were commercially available.

[0132] B16 cells: B16 mouse melanoma cells

[0133] MC38 cells: mouse colon cancer cells

[0134] B16-OVA cells: B16-OVA mouse melanoma cells (OVA gene modified)

[0135] OT-1 cells: CD8 T cells of OT1 mice that have been genetically modified to specifically recognize OVA antigens + T cells

[0136] Anti-PD-L1 antibody: purchased from Roche

[0137] Example 1 Succinic acid promotes tumor antigen-specific CD8 + Increased number of T cells

[0138] 1.1 Knockout of the Sdhb gene

[0139] The Sdhb gene was knocked out in tumor cells B16, MC38, and B16-OVA using CRISPR-Cas9 technology, and the knockout efficiency was verified by biochemical experiments.

[0140] The results are as follows Figure 1 As shown in A, the Sdhb gene was successfully knocked out in all three tumor cells, indicating that the simulated succinate-enriched tumor model was successfully established.

[0141] 1.2Sdhb knockout leads to PD-1 + CD8 + The proportion of T cells in TIL cells increased

[0142] C57 mice were subcutaneously inoculated with Scramble (control group) and Sdhb knockout B16 tumor cells or MC38 tumor cells to observe the tumor antigen-specific PD-1 + CD8 +The proportion of cells in the total infiltrating lymphocytes. Figure 1 As shown in B and C, in the early stage of tumor growth, the expression of tumor antigen-specific PD-1 + CD8 + The proportion of total infiltrating lymphocytes increased in Sdhb knockout B16 or MC38 tumors (mean ± sem (n = 4), t-test, *p < 0.05). This suggests that succinate enrichment in tumors promotes tumor antigen-specific CD8 + T cell infiltration.

[0143] 1.3 Combination of Sdhb knockout and anti-PD-L1 antibody can lead to a significant increase in tumor antigen-specific T cells. Scramble group and Sdhb knockout MC38 tumor-bearing mice received anti-PD-L1 antibody treatment. Starting from day 7, anti-PD-L1 antibody was injected intraperitoneally at a dose of 6 mg / kg every three days. The tumor growth curve during treatment is shown in Figure 2. Figure 1 The results showed that after treatment with anti-PD-L1 antibodies, tumor growth slowed and tumor size began to gradually decrease after about day 13. The tumor reduction was more obvious in Sdhb knockout tumor-bearing mice, indicating that the succinate-enriched MC38 tumor responded to PD-L1 antibody treatment more effectively.

[0144] Detection of tumor antigen-specific T cells (PD-1 + CD8 + ) absolute number. mean±sem (n=5), t-test, *p<0.05, **p<0.01. Figure 1 As shown in E, PD-1 is on the left + The ratio of the total number of CD8+ to the tumor weight, the right side is the ratio of the total number to the volume, the results show that PD-1 + CD8 + The absolute number of T cells was significantly increased in Sdhb knockout MC38 tumors, consistent with the reduction in tumor volume, further indicating that the infiltrating CD8 + The T cell response to anti-PD-L1 antibody treatment was enhanced, and the infiltration number was greater.

[0145] 1.4Sdhb knockout combined with anti-PD-L1 antibody can synergistically inhibit tumor growth

[0146] The Scramble group and Sdhb knockout B16-OVA tumor-bearing mice received anti-PD-L1 antibody treatment. The treatment process was the same as above, and the results were as follows. Figure 1As shown in Figure 1 and Table 2, compared with Sdhb knockout tumor-bearing mice not treated with anti-PD-L1 antibodies and Scramble group tumor-bearing mice treated with anti-PD-L1 antibodies, Sdhb knockout tumor-bearing mice had the most significant tumor growth inhibition effect after anti-PD-L1 antibody treatment.

[0147] Table 1 Average tumor size

[0148]

[0149]

[0150] *The difference Δ in tumor size reduction is relative to the difference Δ in tumor size of the Scramble group.

