Method for inducing and amplifying stem cell-like memory T cells in vitro.

Abstract

This method involves in vitro induction and amplification of stem cell-like memory T cells, obtaining a large number of stem cell-like memory T cells by modifying the T cell culture conditions. The prepared stem cell-like memory T cells possess multidirectional differentiation potential and are suitable for clinical use in all adoptive immunotherapy applications, including tumor immunotherapy, infectious immunotherapy, and autoimmune diseases.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and more particularly to a method for inducing and amplifying stem cell-like memory T cells in vitro. [Background technology]

[0002] T-cell immunotherapy is a novel tumor treatment method following surgery, radiotherapy, and chemotherapy. It aims to treat or alleviate disease by inducing an immune response in the body through autologous transfusion of allogeneic or autologous T cells that have been activated and amplified ex vivo. Currently, it is considered a very promising strategy for tumor treatment. However, this method is currently based primarily on terminal effector T cells, which have difficulty surviving long periods in the body, significantly impacting the clinical efficacy of adoptive cell therapy. Recent studies have shown that poorly differentiated T cells exhibit stronger antitumor effects, particularly in relation to recently reported stem cell-like memory T cells (T cells). SCM Compared to terminal effector T cells, these cells possess stronger proliferative, self-renewal, and survival capabilities, and can differentiate into central memory T cells and effector memory T cells. In a mouse antitumor model, adoptive stem cell-like memory T cells were found to exhibit stronger antitumor effects than adoptive central memory T cells or effector memory T cells.

[0003] Stem cell-like memory T cells are the most ideal cell subpopulation for adoptive T cell therapy, but amplification is difficult under normal culture conditions, and therefore, an effective method for rapidly inducing stem cell-like memory T cells in vitro has not yet been established. It has been reported that transient application of CD3 / CD28 costimulatory molecules, followed by the combined use of cytokines IL-7 and IL-15 with IL-21, improves the induction rate of stem cell-like memory T cells and allows for the culture of a larger number of stem cell-like memory T cells, thus providing an optimal culture method for adoptive T cell therapy. However, when inducing large quantities of stem cell-like memory T cells, the large doses of cytokine drugs significantly increase production costs. At the same time, flow cytometry sorting technology is used to obtain large quantities of stem cell-like memory T cells, but this technology has high demands on equipment and operating environment, making it unsuitable for large-scale cell production, and also carries a high risk of microbial infection. Therefore, it is particularly important to find a method for rapidly preparing stem cell-like memory T cells with high efficiency, low cost, and high safety margin.

[0004] Several studies have shown that T cells can be kept in a poorly differentiated state by appropriately inhibiting T cell metabolism during in vitro amplification. For example, rapamycin can keep T cells in a stem cell state by inhibiting the glycolysis process of T cells. Mannose is an isomer of glucose and is distributed in various tissues such as human blood, skin, and organs. Mannose is involved in the synthesis of glycoproteins and can regulate the function of the autoimmune system. At the same time, mannose can suppress tumor growth by interfering with glucose metabolism and the stability of the PD-L1 protein in tumor cells. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] A technical challenge that those skilled in the art should urgently address is providing a method for rapidly and efficiently inducing and amplifying stem cell-like memory T cells in vitro. [Means for solving the problem]

[0006] In one embodiment, the present invention is S1: A process for activating and amplifying T cells, S2: A step of culturing T cells by adding mannose to the culture medium during or after activation. The present invention provides a method for inducing and amplifying stem cell-like memory T cells in vitro, characterized by including the following:

[0007] Preferably, step S1 above includes coating the cell culture plate with an antibody, followed by activating and amplifying T cells in a cytokine-containing medium.

[0008] Preferably, the antibodies mentioned above are CD3 antibodies and CD28 antibodies.

[0009] Preferably, the cytokines are selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0010] Preferably, the cytokine is IL-2.

[0011] Preferably, the concentration of the above-mentioned CD3 antibody is 1-2 μg / mL.

[0012] Preferably, the concentration of the CD28 antibody is 1-2 μg / mL.

[0013] Preferably, the mannose concentration is 2.0 to 100.0 mM.

[0014] Preferably, the mannose concentration is 5.0 to 50.0 mM.

[0015] Preferably, the above method further includes the following steps.

[0016] S3: The cells are cultured in a medium containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem cell-like memory T cells.

[0017] Preferably, the above cytokine is selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0018] Preferably, the above cytokine is IL-2.

[0019] Preferably, the time for changing the above medium is 1 to 3 days.

[0020] Preferably, the time for changing the above medium is 2 days.

[0021] Preferably, the above lymphocyte population includes CD4 T cells, CD8 T cells, γδ T, TILs, TCR-T, CAR-T, STAR-T.

[0022] Preferably, the above method further includes the following steps.

