A method for in vitro induction of expansion of stem memory T cells

Abstract

The present application relates to a method for in vitro induction and expansion of stem memory T cells, which obtains a large number of stem memory T cells by changing the culture conditions of T cells, and the prepared stem memory T cells have multilineage differentiation potential and are suitable for any clinical adoptive immunotherapy, including tumor and infection immunity and autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method for in vitro induction and expansion of stem memory T cells. Background Technology

[0002] Following surgery, radiotherapy, and chemotherapy, T-cell immunotherapy has emerged as a novel cancer treatment method. It involves reinfusing activated and expanded T cells derived from the patient or from an external source to trigger an immune response, thereby treating or alleviating the disease. It is considered a highly promising cancer treatment strategy. However, most current methods rely on terminal effector T cells, which have difficulty surviving long-term in vivo, significantly impacting the clinical efficacy of adoptive cell therapy. Recent studies have shown that lower-differentiated T cells possess stronger anti-tumor effects, particularly the recently reported stem cell-like memory T cells (T cells). SCM Compared to terminal effector T cells, they possess stronger proliferative, self-renewal, and survival capabilities, and can differentiate into central memory T cells and effector memory T cells. In mouse anti-tumor models, adoptive stem memory T cells have been found to exhibit stronger anti-tumor effects than adoptive central memory T cells or effector memory T cells.

[0003] Stem memory T cells are the most desirable cell subset for adoptive T cell therapy. However, due to their difficulty in expansion under conventional culture conditions, an effective method for rapid in vitro induction of stem memory T cells remains lacking. Previous studies have reported that a brief period of CD3 / CD28 co-stimulatory action, combined with the cytokines IL-7, IL-15, and IL-21, can increase the induction rate of stem memory T cells, resulting in a larger number of cells and thus providing a more optimized culture protocol for adoptive T cell therapy. However, large-scale induction of stem memory T cells requires significant amounts of cytokines, greatly increasing production costs. Furthermore, flow cytometry sorting is necessary to obtain large quantities of stem memory T cells, but this technique has high requirements for equipment and operating environment, making it unsuitable for large-scale cell production, and carries a high risk of microbial infection. Therefore, exploring a rapid, efficient, low-cost, and safe method for preparing stem memory T cells is crucial.

[0004] Studies have shown that during in vitro T cell expansion, appropriately inhibiting T cell metabolism can maintain T cells in a low-differentiation state. For example, rapamycin can maintain the stem state of T cells by inhibiting their glycolysis. Mannose is an isomer of glucose, distributed in human blood and various tissues, such as skin and organs. Mannose can participate in the synthesis of glycoproteins and regulate the function of the immune system. Simultaneously, mannose can inhibit tumor growth by interfering with intracellular glucose metabolism and the stability of PD-L1 protein in tumor cells. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] How to provide a method for rapidly and efficiently inducing and expanding stem memory T cells in vitro is a technical problem that urgently needs to be solved by those skilled in the art.

[0007] Solution for solving the problem

[0008] On one hand, the present invention provides a method for in vitro induction and expansion of stem memory T cells, characterized in that the method includes the following steps:

[0009] S1: Activates and expands T cells;

[0010] S2: Add mannose to the culture medium during or after T cell activation.

[0011] Preferably, step S1 includes: coating a cell culture plate with an antibody and then activating and expanding T cells in a culture medium containing cytokines.

[0012] Preferably, the antibody is a CD3 antibody and a CD28 antibody.

[0013] Preferably, the cytokine is selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21.

[0014] Preferably, the cytokine is IL-2.

[0015] Preferably, the concentration of the CD3 antibody is 1–2 μg / mL.

[0016] Preferably, the concentration of the CD28 antibody is 1–2 μg / mL.

[0017] Preferably, the concentration of mannose is 2.0-100.0 mM.

[0018] Preferably, the concentration of mannose is 5.0-50.0 mM.

[0019] Preferably, the method further includes the following steps:

[0020] S3: The culture medium was changed to one containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem memory T cells.

[0021] Preferably, the cytokine is selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21.

[0022] Preferably, the cytokine is IL-2.

[0023] Preferably, the culture medium is replaced every 1-3 days.

[0024] Preferably, the culture medium is replaced every 2 days.

[0025] Preferably, the lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T, and STAR-T.