[0151] As shown in Table 1, unexpectedly, on day 22, the Sdhb knockout + anti-PD-L1 antibody treatment group showed a synergistic effect in inhibiting the tumor volume of tumor-bearing mice, which was better than the sum of the inhibitory effects of the Sdhb knockout group and the Scramble + anti-PD-L1 antibody treatment group (424.8>128.9+274.8), indicating that succinate-enriched tumors respond better to immune checkpoint therapy.

[0152] 1.5Sdhb knockout combined with anti-PD-L1 antibody synergistically leads to IFNγ + CD8 + Increased T cells

[0153] On day 22 of treatment, lymphocytes isolated from the tumor were stimulated with PMA (10 ng / mL), ionomycin (0.5 ug / mL, brefeldin A (BFA)) and monensin for 4 h, and the expression of cytokine IFNγ was counted. + CD8 + The absolute number of T cells, representing CD8 T cells specifically activated by tumor antigens + T quantity.

[0154] Likewise, if Figure 1 As shown in G and Table 2, after Sdhb knockout + anti-PD-L1 antibody treatment, the most significant IFNγ + CD8 + T cell density and IFNγ + CD8 + The increase in T cell density was much higher than the sum of the Sdhb knockout group and the Scramble + anti-PD-L1 antibody treatment group (i.e., 209.3>10.9+105.7), indicating that succinate-enriched tumors had more tumor antigen-specific activated CD8 + T cells.

[0155] Table 2 IFNγ + CD8 + Average T cell density

[0156]

[0157] *The cell density difference Δ is relative to the cell density difference Δ of the scramble group.

[0158] As shown in Table 3, compared with the Scramble group, the IFNγ + CD8 + T cell density increased to a certain extent. Unexpectedly, in the Sdhb knockout + anti-PD-L1 antibody treatment group, IFNγ + CD8 + The increase in T cell density was much higher than the sum of the Sdhb knockout group (10.9) and the Scramble + anti-PD-L1 antibody treatment group (105.7) (i.e., 209.3>10.9+105.7), showing a significant synergistic effect. + The absolute number of T cells and their ability to respond to anti-PD-L1 antibodies were enhanced in succinate-enriched tumors.

[0159] 1.6 Supplementation of sodium succinate enhances the tumor suppression effect induced by PD-L1 antibody and leads to IFNγ + CD8 + Increased T cells

[0160] Experimental methods: see Figure 1 H,B16-OVA tumor-bearing mice received a single dose of anti-PD-L1 antibody (10 mg / kg) and were subcutaneously injected with sodium succinate (2.77 g / kg daily). Sodium succinate was dissolved in water to a 102.6 mM, pH 7.4 isotonic solution and 2 mL was injected subcutaneously into the neck. A control group received an equal volume of saline.

[0161] Figure 1 Figure 1 shows tumor growth curves in B16-OVA tumor-bearing mice treated with a single anti-PD-L1 antibody treatment (10 mg / kg) followed by subcutaneous injection of sodium succinate (2.77 g / kg daily). The results showed that sodium succinate injection significantly inhibited tumor growth compared to the saline control group, indicating that exogenous succinate supplementation can also enhance anti-tumor immune responses.

[0162] After day 22, the tumor antigen activated T cells (IFNγ + CD8 + ) absolute number, and tumor-infiltrating CD8 T cells that specifically recognize OVA antigens using OVA Tetramer labeling+ Absolute number of T cells. Mean ± sem (n = 5), two-way ANOVA, *p < 0.05, **p < 0.01.

[0163] like Figure 1 J and Table 3, after treatment with sodium succinate, IFNγ in tumors + CD8 + T cells and ova tetramer + The density of T cells increased significantly, by 4.3 times and 2.3 times, respectively, indicating that exogenous supplementation of succinate promoted the infiltration of tumor-specific antigen-specific CD8 + T cell count.

[0164] Table 3 Cell density

[0165]

[0166] *Fold changes are relative to the control group.

[0167] The above results indicate that succinic acid (or its salt) can increase the expression of tumor antigen-specific CD8 + T cell-mediated anti-tumor effects, such as PD-L1 antibody activation, supplementation with succinic acid (or its salt) can significantly inhibit tumor growth and significantly increase IFNγ + CD8 + T cells and ova tetramer + The density or number of T cells.