[0023] S4: Stimulate the T cells obtained in step S3 with an antibody and further culture them.

[0024] Preferably, the above culture is carried out for 12 days.

[0025] Preferably, the above step S4 is carried out cyclically two or more times.

[0026] Preferably, the above antibody is a CD3 antibody and / or a CD28 antibody.

[0027] Preferably, the above antibody is a CD3 antibody.

[0028] Preferably, the concentration of the above antibody is 0.5 to 2 μg / mL.

[0029] Preferably, the concentration of the above antibody is 1 μg / mL.

[0030] Preferably, the species origin of the above T cells includes human-derived T cells or mouse-derived T cells.

[0031] In another embodiment, the present invention provides a population of lymphocytes prepared by the method described above.

[0032] In another embodiment, the present invention provides a pharmaceutical composition comprising the above-mentioned lymphocyte population.

[0033] Preferably, the above pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0034] Preferably, the above pharmaceutical composition further comprises additional therapeutic agents.

[0035] In another embodiment, the present invention provides the use of a lymphocyte population prepared by the above method, the above lymphocyte population, or the above composition in the preparation of a pharmaceutical for adoptive immunotherapy.

[0036] Preferably, the adoptive immunotherapy described above is antitumor immunotherapy, anti-infective immunotherapy, and autoimmune disease therapy.

[0037] In another aspect, the present invention provides the use of mannose in the induction and amplification of stem cell-like memory T cells in vitro. [Effects of the Invention]

[0038] 1. The compounds used in this invention are relatively simple and inexpensive, and are characterized by low production costs and high production safety. By changing the culture conditions of T cells, a large number of stem cell-like memory T cells can be obtained, thereby reducing the use of other cytokines, significantly reducing toxic side effects resulting from cytokine use, and lowering the production cost of stem cell-like memory T cells.

[0039] 2. The stem cell-like memory T cells obtained in this invention have multidirectional differentiation potential. Differentiated T cells can be efficiently recruited to local areas to perform their function of capturing and eliminating antigens or antigen-carrying tumor cells or pathogens while maintaining the characteristics of stem cell-like T cells in vivo. This creates conditions for further differentiation into effector T cells, extends the in vivo persistence of adoptive T cells in adoptive immunotherapy, and provides long-term protective effects.

[0040] 3. The stem cell-like memory T cells obtained in this invention are suitable for all clinical adoptive immunotherapy, including but not limited to tumor immunity, infection immunity, and autoimmune diseases. [Brief explanation of the drawing]

[0041] [Figure 1] This is a schematic diagram illustrating the induction and amplification of stem cell-like memory T cells in PBMCs. [Figure 2] This study demonstrates that mannose treatment significantly promotes the formation of stem cell-like memory T cells in PBMCs. Here, A and B show the percentages of CD62L+CCR7+(TSCM) and CD45ROCD27+(TSCM) T cells when gated to CD8, and C shows the mean fluorescence intensity of the T cell stem cell transcription factor TCF-1 when gated to CD8. [Figure 3] This study demonstrates that mannose treatment significantly promotes the formation of mouse stem cell-like memory T cells. Here, A represents the percentage of CD44+CD62L+(TCM) T cells when gated to CD8, B represents the average fluorescence intensity of the T cell stem cell transcription factor TCF-1 when gated to CD8, C represents the effect of mannose on the rate of T cell apoptosis, and D represents the expression levels of T cell stem cell-related transcription factors in transcriptome sequencing results of T cells treated with mannose (20 mM). [Figure 4]This study demonstrates that treatment with mannose (DM, 50 mM) significantly promotes the formation of stem cell-like memory T cells in 1G4 TCR T cells. Here, A shows the average fluorescence intensity of TCF-1 when gated to CD8, and B shows the average fluorescence intensity of TCF-1 when gated to CD4. [Figure 5] This study demonstrates that long-term in vitro treatment with mannose (DM, 50 mM) significantly promotes the formation of stem cell-like memory T cells. Here, A is an experimental flowchart of T cells treated with mannose for a long period. B shows the percentage of CD45RO-CD27+ stem cell-like memory T cells when gated to CD8, and C shows the average fluorescence intensity of the T cell stem cell transcription factor TCF-1 when gated to CD8. [Figure 6] This study shows that long-term in vitro treatment with mannose (DM, 50 mM) significantly increases the proportion of 1G4 TCRT cells. Here, A represents the proportion of CD45RO-CD27+ stem cell-like memory T cells when gated to CD8, and B represents the average fluorescence intensity of the T cell stem cell transcription factor TCF-1 when gated to CD8. [Figure 7] This study demonstrates that long-term in vitro treatment with mannose (DM, 50 mM) significantly promotes the formation of stem cell-like memory T cells in TIL cells. The figure shows the average fluorescence intensity of the T cell stem cell transcription factor TCF-1 when gated to CD8. [Figure 8] T cells (CE12 / ME12 / CE36 / ME36) treated with mannose (DM, 50 mM) for an extended period showed strong in vivo antitumor activity. Here, A is a schematic diagram of an in vivo experiment in HepG2-NYESO tumor-bearing mice, and B and C are growth curves and tumor volume statistics of HepG2-NYESO tumors after adoptive transfer of 1G4 TCR T cells (CE12 / ME12 / CE36 / ME36) from different treatment groups, respectively. [Figure 9]T cells treated with mannose (DM, 20 mM) showed strong in vivo antitumor activity and persistence. Here, A is a schematic diagram of an in vivo experiment in B16-OVA tumor-bearing mice, and B, C, D, and E show the proportion of adoptive T cells in the mouse tumor, tumor-perfusion lymph nodes (TDLN), blood, and spleen after adoptive transfer of OT-IT cells from different treatment groups (control group and mannose-treated group), respectively. F shows the growth curves of the B16-OVA tumor after adoptive transfer of OT-IT cells from different treatment groups (control group and mannose-treated group). [Modes for carrying out the invention]