[0026] Preferably, the T cell species source includes human T cells or mouse-derived T cells.

[0027] On the other hand, the present invention provides a lymphocyte population prepared by the above method.

[0028] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned lymphocyte population.

[0029] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0030] Preferably, the pharmaceutical composition further comprises an additional therapeutic agent.

[0031] On the other hand, the present invention provides the use of the lymphocyte populations prepared by the above method, the above lymphocyte populations, or the above composition in the preparation of adoptive immunotherapy drugs.

[0032] Preferably, the adoptive immunotherapy is anti-tumor immunotherapy, anti-infective immunotherapy, or autoimmune disease treatment.

[0033] On the other hand, the present invention provides the application of mannose in in vitro induction and expansion of stem memory T cells.

[0034] The effects of the invention

[0035] 1. The compounds used in this invention are relatively simple and inexpensive, featuring low price and high production safety. A large number of stem memory T cells can be obtained by modifying the culture conditions of T cells, reducing the use of other cytokines and significantly lowering the toxic side effects of cytokine application, thereby reducing the production cost of stem memory T cells.

[0036] 2. The stem memory T cells obtained by this invention have multi-directional differentiation potential. The differentiated T cells can be efficiently recruited to the sites where they function, capture and clear antigens or tumor cells or pathogens carrying antigens, and maintain their stem T cell characteristics in vivo, creating conditions for further differentiation into effector T cells. This prolongs the in vivo long-term effect of adoptive T cells in adoptive immunotherapy, thereby producing a long-lasting protective effect.

[0037] 3. The stem memory T cells obtained by this invention are applicable to any adoptive immunotherapy in clinical practice, including but not limited to tumors, infections, and autoimmune diseases. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the induced expansion of stem memory T cells in PBMCs.

[0039] Figure 2 Mannose treatment significantly increased the proportion of stem memory T cells in PBMCs; among them, A and B are CD62L cells under the CD8 phylum. + CCR7 + (T SCM ) and CD45RO - CD27 + (T SCM The proportion of T cells; C represents the proportion of CD62L cells under the CD4 phylum. + CCR7 + (T SCM The proportion of T cells; D represents the average fluorescence intensity of T cell stem transcription factor TCF-1, which regulates T cells under the CD8 and CD4 gates, respectively.

[0040] Figure 3 Mannose treatment significantly increased the proportion of stem memory T cells in mice; where A represents CD44 under the CD8 phylum. + CD62L + (T CM A) The proportion of T cells; B) The mean fluorescence intensity of T cell stemness-regulating transcription factor TCF-1 under CD8; C) The effect of mannose on T cell apoptosis; D) The expression level of T cell stemness-related transcription factors in the transcriptome sequencing results of T cells treated with mannose (20mM).

[0041] Figure 4 Treatment with mannose (DM, 50 mM) significantly increased the proportion of 1G4 TCR T cells; where A represents the mean fluorescence intensity of T cell stem transcription factor TCF-1 regulated under the CD8 phylogenetic tree; and B represents the mean fluorescence intensity of T cell stem transcription factor TCF-1 regulated under the CD4 phylogenetic tree.

[0042] Figure 5T cells treated with mannose (DM, 20 mM) exhibited strong and persistent antitumor activity in vivo. Figure A shows a schematic diagram of the in vivo experiment in B16-OVA tumor-bearing mice; Figures B, C, D, and E show the proportions of adopted T cells in mouse tumors, tumor draining lymph nodes (TDLNs), blood, and spleen after adopting OT-I T cells from different treatment groups (control group and mannose-treated group); Figure F shows the growth curves of B16-OVA tumors after adopting OT-I T cells from different treatment groups (control group and mannose-treated group). Detailed Implementation

[0043] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0044] On one hand, the present invention provides a method for in vitro induction and expansion of stem memory T cells, characterized in that the method includes the following steps:

[0045] S1: Activates and expands T cells;

[0046] S2: Add mannose to the culture medium during or after T cell activation.

[0047] In some implementations, step S1 includes: activating and expanding T cells in a culture medium containing cytokines after coating a cell culture plate with an antibody.

[0048] In some implementations, the antibodies are CD3 antibodies and CD28 antibodies.

[0049] In some embodiments, the cytokine is selected from one or more of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0050] In some implementations, the cytokine is selected from IL-2.