[0168] Example 2 Sodium Homosuccinate Promotes T Cell Survival

[0169] 2.1Sdhb knockout in tumor supernatant resulted in a significant decrease in the apoptotic rate of co-cultured T cells

[0170] according to Figure 2 After 24 hours of co-culture, the percentage of apoptotic T cells was determined and statistically analyzed. (A) Mean ± SEM (n = 5), t-test, ****p < 0.001. The supernatant from Sdhb-knockout tumors was enriched in succinate.

[0171] The results are as follows Figure 2 As shown in B, the apoptotic proportion of T cells cultured in the supernatant of Sdhb knockout tumors was significantly lower than that in the Scramble treatment group, indicating that the enrichment of succinate inhibited T cell apoptosis induced by antigen stimulation.

[0172] 2.2 Succinate treatment leads to a significant decrease in the proportion of T cell apoptosis

[0173] like Figure 2As shown in Figure C, OT-1 cells were treated with 40 mM sodium succinate. Mixed OT1 mouse spleen cells were activated and cultured for 2 days in medium supplemented with OVA peptide (1 μM), IL-2 (10 ng / mL), control medium, or medium supplemented with succinate. Dead cells were removed by Ficoll and plated on CD3 / CD28-coated plates (both at 0.5 μg / mL). The cells were activated and expanded for another 2 days before the medium was changed and passaged. After 6 days of succinate treatment, dead cells were removed by Ficoll, and the remaining viable cells were cocultured with B16-OVA for 24 hours. The percentage of apoptotic T cells was determined and statistically analyzed. (Mean ± SEM (n = 5), t-test. **p < 0.001).

[0174] The results are as follows Figure 2 D shows Annexin V + PI - Characterization of early apoptotic populations, Annexin V + PI + To characterize the late apoptotic population, the sum of the early apoptotic and late apoptotic populations was counted in this figure. The results showed that the overall apoptotic ratio of T cells in the succinate pretreatment group was significantly lower than that in the control group.

[0175] 2.3 Succinate treatment leads to a significant decrease in the proportion of T cell apoptosis

[0176] Figure 2 E shows the succinate treatment conditions and procedures. Specifically, naive CD8 T cells were purified from the spleen of OT1 mice using a kit and plated onto CD3 (2 μg / mL) / CD28 (1 μg / mL)-coated culture plates. IL-2 was added for activation and culture in the indicated culture medium, and cells were passaged and expanded every two days. On day 6, the survival rate of cells was analyzed. Additionally, dead cells were removed using Ficoll, and either IL-2 or IL-7 was added to the two culture medium conditions. Culture was continued for another 24 hours, and the apoptosis rate was measured.

[0177] The results are as follows Figure 2 F shows that the dead cell population was marked with the dead cell dye FD510, and FD510 was counted. - The results of the population expression of live cell population showed that the survival rate of T cells in the succinate group increased significantly by about 20% on the 6th day, and the apoptosis of T cells in the succinate group was significantly reduced with or without the addition of IL-2 and IL-7. Figure 2 G, mean ± sem (n = 5), t-test, ****p < 0.001. This indicates that succinate inhibits T cell apoptosis independently of IL-2 or IL-7 mediated pathways.

[0178] In addition, use Figure 2Cells were treated with the protocol shown in Figure 3 for 6 days and then restimulated with 1 μM OVA peptide for 24 hours. The overall apoptotic rate was measured. The results also showed that succinate-enriched T cells inhibited apoptosis induced by re-exposure to strong antigen. Mean ± SEM (n = 3), one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. These results demonstrate that direct treatment of CD8 T cells with succinate enhances their anti-apoptotic capacity, independent of the IL2 / 7 signaling pathway.

[0179] Example 3 Succinate promotes T cell stemness

[0180] In this example, the effect of succinate pretreatment on T cell stemness was further tested.

[0181] like Figure 2 Murine OT1 cells were cultured as described in C for 6 days, and the expression of TCF-1, a marker for memory stem cells, was assessed by flow cytometry. TCF-1 is a key transcription factor that promotes the formation of memory T cells and activates the transcription of a series of downstream stemness-related genes, such as Ccr7. Mean ± SEM (n = 5), t-test, ****p < 0.001. Compared with the control group, the expression of TCF-1, a marker for stem cells, was significantly higher in the succinate-treated group, approximately double that of the control group, indicating that succinate treatment promotes the expression of stemness-related transcription factors in cultured murine T cells.