[0042] To make the technical means and beneficial effects of the present invention clearer and easier to understand, the following detailed description is provided through specific embodiments. Herein, the accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of local features. Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood in the art to which the present invention pertains.

[0043] In one embodiment, the present invention is S1: A process for activating and amplifying T cells, S2: A step of culturing T cells by adding mannose to the culture medium during or after activation. The present invention provides a method for inducing and amplifying stem cell-like memory T cells in vitro, characterized by including the following:

[0044] In some embodiments, step S1 above includes coating a cell culture plate with an antibody, followed by activating and amplifying T cells in a cytokine-containing medium.

[0045] In some embodiments, the antibodies described above are CD3 antibodies and CD28 antibodies.

[0046] In some embodiments, the cytokines described above are selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0047] In some embodiments, the above cytokines are selected from IL-2.

[0048] In some embodiments, the concentration of the above-mentioned CD3 antibody is 1-2 μg / mL.

[0049] In some embodiments, the concentration of the above-mentioned CD3 antibody is approximately 1 μg / mL.

[0050] In some embodiments, the concentration of the above-mentioned CD3 antibody is approximately 1.5 μg / mL.

[0051] In some embodiments, the concentration of the above-mentioned CD3 antibody is approximately 2 μg / mL.

[0052] In some embodiments, the concentration of the above-mentioned CD28 antibody is 1-2 μg / mL.

[0053] In some embodiments, the concentration of the above-mentioned CD28 antibody is approximately 1 μg / mL.

[0054] In some embodiments, the concentration of the above-mentioned CD28 antibody is approximately 1.5 μg / mL.

[0055] In some embodiments, the concentration of the above-mentioned CD28 antibody is approximately 2 μg / mL.

[0056] In some embodiments, the concentration of mannose is 2.0 to 100.0 mM.

[0057] In some embodiments, the mannose concentration is 5.0 to 50.0 mM.

[0058] In some embodiments, the above mannose concentrations are approximately 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 11.0 mM, 12.0 mM, 13.0 mM, 14.0 mM, 15.0 mM, 16.0 mM, 17.0 mM, 18.0 mM, 19.0 mM, 20.0 mM, 21.0 mM, 22.0 mM, 23.0 mM, 24.0 mM, 25.0 mM, and 26.0 mM. These are approximately 27.0mM, 28.0mM, 29.0mM, 30.0mM, 31.0mM, 32.0mM, 33.0mM, 34.0mM, 35.0mM, 36.0mM, 37.0mM, 38.0mM, 39.0mM, 40.0mM, 41.0mM, 42.0mM, 43.0mM, 44.0mM, 45.0mM, 46.0mM, 47.0mM, 48.0mM, 49.0mM, and 50.0mM.

[0059] In some embodiments, the above method further includes the following steps. S3: The cells are cultured in a medium containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem cell-like memory T cells.

[0060] In some embodiments, the cytokines described above are selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21.

[0061] In some embodiments, the above cytokines are selected from IL-2.

[0062] In some embodiments, the time for changing the culture medium is 1 to 3 days.

[0063] In some embodiments, the time for changing the culture medium is 2 days.

[0064] In some embodiments, the lymphocyte population described above includes CD4 T cells, CD8 T cells, γδ T cells, TILs, TCR-T cells, CAR-T cells, and STAR-T cells.

[0065] In some embodiments, the above method further includes the following steps. S4: The T cells obtained in step S3 are stimulated with antibodies and further cultured.

[0066] In some embodiments, the above culture is carried out for 12 days.

[0067] In some embodiments, step S4 described above is performed cyclically two or more times.