[0051] In some embodiments, the concentration of the CD3 antibody is 1–2 μg / mL.

[0052] In some implementations, the concentration of the CD3 antibody is approximately 1 μg / mL.

[0053] In some implementations, the concentration of the CD3 antibody is approximately 1.5 μg / mL.

[0054] In some implementations, the concentration of the CD3 antibody is approximately 2 μg / mL.

[0055] In some embodiments, the concentration of the CD28 antibody is 1–2 μg / mL.

[0056] In some embodiments, the concentration of the CD28 antibody is about 1 μg / mL.

[0057] In some implementations, the concentration of the CD28 antibody is approximately 1.5 μg / mL.

[0058] In some embodiments, the concentration of the CD28 antibody is about 2 μg / mL.

[0059] In some embodiments, the concentration of the mannose is 2.0-100.0 mM.

[0060] In some embodiments, the concentration of mannose is 5.0-50.0 mM.

[0061] In some embodiments, the concentration of the mannose is about 5.0 mM, about 6.0 mM, about 7.0 mM, about 8.0 mM, about 9.0 mM, about 10.0 mM, about 11.0 mM, about 12.0 mM, about 13.0 mM, about 14.0 mM, about 15.0 mM, about 16.0 mM, about 17.0 mM, about 18.0 mM, about 19.0 mM, about 20.0 mM, about 21.0 mM, about 22.0 mM, about 23.0 mM, about 24.0 mM, about 25.0 mM, about 26.0 mM, about... 27.0mM, approximately 28.0mM, approximately 29.0mM, approximately 30.0mM, approximately 31.0mM, approximately 32.0mM, approximately 33.0mM, approximately 34.0mM, approximately 35.0mM, approximately 36.0mM, approximately 37.0mM, approximately 38.0mM, approximately 39.0mM, approximately 40.0mM, approximately 41.0mM, approximately 42.0mM, approximately 43.0mM, approximately 44.0mM, approximately 45.0mM, approximately 46.0mM, approximately 47.0mM, approximately 48.0mM, approximately 49.0mM, approximately 50.0mM.

[0062] In some implementations, the method further includes the following steps:

[0063] S3: The culture medium was changed to one containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem memory T cells.

[0064] In some embodiments, the cytokine is selected from one or more of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0065] In some implementations, the cytokine is selected from IL-2.

[0066] In some implementations, the culture medium is replaced every 1-3 days.

[0067] In some implementations, the culture medium is replaced every 2 days.

[0068] In some embodiments, the lymphocyte population includes CD4 T cells, CD8 T cells, γδT, TILs, TCR-T, CAR-T, and STAR-T.

[0069] In some implementations, the T cell species source includes human T cells or mouse-derived T cells.

[0070] In some embodiments, the present invention provides a method for in vitro expansion of stem memory T cells, comprising the following steps: separating and obtaining human peripheral blood mononuclear cells (PBMCs) using a dextran-diatrizoate meglumine mixture. The obtained PBMCs are then seeded in 24-well culture plates pre-coated with human CD3 antibody, and free human CD28 antibody and cytokines such as rhIL-2 are added for activation and culture. At any time during or within the first 3 days of PBMC activation, mannose is added to the culture medium, and the cells are cultured at 37°C, 5% CO2, and saturated humidity. Three days after T cell activation, half the medium is replaced, and the corresponding amount of rhIL-2 is added. Subsequently, depending on the T cell growth status, an equal amount of culture medium containing rhIL-2 and mannose is added every 2 days to maintain the T cell density at 0.5-2 × 10⁻⁶ cells / well. 6 / ml. Stem memory T cells can be obtained after 9-15 days of culture.

[0071] On the other hand, the present invention provides a lymphocyte population prepared by the above method.

[0072] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned lymphocyte population.

[0073] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0074] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0075] On the other hand, the present invention provides the use of the lymphocyte populations prepared by the above method, the above lymphocyte populations, or the above composition in the preparation of adoptive immunotherapy drugs.

[0076] In some implementations, the adoptive immunotherapy is antitumor immunotherapy, anti-infective immunotherapy, or autoimmune disease treatment.

[0077] This invention provides a method for processing individuals, which may include:

[0078] The expanded lymphocyte population described above is then administered to individuals in need.