[0182] In addition, human peripheral blood T cells were activated with CD3 / CD28 and cultured without or with 40 mM sodium succinate for 7 days. TCF-1 expression was analyzed by flow cytometry. Mean ± SEM (n = 3), t-test, ****p < 0.0001. TCF-1 expression showed a significant fold increase ( Figure 3 B) Human peripheral blood isolated T cells were treated with 40 mM sodium succinate for 7 days, and CD62L was detected and counted by flow cytometry. + CCR7 + The results showed that after treatment with sodium succinate, CD62L + CCR7 + The proportion of labeled stem memory T cells increased more than doubled from 13.5% to 28.4% compared with the control group ( Figure 3 C), showing that succinate treatment also promotes the stemness phenotype of human T cells cultured in vitro.

[0183] like Figure 2C. Culture mouse OT1 cells to day 6, collect two groups of living cells to extract RNA and sequence, and analyze the expression of memory and effector T cell related genes by heat map. Volcano map of differentially expressed genes by RNA sequencing ( Figure 3 D) and heat map ( Figure 3 E) Analysis showed that after treatment with succinate, the expression of memory-related genes increased, while the expression of effector-related genes decreased. Surface succinate treatment promoted the transcriptional characteristics of stem T cells.

[0184] like Figure 3 As shown in Figure F, T cells were first activated in the absence of succinate, and then treated with succinate. The expression of TCF-1 was detected by flow cytometry, and the expression of TCF-1 (gene name: Tcf7) was detected by qPCR. After treatment with succinate, the expression of TCF-1 ( Figure 3 G), Tcf7 gene transcription levels also increased ( Figure 3 H), indicating that succinate can also induce the stemness of activated T cells.

[0185] The above results indicate that high succinate treatment during in vitro T cell culture induces stem cell differentiation of human and mouse T cells.

[0186] Example 4 Succinate induces T cell mitochondrial metabolism and epigenetic changes

[0187] Flow cytometry was used to detect changes in mitochondrial mass in succinate-treated T cells under continuous antigen stimulation in vitro. Figure 2 Mouse spleen OT1 cells were activated under control conditions or succinate-added conditions as described in C. After removing dead cells on day 3, OVA (1 μM) was added to continue stimulating T cells and continued to be treated under control or succinate-added conditions for 4 days. Flow cytometry and statistical analysis of the changes in T cell mitochondrial membrane potential (A) and reactive oxygen species (ROS) under continuous stimulation conditions were performed. Mean ± sem (n = 3), one-way ANOVA, ***p < 0.001. Figure 4 As shown in A, compared with the control group, after OVA stimulation, the mitochondrial membrane potential marked by TMRE was significantly reduced, while the mitochondrial reactive oxygen species marked by Mito-SOX was significantly increased, indicating that the number of damaged mitochondria increased. The addition of succinate inhibited the loss of membrane potential and the generation of reactive oxygen species ( Figure 4 AB). This indicates that succinate treatment significantly protected T cells from in vitro antigen stimulation, and that continuous antigen stimulation significantly improved the quality of T cell mitochondria.

[0188] Furthermore, the seahorse method was used to detect changes in mitochondrial oxidative phosphorylation capacity of T cells after 6 days of succinate treatment under non-continuous stimulation conditions, and the changes in basal oxygen consumption and maximum oxygen consumption of the two groups of cells were statistically compared; the spare respiratory capacity was calculated; and the changes in mitochondrial ATP production were calculated by subtracting the minimum oxygen consumption from the basal oxygen consumption. Among them, mitochondrial respiratory potential is closely related to the formation of memory T cells. The results showed that after treatment with succinate, the basal oxygen consumption and maximum oxygen consumption of T cells increased significantly, the spare respiratory potential doubled, and the mitochondrial ATP production also increased significantly ( Figure 4 C to Figure 4 F), indicating that succinate treatment improves mitochondrial metabolic adaptability.