[0068] In some embodiments, the antibodies described above are CD3 antibodies and / or CD28 antibodies.

[0069] In some embodiments, the antibodies described above are CD3 antibodies and CD28 antibodies.

[0070] In some embodiments, the antibody described above is a CD3 antibody.

[0071] In some embodiments, the antibody described above is a CD28 antibody.

[0072] In some embodiments, the concentration of the antibody is 0.5 to 2 μg / mL.

[0073] In some embodiments, the concentration of the antibody is 0.5 μg / mL, or 0.6 μg / mL, or 0.7 μg / mL, or 0.8 μg / mL, or 0.9 μg / mL, or 1 μg / mL, or 1.1 μg / mL, or 1.2 μg / mL, or 1.3 μg / mL, or 1.4 μg / mL, or 1.5 μg / mL, or 1.6 μg / mL, or 1.7 μg / mL, or 1.8 μg / mL, or 1.9 μg / mL, or 2 μg / mL.

[0074] In some embodiments, the concentration of the antibody is 1 μg / mL.

[0075] In some embodiments, the species origin of the T cells described above includes human-derived T cells or mouse-derived T cells.

[0076] In some embodiments, the present invention provides a method for in vitro amplification of stem cell-like memory T cells, comprising the step of isolating them with a dextran-meglumine diatrizoate mixture to obtain human peripheral blood mononuclear cells (PBMCs). The obtained PBMCs are then inoculated into 24-well plates pre-coated with human CD3 antibody, and activated culture is performed by adding free human CD28 antibody and cytokines such as rhIL-2. At any point during or within the first three days of activation of the PBMC cells, 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 of the medium is replaced and the corresponding rhIL-2 is added. Thereafter, depending on the growth state of the T cells, equal volumes of medium containing rhIL-2 and mannose are added every two days, and the T cell density is increased from 0.5 to 2 × 10⁶. 6 The cells were maintained at a concentration of / ml. Stem cell-like memory T cells were obtained after 9-15 days of culture. The T cells cultured until day 12 were continuously stimulated with human CD3 antibody for 24 hours, transferred to a new cell culture plate, and cultured further until day 24. The T cells cultured until day 24 were continuously stimulated with human CD3 antibody for 24 hours, transferred to a new cell culture plate, and cultured further until day 36.

[0077] In another embodiment, the present invention provides a lymphocyte population prepared by the method described above.

[0078] In another embodiment, the present invention provides a pharmaceutical composition comprising the above-mentioned lymphocyte population.

[0079] In some embodiments, the above-described pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0080] In some embodiments, the above-described pharmaceutical composition further comprises additional therapeutic agents.

[0081] In another embodiment, the present invention provides the use of a lymphocyte population prepared by the above method, the above lymphocyte population, or the above composition in the preparation of a pharmaceutical for adoptive immunotherapy.

[0082] In some embodiments, the above adoptive immunotherapy is an antitumor immunotherapy, an anti-infective immunotherapy, or an autoimmune disease therapy.

[0083] The present invention The present invention provides a method for treating an individual, which involves administering the amplified lymphocyte population described above to an individual in need.

[0084] Lymphocyte populations can be administered intravenously to an individual, for example, by infusion.

[0085] In another aspect, the present invention provides the use of mannose in the induction and amplification of stem cell-like memory T cells in vitro.

[0086] Unless otherwise defined herein, technical and scientific terms used herein shall have the same meanings as those generally understood by those skilled in the art in which the present invention pertains, by reference to the content of the present invention.

[0087] The terms “one / one type (a)” or “one / one type (an)” refer to one / one type or multiple / one type, and for example, “one / one type of molecule” should be understood to refer to one / one type or multiple / multiple types of molecules. Therefore, the terms “one / one type (a)” or “one / one type (an),” “one / one type or multiple / multiple types,” and “at least one / at least one type” can be used interchangeably in this specification.

[0088] In the claims and specification of the present invention, unless otherwise required by the language of expression or by necessary suggestion in the context, the term “comprise” or variations such as “comprises” or “comprising” shall be used to mean “comprise,” that is, to specify the presence of such feature, but shall not preclude the presence or addition of other features in each embodiment of the present invention.

[0089] The term "approximately" encompasses values ​​within a range of approximately ±25% of the specified value. In other embodiments, the term "approximately" encompasses values ​​within a range of approximately ±20%, ±15%, ±10%, or ±5% of the specified value. For example, "approximately 3 grams" refers to values ​​such as 2.7 to 3.3 grams (i.e., 3 grams ± 10%).

[0090] The term "T cell" refers to a cell with a TCR on its surface or a cell with a genetically edited or modified TCR. In this specification, "T cell" may also refer to a cell population that includes other cell types besides T cells themselves, for example, a cell population that includes 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more T cells. Furthermore, T cells may include T cells at all stages of differentiation and may also include T cells that have antigens or markers on their cell surface.