[0079] Lymphocyte populations can be administered intravenously, for example, by infusion into an individual.

[0080] On the other hand, the present invention provides the application of mannose in in vitro induction and expansion of stem memory T cells.

[0081] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art upon reference to the published text.

[0082] The terms “a” or “an” refer to one or more molecules, for example, “a molecule” should be understood to represent one or more molecules. Therefore, the terms “a” or “an”, “one or more”, and “at least one” are used interchangeably in this document.

[0083] In the claims and description of this invention, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” are used in the sense of inclusion, that is, specifying the presence of the said feature, but not excluding the presence or addition of other features in various embodiments of the invention.

[0084] The term "about" covers a range of values ​​within ±25% of a given value. In other embodiments, the term "about" covers a range of values ​​within ±20%, ±15%, ±10%, or ±5% of a given value. For example, "about 3 grams" means a value of 2.7-3.3 grams (i.e., 3 grams ± 10%).

[0085] The term "T cell" refers to a cell with a TCR on its surface or a TCR that has been genetically edited or modified. In this specification, "T cell" can refer to a cell population containing other cell types besides T cells themselves, such as a cell population containing more than 60%, 70%, 80%, 90%, or 95% T cells. Furthermore, T cells include T cells at all stages of differentiation and can be T cells that carry antigens or markers on their cell surface.

[0086] The terms "stem memory T cells" and "memory stem cell-like T cells" generally refer to cells in the early differentiation stage of memory T cells, possessing stem cell characteristics and strong multi-lineage differentiation potential (T memory stem cells, T cells). SCM ). T SCM After responding to antigen stimulation, cells can differentiate into central memory T cells (T cells). CM ), effector memory T cells (T cells) EM ) and effector T cells (T cells) EF This leads to the generation of a large number of IFN-γ effectors, resulting in a more powerful lethality. While possessing differentiation potential, it also has a strong self-renewal capability.

[0087] The term "mannose" generally refers to a type of sugar. For example, the CAS number for mannose can be 3458-28-4. In this application, mannose can refer to any derivative of mannose, which may be naturally derived and / or artificially synthesized.

[0088] The term "culture medium" generally refers to a medium that enables stem cells to grow normally. In some embodiments, the stem cell culture medium also keeps the stem cells in suspension. For example, in this application, the stem cell culture medium may comprise DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.

[0089] The term "CD3 antibody" generally refers to an antigen-binding molecule that specifically binds to CD3. For example, CD3 antibodies can contain T3, I, eu4, and HCHT1. CD3 molecules are linked to T cell antigen receptors via salt bridges and participate in T cell signal transduction.

[0090] The term "CD28 antibody" generally refers to an antigen-binding molecule that specifically binds to CD28. Human CD28 is located at 2q33 and has similar exons and introns to CTLA4. Both have ligands from the B7 family, including B7-1 (CD80) and B7-2 (CD86). CD28 and CD80 have high aggregation affinity and can interact with a variety of signaling molecules or kinases, such as PI-3K, GRB-2 / s, and tyrosine kinase ITK.

[0091] The term "cytokine" generally refers to a class of small-molecule proteins with broad biological activity synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. These cytokines can have multiple functions, including regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cell function, and tissue repair. In this application, the cytokines may include interleukins, interferons, the tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. For example, the cytokine may be an interleukin.

[0092] The term "interleukin" generally refers to secreted proteins or signaling molecules that promote the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes, as well as by monocytes, macrophages, and endothelial cells. As used herein, interleukin (IL) can be selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL -18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. As used herein, the term "interleukin" may comprise a full-length interleukin or a fragment (e.g., a truncated form) or a variant thereof that substantially retains the biological activity of the corresponding wild-type interleukin (e.g., having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even at least 100% of the biological activity of the corresponding wild-type interleukin). Interleukins as used herein may be derived from any mammalian species. In some embodiments, interleukins are derived from species selected from humans, horses, cattle, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In some embodiments, interleukins may be mutant forms. For example, an interleukin may be superIL-2 (also known as sIL2, see Nature 484, 529-533, 2612), which can be obtained by modifying IL-2 to increase its binding affinity for IL-2Rβ. Mutations in sIL-2 are primarily core cytokines. Molecular dynamics simulations indicate that evolutionary mutations stabilize IL-2, reducing the flexibility of the helix at the IL-2Rβ binding site to a conformation similar to the optimized receptor-binding conformation when binding to CD25. Compared to IL-2, sIL-2 induces superior expansion of cytotoxic T cells, leading to improved in vivo antitumor responses, less expansion of regulatory T cells, and reduced pulmonary edema. For example, in this application, the cytokines comprise one or more selected from the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0093] The term "pharmaceutical composition" refers to a lymphocyte population containing the stem memory T cells or predominantly stem memory T cells as described herein, formulated for administration to an individual. Preferably, the pharmaceutical composition is sterile. In one embodiment, the pharmaceutical composition is pyrogen-free.