[0189] Activated T cells were co-treated with succinate and the mitochondrial ATP synthase inhibitor oligomycin for 4 days, and TCF-1 expression was detected and counted by flow cytometry. Mean ± sem (n = 3), t-test, ***p < 0.001, ****p < 0.0001. The results showed that compared with the control group, TCF-1 expression remained at a higher level after co-treatment of T cells with succinate and the mitochondrial ATP synthase inhibitor oligomycin ( Figure 4 G), indicating that succinate-enhanced mitochondrial metabolism is not sufficient to induce elevated expression of the stemness molecule TCF-1.

[0190] Figure 4 H is a schematic diagram of the tricarboxylic acid cycle. Metabolic mass spectrometry was used to examine changes in the levels of succinate and its upstream molecule, α-ketoglutarate, in T cells following 6 days of succinate treatment. Mean ± sem (n = 4), t-test, ns, no difference, ****p < 0.0001. This indicates that succinate treatment significantly increased the level of succinate in T cells, while the level of its upstream molecule, α-ketoglutarate, did not change significantly ( Figure 4 I), the ratio of succinate to α-ketoglutarate content was more than 4 times that of the control group, suggesting that the enrichment of succinate may inhibit the activity of demethylases using α-ketoglutarate as a substrate and participate in epigenetic changes.

[0191] CUT&Tag sequencing results analysis ( Figure 4J), using CUT&Tag sequencing technology to analyze the modification levels of histone H3 lysine 3 and lysine 27 trimethylation on genes related to T cell differentiation. Among them, H3K4me3 modification promotes gene open expression, while H3K27me3 modification inhibits gene open expression. GO analysis of the intersection of genes with increased H3K4me3 modification and genes with decreased H3K27me3 modification in the sodium succinate treatment group showed that they were related to T cell differentiation. Among them, H3K4me3 binding to stemness-related transcription factors TCF7 and ID3 was enhanced in the succinate treatment group, while H3K27me3 binding was reduced ( Figure 4 K). This indicates that succinate treatment regulates changes in histone H3K4me3 and H3K27me3 modifications, leading to the open expression of stemness-related genes.

[0192] The above results indicate that succinate treatment promotes T cell survival by promoting mitochondrial metabolism and epigenetic chromatin opening of stemness-related molecules, thereby promoting their expression.

[0193] Example 5 Succinate pretreatment of T cells for anti-tumor function

[0194] 5.1 Methods

[0195] CD45.2 + B16-OVA tumor-bearing mice were injected with PBS or adoptively transferred with CD45.1 + OT1 cells were treated with control group or succinate pretreatment, and the tumor growth curve was followed ( Figure 5 A). Mean ± sem (n = 5), one-way ANOVA, **p < 0.01. 14 days after adoptive transfer, tumor tissues were harvested and flow cytometry was used to analyze the percentage and absolute number of tumor-infiltrating OT1 cells. The percentage of terminally exhausted tumor-infiltrating OT1 cells (PD1 + TIM3 + ), and the proportion of “stem-like” exhausted precursor T cells (TCF1 + ). mean±sem(n=5), t-test, *p<0.01, **p<0.01, ***p<0.001.

[0196] 5.2 Results

[0197] like Figure 4 As shown in A, CD45.1 + T cells are a hallmark of adoptively transferred T cells. Adoptive transfer of succinate-pretreated T cells had the most significant inhibitory effect on tumor growth. The proportion of tumor-infiltrating OT1 cells (89.9%) was significantly higher than that in the control group (46.6%), and the absolute number of OT1 cells increased by more than two times ( Figure 5 B), PD1 + TIM3+ The terminal exhaustion ratio of the control group was reduced to a certain extent ( Figure 5 C).

[0198] like Figure 5 As shown in D, the proportion of "stem-like" exhausted precursor T cells (TCF1 + )(19.4%) was more than doubled compared to the control group (8.20%).

[0199] Figure 5 E is a schematic diagram of long-term co-transfer, CD45.1 + CD45.2 + Control group and CD45.1 + The succinate pre-treated group was transferred into B16-OVA tumor-bearing mice for 28 days, and the proportions and phenotypes of the two groups of cells infiltrating the tumor before and 28 days after adoptive transfer were analyzed by flow cytometry.