[0091] The terms "stem cell-like memory T cells" and "memory stem cell-like T cells" usually refer to cells that are in the early differentiation stage of memory T cells, possess stem cell characteristics, and have strong multidirectional differentiation potential. SCM ) refers to T SCM Cells respond to antigen stimulation by becoming central memory T cells (T CM ), effector memory T cells (T EM ), effector T cells (T EFIt differentiates into ), and further produces numerous effectors such as IFN-γ, resulting in stronger killing ability, and possesses both differentiation ability and strong self-renewal ability.

[0092] The term "mannose" usually refers to a carbohydrate. For example, mannose may have a CAS number of 3458-28-4. In this application, mannose may refer to any of the derivatives of mannose, which may be naturally extracted and / or artificially synthesized.

[0093] The term "culture medium" typically refers to a medium that allows stem cells to grow normally. In some embodiments, the stem cell culture medium may also suspend the stem cells. For example, in this application, the stem cell culture medium includes DMEM medium, 1640 medium, MEM medium, and X-VIVO medium.

[0094] The term "CD3 antibody" typically refers to an antigen-binding molecule that specifically binds to CD3. For example, CD3 antibodies may include T3, I, eu4, and HCHTl. The CD3 molecule binds to T cell antigen receptors via salt bridges and is involved in T cell signaling.

[0095] The term "CD28 antibody" typically 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, and both ligands belong to the B7 family, including B7-1 (CD80) and B7-2 (CD86). CD28 and CD80 have high aggregation affinity and can interact with various signaling molecules or kinases such as PI-3K, GRB-2 / s, and tyrosine kinase ITK.

[0096] The term "cytokine" typically refers to a type of small molecule protein with broad biological activity that is synthesized and secreted in response to stimulation by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and some non-immune cells (e.g., endothelial cells, epidermal cells, fibroblasts, etc.). These cytokines may have various functions, including regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotency, and repairing damaged tissue. In this application, these cytokines may include interleukins, interferons, tumor death factor superfamily, colony-stimulating factors, chemokines, and growth factors. For example, the cytokine described above is an interleukin.

[0097] The term "interleukin" typically 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 from helper CD4 T lymphocytes, as well as from monocytes, macrophages, and endothelial cells. As used herein, interleukins (ILs) include 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 include full-length interleukins or fragments (e.g., deletions) or variants thereof that substantially retain the biological activity corresponding to wild-type interleukin (e.g., biological activity corresponding to wild-type interleukin having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the biological activity). Interleukins as used herein may be derived from any mammal. In some embodiments, the interleukin is derived from a species selected from humans, horses, cattle, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, and non-human primates. In some embodiments, the interleukin may be a mutant. For example, the interleukin may be super IL-2 (also known as sIL2; see Nature 484, 529–533, 2612), which is obtained by modifying IL-2 to increase its binding affinity to IL-2Rβ. Mutations in sIL-2 are often found in the core of the cytokine, and molecular dynamics simulations suggest that evolutionary mutations stabilize IL-2, reducing the flexibility of the helix at the IL-2Rβ binding site, as well as allowing the optimized receptor binding conformation to bind to CD25.Compared to IL-2, sIL-2 induces superior amplification of cytotoxic T cells, resulting in improved antitumor responses in vivo, reduced swelling of T regulatory cells, and alleviation of pulmonary edema. For example, in this application, the above cytokines include one or more selected from the group consisting of I-L2, IL-7, IL-15, IL-17, and IL-21.

[0098] The term "pharmaceutical composition" refers to a lymphocyte population formulated for administration to an individual and containing or primarily composed of stem cell-like memory T cells as described herein. Preferably, the pharmaceutical composition is sterile. In one embodiment, the pharmaceutical composition is pyrogen-free.

[0099] Stem cell-like memory T cells or lymphocyte populations primarily composed of stem cell-like memory T cells are formulated and administered according to appropriate medical practices. Factors considered in this process include the type of disease being treated, the specific individual being treated, the individual's clinical condition, the administration site, the method of administration, the administration schedule, possible side effects, and factors known to the physician.

[0100] The term "effective dose" refers to the amount of stem cell-like memory T cells or a population of lymphocytes primarily composed of stem cell-like memory T cells that is effective in treating an individual's disease state, disease, or illness.

[0101] The term "tumor" usually refers to a physiological condition in mammals characterized by uncontrolled cell proliferation. In this application, tumors may include lymphomas, germ tumors, sarcomas, and leukemias. For example, tumors 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 gastric cancer (including gastrointestinal cancer). This may include 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 renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade small non-incisional NHL, AIDS-related lymphoma, Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), pilosarcocyte leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD)).