[0094] Stem memory T cells, or a predominantly stem memory T cell lymphocyte population, will be formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the type of specific disease being treated, the specific individual being treated, the individual's clinical condition, the site of administration, the method of administration, the timing of administration, possible side effects, and other factors known to the physician.

[0095] The term "effective dose" refers to the amount of stem memory T cells or a lymphocyte population dominated by stem memory T cells that effectively treat an individual's disease condition, disease, or symptom.

[0096] The term "tumor" generally refers to a physiological state in mammals characterized by unregulated cell growth. In this application, a tumor may include lymphoma, blastoma, sarcoma, and leukemia. For example, it may include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma). Golden lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate-grade diffuse NHL, advanced immunoblastic NHL, advanced lymphocytic NHL, advanced small uncut cell NHL, AIDS-associated lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorder (PTLD).

[0097] The term "infectious disease" generally refers to a disease caused by a virus invading the body through various routes and multiplying in susceptible host cells. For example, viral infectious diseases can include upper respiratory tract infections, bronchitis, viral pneumonia, viral enteritis, viral myocarditis, measles, chickenpox, rubella, roseola infantum, hand-foot-and-mouth disease, mumps, hepatitis B, SARS, HIV, AIDS, avian influenza, and hepatitis A.

[0098] The term "autoimmune disease" generally refers to diseases caused by damage to the body's own tissues due to an immune response to self-antigens. Autoimmune diseases can be divided into organ-specific autoimmune diseases and systemic autoimmune diseases. For example, these autoimmune diseases may include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, goodpasture syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, polyarteritis nodosa, and Wegener's granulomatosis.

[0099] The term "individual" refers to a mammal. A mammal can be a primate, particularly a human, or it can be livestock, zoo animals, or companion animals. In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals can be used, particularly mammals commonly used as models for demonstrating therapeutic efficacy in humans (e.g., rodents, primates, pigs, dogs, or rabbits).

[0100] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0101] In this invention, the PBMCs are obtained, including but not limited to those obtained by density gradient centrifugation; the final concentration of rhIL-2 is 50-100 IU; the cytokines include, but are not limited to, any cytokines used for in vitro T cell induction such as IL-7, IL-15, and IL-21; the concentration of added mannose can be between 2.0-100.0 mM, with a recommended concentration range of 10.0-50.0 mM.

[0102] Example 1: An in vitro expansion method using human stem memory T cells as the main cell population.

[0103] This embodiment includes the following steps:

[0104] 1. Coat cell culture plates with human CD3 antibody at a final concentration of 1 μg / ml;

[0105] 2. Collect 10 ml of peripheral blood from a healthy individual into a centrifuge tube. Dilute the peripheral blood with phosphate-buffered saline (PBS) at a 1:1 ratio and mix thoroughly. Take another 50 mL centrifuge tube, add 20 mL of lymphocyte separation medium (Ficoll), and then slowly add the mixed blood diluent along the tube wall to the upper layer of the lymphocyte separation medium at a Ficoll:blood dilution volume ratio of 1:1, so that the two can form clear layers. Centrifuge at 800 g with no acceleration and no deceleration for 20 min.

[0106] 3. After centrifugation, transfer the mononuclear cell layer to a 50mL centrifuge tube, wash once with 20mL PBS, centrifuge at 800g for 5min, and discard the supernatant; then resuspend the cells in T cell culture medium for counting and dilute the cells to 1 million / ml.