[0200] Statistical results showed that the cells in the succinate pretreatment group still showed a higher survival advantage 28 days after being adopted into the host body, and the survival rate in tumor tissue was increased by more than 3 times compared with the control group ( Figure 5 F).

[0201] The spleen of the succinate pretreatment group increased by more than 1.5 times compared with the control group, and the proportion in the draining lymph nodes increased by more than 2 times ( Figure 5 G), indicating that succinate pretreatment increases the ability of adoptive cells to maintain long-term in the host.

[0202] Statistics on the absolute number of adoptive cells in tumor tissues showed that the density of the succinate pretreatment group was 3.89 times higher than that of the control group. + The density of exhausted precursor T cells was three times higher on average, and the density of T cells that could secrete multiple cytokines (IFNγ, TNFα, and IL-2) also increased by more than 2.7 times ( Figure 5 H). This indicates that adoptive succinate pretreatment of cells can significantly enhance their long-term maintenance and long-term anti-tumor ability in tumor tissues.

[0203] B16-OVA tumor-bearing mice were injected with PBS or adoptively transferred into the control group or OT1 cells pretreated with succinate through the tail vein. Five days later, they were treated with anti-PD-L1 antibody (6 mg / kg intraperitoneally every three days) and the tumor growth curve was tracked.

[0204] The results of tumor inhibition are as follows Figure 5 I and Table 5.

[0205] Table 5 Tumor volume

[0206]

[0207] *The difference Δ in tumor size reduction is relative to the difference Δ in tumor size of the control group.

[0208] Unexpectedly, on day 27, the succinate + anti-PD-L1 antibody treatment group showed a synergistic effect in inhibiting the tumor size of tumor-bearing mice, which was superior to the sum of the inhibitory effects of the succinate treatment group and the control group + anti-PD-L1 antibody treatment group (i.e., 468.2>236.4+154.4mm 3 ). This suggests that adoptively transferred succinate-pretreated antigen-specific T cells are more responsive to immune checkpoint blockade therapy.

[0209] In addition, draining lymph nodes and tumor tissues were collected 15 days after adoptive transfer, and the absolute number of adoptively transferred OT1 cells, CD45.1 + T cells are a marker of adoptively transferred T cells. Mean ± SEM (n = 6), t-test, *p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0210] The results are as follows Figure 5 J and Table 6.

[0211] Table 6CD45.1 + Average number of T cells

[0212]

[0213] *The cell density difference Δ is relative to the cell density difference Δ of the control group.

[0214] Surprisingly, the absolute number and density of adoptively transferred succinate-pretreated T cells in tumor tissue significantly increased after combined treatment with an anti-PD-L1 antibody, and the increase in cell density was much higher than the combined value of the succinate-treated group and the control group plus anti-PD-L1 antibody treatment (295.5 > 166.1 + 67.2). Succinate-pretreatment of antigen-specific T cells in vitro significantly enhanced their ability to respond to immune checkpoint blockade therapy and control tumors, potentially improving the long-term anti-tumor efficacy of adoptively transferred tumor antigen T cells, CAR-T cells, or TCR-T cells, and the efficacy of combined immune checkpoint blockade therapy in clinical practice.

[0215] discuss

[0216] Currently based on CD8 + T cell anti-tumor methods such as adoptive transfer of CAR-T, TCR-T or immune checkpoint therapy have made great progress in clinical practice, but they still face problems such as low response efficiency and inability to effectively control tumors in the long term. Improving the stemness of tumor antigen-specific T cells is an effective means to increase the effect of the above immunotherapy.+ Whether it is T cells or modified CAR-T or TCR-T cells, T cells need to be expanded and cultured in vitro, but the stemness of conventionally cultured T cells is low and they are rapidly exhausted after adoptive transfer. The present invention proposes an in vitro expansion method for obtaining a high proportion of stem memory T cells, and the addition of high concentrations of succinic acid (salt or ester) during the in vitro culture of T cells. It has been observed in both human and mouse T cells that succinate treatment enhances the memory stemness phenotype of T cells. In addition, the mitochondrial metabolic capacity of T cells cultured using this method is enhanced, and the expression of memory stem differentiation-related genes such as TCF-1 is increased. Not only is apoptosis reduced in the in vitro depletion model, but pretreatment can also enhance the long-term anti-tumor ability of adoptively transferred T cells in vivo, and the ability to respond to immune checkpoint therapy.