[0102] The term "infectious" disease typically refers to illnesses caused by viruses entering the body through multiple routes and replicating within susceptible host cells. Examples of viral infections include upper respiratory tract infections, bronchitis, viral pneumonia, viral gastroenteritis, viral myocarditis, measles, chickenpox, rubella, roseola in children, hand, foot, and mouth disease, mumps, hepatitis B, severe acute respiratory syndrome (SARS), HIV, AIDS, avian influenza, and hepatitis A.

[0103] The term "autoimmune disease" generally refers to diseases in which the body's immune response to self-antigens causes damage to its own tissues. Autoimmune diseases are divided into organ-specific autoimmune diseases and systemic autoimmune diseases. For example, the autoimmune diseases listed above 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, bullous pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuropathy, systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, polyarteritis nodosa, and Wegener's granulomatosis.

[0104] The term "individual" refers to a mammal. A mammal may be a primate, particularly a human, or it may be a livestock, zoo animal, or companion animal. In some preferred embodiments, the individual is a human. In other preferred embodiments, a non-human mammal may be employed, in particular a mammal that has been conventionally used as a model to demonstrate therapeutic effects in humans (e.g., mouse, primate, pig, canid, or rabbit).

[0105] The method of the present invention will be described below through the following specific examples. These examples are intended to illustrate the basic principles, main features, and advantages of the present invention, and it should be understood that the scope of the present invention is not limited by these examples. The conditions used in the examples can be further adjusted according to specific requirements, and the conditions not specified are those commonly used in conventional experiments.

[0106] In the present invention, the above PBMC is obtained by means including but not limited to density gradient centrifugation, the final concentration of the above rhIL-2 is 50-100 IU, the above cytokines include but are not limited to any cytokines for in vitro T cell induction such as IL-7, IL-15, and IL-21, and the above mannose is added in a concentration range of 2.0-100.0 mM, with a recommended concentration range of 10.0-50.0 mM.

[0107] <Example 1: In vitro amplification method using human-derived stem cell-like memory T cells as the main cell population> This embodiment is, 1. A step of coating a cell culture plate with human CD3 antibody at a final concentration of 1 μg / ml, 2. Collect 10 ml of peripheral blood from a healthy person in a centrifuge tube, dilute it with phosphate buffer (PBS) in a 1:1 ratio and mix uniformly, add 20 ml of lymphocyte separation solution (ficoll) to another new 50 ml centrifuge tube, and then slowly add the uniformly mixed ficoll:blood diluent, along the tube wall, to the upper layer of lymphocyte separation solution so that a clear layer forms between the two. Centrifuge for 20 minutes at a rotation speed of 800 g, with acceleration and deceleration set to 0. 3. Once centrifugation is complete, transfer the cells from the monocyte layer to a 50 mL centrifuge tube using a pipette, wash once with 20 mL of PBS, centrifuge at 800 g for 5 minutes, discard the supernatant, and then resuspend in T cell medium and dilute to 1 million / ml while counting the cells. 4. Next, add human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml to the T cell suspension, mix and suspend uniformly, inoculate the cells into a pre-coated 24-well plate, add mannose at the corresponding concentration by blowing until uniform, and then place it in an incubator for culture. Depending on the growth state of the T cells, add a medium containing rhIL-2 and mannose in equal amounts every two days to maintain the T cell density at 0.5 - 2×106 / ml. On the 12th day of culture, use flow cytometry to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1 that controls the differentiation of stem cell-like memory T cells, and 5. As a result, as shown in Figure 2, on the 12th day of culture, in the mannose-treated group, CCR7 + CD62L + and CD45RO CD27 + the proportion of stem cell-like memory T cells is significantly higher than that of the control group under normal culture conditions. On the other hand, the expression of the transcription factor TCF-1 that controls the stem cell-like characteristics of T cells in mannose-treated T cells is also significantly higher than that of the control group, and is included.

[0108] <Example 2: In vitro amplification method of a cell population mainly composed of mouse-derived stem cell-like memory T cells> This example 1. First, pre-coat a cell culture plate with mouse CD3 antibody at a final concentration of 2 μg / ml, and 2. Isolate mouse spleen cells, obtain mouse T cells by nylon fiber column or magnetic bead sorting, and dilute them to 1 million / ml in a T cell medium. 3. Add mouse CD28 antibody at a final concentration of 1 μg / ml and rmIL-2 at 20 - 50 U / ml to the T cells, add mannose at the corresponding concentration by blowing until uniform, inoculate the cells into a cell culture plate coated with mouse CD3 antibody, and place it in an incubator for culture. 4. Next, depending on the growth state of the T cells, add a medium containing mouse IL-2 recombinant protein and mannose in equal amounts every 24 hours to maintain the cell density at 0.5 - 2×106 Add to maintain the concentration at / ml. On day 6 of T cell culture, flow cytometry is used to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells. 5. As a result, as shown in Figure 3, on day 6 of culture, CD44 was found in the mannose-treated group. + CD62L + The proportion of stem cell-like memory T cells increased from approximately 15.0% to approximately 77.8%, which is significantly higher than that of T cells under normal culture conditions. Furthermore, the expression of the transcription factor TCF-1, which controls the stem cell-like characteristics of T cells, and the transcription levels of T cell stem cell-related transcription factors were significantly higher in mannose-treated T cells compared to the control group. Includes.