[0107] 4. Next, add human CD28 antibody to a final concentration of 1 μg / ml and rhIL-2 to 300 U / ml of T cell suspension, mix well, seed the cells in pre-coated 24-well plates, add the appropriate concentration of mannose, mix well by pipetting, and then place in an incubator for culture. Depending on the T cell growth status, supplement with an equal volume of culture medium containing rhIL-2 and mannose every 2 days to maintain the T cell density at 0.5-2 × 10⁻⁶ cells / well. 6 / ml. After 12 days of culture, the proportion of stem memory T cells in the culture and the expression level of TCF-1, a transcription factor that regulates the differentiation of stem memory T cells, were detected by flow cytometry.

[0108] 5. Results are as follows Figure 2 As shown: On day 12 of culture, in the mannose-treated group, CCR7... + CD62L + and CD45RO - CD27 + The proportion of stem memory T cells in the treated T cells was significantly higher than that in the control group under normal culture conditions. At the same time, the expression of TCF-1, a transcription factor that regulates the stemness of T cells, was also significantly higher in the mannose-treated T cells than in the control group.

[0109] Example 2: An in vitro expansion method using mouse stem memory T cells as the main cell population.

[0110] This embodiment includes the following steps:

[0111] 1. First, the cell culture plate was pre-coated with mouse CD3 antibody at a final concentration of 2ug / ml.

[0112] 2. Mouse spleen cells were isolated and mouse T cells were obtained by sorting with nylon hairs or magnetic beads, and then diluted to 1 million / ml with T cell culture medium.

[0113] 3. Add mouse CD28 antibody to T cells at a final concentration of 1 μg / ml, rm IL-2 at a final concentration of 20-50 U / ml, and mannose at the appropriate concentration. Mix well by pipetting. Seed the cells in cell culture plates coated with mouse CD3 antibody and incubate them in an incubator.

[0114] 4. Then, based on the T cell growth status, add an equal amount of culture medium containing recombinant mouse IL-2 protein and mannose every 24 hours to maintain the cell density at 0.5-2 × 10⁻⁶ cells / day. 6 / ml. On day 6 of T cell culture, flow cytometry was used to detect the proportion of stem memory T cells in the culture and the expression level of TCF-1, a transcription factor that regulates the differentiation of stem memory T cells.

[0115] 5. Results are as follows Figure 3 As shown: On day 6 of culture, CD44 was the most dominant cell type in the mannose-treated group. + CD62L + The proportion of stem memory T cells increased from about 15.0% to about 77.8%, which was significantly higher than that of T cells under normal culture conditions. At the same time, in T cells treated with mannose, the expression of TCF-1, a transcription factor that regulates T cell stemness, and the transcriptional levels of transcription factors related to T cell stemness were significantly higher than those in the control group.

[0116] Example 3: A method for in vitro expansion of 1G4 TCR T cells with stem memory T cells as the main cell population.

[0117] 1. Coat cell culture plates with human CD3 antibody at a final concentration of 1 μg / ml.

[0118] 2. Collect 10 ml of peripheral blood from a healthy individual into a centrifuge tube. Dilute the peripheral blood with phosphate-buffered saline (PBS) at a 1:1 ratio and mix thoroughly. Take another 50 mL centrifuge tube, add 20 mL of lymphocyte separation medium (Ficoll), and then slowly add the mixed blood diluent along the tube wall to the upper layer of the lymphocyte separation medium at a Ficoll:blood dilution volume ratio of 1:1, so that the two can form clear layers. Centrifuge at 800 g with no acceleration and no deceleration for 20 min.

[0119] 3. After centrifugation, transfer the mononuclear cell layer to a 50mL centrifuge tube, wash once with 20mL PBS, centrifuge at 800g for 5min, and discard the supernatant; then resuspend the cells in T cell culture medium for counting and dilute the cells to 1 million / ml.

[0120] 4. Then, add human CD28 antibody to a final concentration of 1ug / ml and rhIL-2 to 300U / ml to the T cell suspension, mix well, seed the cells in a pre-coated 24-well plate, add the corresponding concentration of mannose, mix well by pipetting, and then place in an incubator for culture.

[0121] 5. After T cells have been cultured for 48 hours, PBMCs were infected with 1G4 TCRs using viral centrifugation.