[0217] In addition, some malignant tumor patients carry inactivation mutations of the SDH complex, including one or more inactivation mutations of SDHA, SDHB, SDHC, SDHD subunits and SDHAF2 assembly factors, which lead to a large amount of succinate enrichment in the tumor microenvironment. However, the enrichment of succinate has a negative impact on CD8 + The anti-tumor effect of T cells is still controversial. Here, we constructed Sdhb knockout to simulate succinate-rich tumors and found that Sdhb knockout tumors responded better to PD-L1 antibody treatment, and tumor antigen-specific CD8 + Therefore, it is proposed that tumors with inactivation mutations of SDH-related subunits have enhanced responses to immune checkpoint therapy.

[0218] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing stem memory T cells in vitro, characterized in that: Including steps: (a) culturing a first cell population containing T cells in vitro in the presence of succinic acid or a pharmaceutically acceptable salt or ester thereof to obtain a second cell population containing stem memory T cells.

2. The method according to claim 1, wherein The first cell population is a primary or passaged T cell population isolated from a human or non-human mammal.

3. The method according to claim 1, wherein The first cell population is an activated T cell population.

4. The method according to claim 1, wherein In in vitro culture, the concentration of succinic acid or a pharmaceutically acceptable salt or ester thereof is about 5-200 mM, preferably about 10-100 mM, more preferably about 15-80 mM, and most preferably about 20-70 mM, calculated as succinic acid.

5. The method according to claim 1, wherein The method further comprises: (a1) culturing the first cell population containing T cells in vitro under the same culture conditions but in the absence of succinic acid or a pharmaceutically acceptable salt or ester thereof, thereby obtaining a third cell population containing T cells.

6. The method according to claim 1, wherein The method comprises: (t1) After coating the plate with CD3 antibodies, T cells are activated and expanded using CD28 antibodies, IL-2, and other cytokines; (t2) during T cell activation and culture, adding a sodium succinate solution having a predetermined concentration (e.g., 20-100 mM, preferably about 40±10 mM) to the T cell culture medium; (t3) supplementing the culture medium with IL-2 and a sodium succinate solution at a predetermined concentration (e.g., 20-100 mM, preferably about 40±10 mM) every 2-3 days; (t4) After culturing for a period of time (e.g., about 4-8 days, preferably 5-7 days, such as 6 days), a lymphocyte population mainly composed of memory stem T cells can be obtained.

7. A use of succinic acid or a pharmaceutically acceptable salt or ester thereof, characterized in that: For preparing a preparation or composition for one or more uses selected from the group consisting of: (a) Promote tumor antigen-specific CD8 + Increased number of T cells; (b) preparing or expanding stem memory T cells in vitro; (c) increasing T cell activity or reducing T cell apoptosis rate; (d) improving the stemness of T cells; (e) Increase the basal and maximal oxygen consumption of T cells; (f) Increase mitochondrial ATP production in T cells; (g) Promotes chromatin opening and expression of stemness-related molecules in T cells.

8. An active ingredient combination, characterized in that The active ingredient combination includes: (Z1) succinic acid or a pharmaceutically acceptable salt or ester thereof; and (Z2) Drugs targeting immune checkpoints.

9. A medicine box, characterized in that: The medicine box comprises: (Y1) a first pharmaceutical composition comprising (Y1a) succinic acid or a pharmaceutically acceptable salt or ester thereof; and (Y1b) a pharmaceutically acceptable carrier; (Y2) a second pharmaceutical composition comprising (Y2a) an immune checkpoint active ingredient; and (Y2b) a pharmaceutically acceptable carrier.

10. Use of the active ingredient combination according to claim 8, characterized in that: Used for preparing drugs for treating tumors.

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