[0109] <Example 3: In vitro amplification method for 1G4 TCR T cells from a cell population mainly composed of stem cell-like memory T cells> This embodiment is, 1. A step of coating a cell culture plate with human CD3 antibody at a final concentration of 1 μg / ml, 2. Collect 10 ml of peripheral blood from a healthy person in a centrifuge tube, dilute it with phosphate buffer (PBS) in a 1:1 ratio and mix uniformly, add 20 ml of lymphocyte separation solution (ficoll) to another new 50 ml centrifuge tube, and then slowly add the uniformly mixed ficoll:blood diluent, along the tube wall, to the upper layer of lymphocyte separation solution so that a clear layer forms between the two. Centrifuge for 20 minutes at a rotation speed of 800 g, with acceleration and deceleration set to 0. 3. Once centrifugation is complete, transfer the cells from the monocyte layer to a 50 mL centrifuge tube using a pipette, wash once with 20 mL of PBS, centrifuge at 800 g for 5 minutes, discard the supernatant, and then resuspend in T cell medium and dilute to 1 million / ml while counting the cells. 4. Next, human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml are added to the T cell suspension, mixed uniformly, and the cells are inoculated into a pre-coated 24-well plate. Mannose at the corresponding concentration is added by bubbling until uniform, and then the plate is placed in an incubator for culture. 5. After culturing T cells for 48 hours, the 1G4 TCR is transmitted to PBMCs by viral centrifugation infection. 6. After viral infection of T cells, the cells are cultured further with a mannose solution of the corresponding concentration, and depending on the growth stage of the T cells, a medium containing equal amounts of rhIL-2 and mannose is added every two days to maintain the T cell density at 0.5 to 2 × 10⁶ / ml. On day 12 of culture, the expression level of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells in the culture, is detected using flow cytometry. 7. Flow cytometry revealed that on day 12 of culture, a lymphocyte population mainly composed of the stem cell-like memory T cells described above was obtained. As a result, as shown in Figure 4, the treatment with mannose significantly increased the expression of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells, compared to the control group. Includes.

[0110] <Example 4: Long-term in vitro amplification method for a cell population mainly composed of human-derived stem cell-like memory T cells> This embodiment is, 1. A step of coating a cell culture plate with human CD3 antibody at a final concentration of 1 μg / ml, 2. Collect 10 ml of peripheral blood from a healthy person in a centrifuge tube, dilute it with phosphate buffer (PBS) in a 1:1 ratio and mix uniformly, add 20 ml of lymphocyte separation solution (ficoll) to another new 50 ml centrifuge tube, and then slowly add the uniformly mixed ficoll:blood diluent, along the tube wall, to the upper layer of lymphocyte separation solution so that a clear layer forms between the two. Centrifuge for 20 minutes at a rotation speed of 800 g, with acceleration and deceleration set to 0. 3. Once centrifugation is complete, transfer the cells from the monocyte layer to a 50 mL centrifuge tube using a pipette, wash once with 20 mL of PBS, centrifuge at 800 g for 5 minutes, discard the supernatant, and then resuspend in T cell medium and dilute to 1 million / ml while counting the cells. 4. Next, human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml are added to the T cell suspension, mixed uniformly, and the cells are inoculated into a pre-coated 24-well plate. Mannose at the corresponding concentration is added by bubbling until uniform, and then the plate is placed in an incubator for culture. 5. Depending on the growth stage of the T cells, every two days, prepare a medium containing equal amounts of rhIL-2 and mannose to increase the T cell density from 0.5 to 2 × 10⁻⁶. 6 Add to maintain the concentration at / ml. On day 12 of culture (CE12&ME12), flow cytometry is used to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells. 6. To further investigate the long-term amplification of stem cell-like memory T cells and the retention of stem cell-like characteristics by mannose, T cells cultured up to day 12 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody. The following day, the cells were transferred to a new cell culture plate and cultured for another 24 days. Next, T cells cultured up to day 24 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody, transferred to a new cell culture plate, and cultured until day 36 (CE36 & ME36). Flow cytometry was used to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1, which controls T cell differentiation. 7. Flow cytometry revealed that lymphocyte populations mainly composed of the stem cell-like memory T cells described above were obtained on days 12 and 36 of culture. As a result, as shown in Figure 5, CD45RO was obtained in the mannose-treated group. + CD27 +The proportion of stem cell-like memory T cells was significantly higher in the mannose-treated T cells compared to the control group under normal culture conditions, while the expression of TCF-1, a transcription factor that regulates the stem cell-like characteristics of T cells, was also significantly higher in the mannose-treated T cells compared to the control group. Includes.