[0122] 6. After T-cell viral infection, add mannose solution of appropriate concentration and continue culturing. Depending on the T-cell growth status, supplement with an equal volume of culture medium containing rhIL-2 and mannose every two days to maintain T-cell density at 0.5-2 × 10⁻⁶ cells / day. 6 / ml. After 12 days of culture, the expression level of TCF-1, a transcription factor that regulates the differentiation of stem memory T cells, in the culture was detected by flow cytometry.

[0123] 7. Flow cytometry analysis revealed that a lymphocyte population primarily composed of stem memory T cells was obtained after 12 days of culture. Results are as follows... Figure 4 As shown, compared with the control group, mannose treatment significantly increased the expression of TCF-1, a transcription factor that regulates the differentiation of dry memory T cells.

[0124] Example 4: Detection of the antitumor function of mannose-induced mouse stem memory T cells in vivo

[0125] This embodiment includes the following steps:

[0126] 1. The antitumor effect of mannose-induced stem memory T cells in mice was conducted in 4-8 week old C57BL6 / N mice. To demonstrate that mannose-treated memory stem T cells possess better antitumor function and persistence compared to normally treated T cells, this invention aims to utilize the OT-I T cell model to investigate the in vivo antitumor function of mannose-induced stem memory T cells.

[0127] 2. First, subcutaneously inoculate C57BL6 / N mice with 2×10⁻⁶ cells per muscle at the axilla of the right hind limb. 5 B16-OVA cells were used to grow tumor cells subcutaneously in mice for approximately 8-10 days. Tumor volume was then measured using calipers. When the tumor volume reached 75 mm², the tumor was considered complete. 3 -150mm 3 At that time, C57BL6 / N tumor-bearing mice were injected intravenously with 4×10 6OT-1 T cells were treated with or not treated with mannose. Tumor volume was measured every 2 days. After about 14 days (the specific operation time depends on the experimental results), spleen and tumor tissue of C57BL6 / N mice were obtained. The mouse tumor tissue was ground and then centrifuged by Percoll density gradient centrifugation, and analyzed by flow cytometry.

[0128] 3. Results are as follows Figure 5 As shown, firstly, statistical analysis of tumor growth volume revealed that, compared to the control group, the tumor growth rate in the experimental group with mannose-treated T cells was significantly slowed down. Furthermore, flow cytometry results showed that the proportion of mannose-treated OT-IT cells in the tumor tissue of tumor-bearing mice increased from approximately 1.0% to approximately 10%, while the proportion of infiltrating T cells in the spleen also significantly increased. This indicates that mannose-treated OT-IT cells exhibit stronger and more persistent anti-tumor effects in the mouse adoptive model.

[0129] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for in vitro induction and expansion of stem memory T cells, characterized in that, The method includes the following steps: S1: Activates and expands T cells; S2: Add mannose to the culture medium during or after T cell activation.

2. The method according to claim 1, characterized in that, Step S1 includes: after coating a cell culture plate with an antibody, activating and expanding T cells in a culture medium containing cytokines.

3. The method according to claim 2, characterized in that, The antibodies are CD3 antibody and CD28 antibody.

4. The method according to claim 2, characterized in that, The cytokines are selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21.

5. The method according to claim 4, characterized in that, The cytokine in question is IL-2.

6. The method according to claim 3, characterized in that, The concentration of the CD3 antibody is 1~2 μg / mL, and the concentration of the CD28 antibody is 1~2 μg / mL.

7. The method according to any one of claims 1-6, characterized in that, The concentration of mannose is 2.0-100.0 mM.

8. The method according to claim 7, characterized in that, The concentration of mannose is 5.0-50.0 mM.

9. The method according to claim 1, characterized in that, The method further includes the following steps: S3: Change the culture medium to one containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem memory T cells; the cytokines are selected from one or more of IL-2, IL-7, IL-15, IL-17 and IL-21.

10. The method according to claim 9, characterized in that, The cytokine in question is IL-2.

11. The method according to claim 9, characterized in that, The culture medium should be changed every 1-3 days.

12. The method according to claim 11, characterized in that, The culture medium is changed every 2 days.

13. The method according to claim 9, characterized in that, The lymphocyte population includes CD4 T cells, CD8 T cells, γδ T cells, TILs, TCR-T cells, CAR-T cells, and STAR-T cells.

14. The method according to claim 1, characterized in that, The T cell species include human T cells or mouse-derived T cells.

15. Application of mannose in in vitro induction and expansion of stem memory T cells.

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