[0111] <Example 5: Long-term in vitro amplification method for 1G4 TCR T cells from a cell population mainly composed of stem cell-like memory T cells> This embodiment is, 1. A step of coating a cell culture plate with human CD3 antibody at a final concentration of 1 μg / ml, 2. Collect 10 ml of peripheral blood from a healthy person in a centrifuge tube, dilute it with phosphate buffer (PBS) in a 1:1 ratio and mix uniformly, add 20 ml of lymphocyte separation solution (ficoll) to another new 50 ml centrifuge tube, and then slowly add the uniformly mixed ficoll:blood diluent, along the tube wall, to the upper layer of lymphocyte separation solution so that a clear layer forms between the two. Centrifuge for 20 minutes at a rotation speed of 800 g, with acceleration and deceleration set to 0. 3. Once centrifugation is complete, transfer the cells from the monocyte layer to a 50 mL centrifuge tube using a pipette, wash once with 20 mL of PBS, centrifuge at 800 g for 5 minutes, discard the supernatant, and then resuspend in T cell medium and dilute to 1 million / ml while counting the cells. 4. Next, human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml are added to the T cell suspension, mixed uniformly, and the cells are inoculated into a pre-coated 24-well plate. Mannose at the corresponding concentration is added by bubbling until uniform, and then the plate is placed in an incubator for culture. 5. After culturing T cells for 48 hours, the 1G4 TCR is transmitted to PBMCs by viral centrifugation infection. 6. After viral infection of T cells, add a mannose solution of the corresponding concentration and continue culturing. Depending on the growth stage of the T cells, add a medium containing equal amounts of rhIL-2 and mannose every two days to increase the T cell density from 0.5 to 2 × 10⁻⁶. 6Add to maintain the concentration at / ml. On day 12 of culture (CE12&ME12), flow cytometry is used to detect the expression level of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells in the culture. 7. To further investigate the long-term amplification of stem cell-like memory T cells and the retention of stem cell-like characteristics by mannose, T cells cultured up to day 12 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody. The following day, the cells were transferred to a new cell culture plate and cultured for another 24 days. Next, T cells cultured up to day 24 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody, transferred to a new cell culture plate, and cultured until day 36 (CE36 & ME36). Flow cytometry was used to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1, which controls T cell differentiation. 8. Flow cytometry revealed that lymphocyte populations mainly composed of the above-mentioned stem cell-like memory T cells were obtained on days 12 and 36 of culture. As a result, as shown in Figure 6, the treatment with mannose significantly increased the expression of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells, compared to the control group. Includes.

[0112] <Example 6: Long-term in vitro amplification method for TIL cells from a cell population mainly composed of stem cell-like memory T cells> This embodiment is, 1. A step of coating a cell culture plate with human CD3 antibody at a final concentration of 1 μg / ml, 2. Collect 10 ml of peripheral blood from a liver cancer patient in a centrifuge tube, dilute it with phosphate buffer (PBS) in a 1:1 ratio and mix uniformly, add 20 ml of lymphocyte separation solution (ficoll) to another new 50 ml centrifuge tube, and then slowly add the uniformly mixed ficoll:blood diluent, along the tube wall, to the upper layer of lymphocyte separation solution so that a clear layer forms between them, and centrifuge for 20 minutes at a rotation speed of 800 g, with acceleration and deceleration at 0. 3. Once centrifugation is complete, transfer the cells from the monocyte layer to a 50 mL centrifuge tube using a pipette, wash once with 20 mL of PBS, centrifuge at 800 g for 5 minutes, discard the supernatant, and then resuspend in T cell medium and dilute to 1 million / ml while counting the cells. 4. Next, human CD28 antibody at a final concentration of 1 μg / ml and rhIL-2 at 300 U / ml are added to the T cell suspension, mixed uniformly, and the cells are inoculated into a pre-coated 24-well plate. Mannose at the corresponding concentration is added by bubbling until uniform, and then the plate is placed in an incubator for culture. 5. After culturing T cells for 48 hours, depending on the growth stage of the T cells, a medium containing equal amounts of rhIL-2 and mannose is added every two days to increase the T cell density from 0.5 to 2 × 10⁻⁶. 6 Add to maintain the concentration at / ml. On day 12 of culture (CE12&ME12), flow cytometry is used to detect the expression level of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells in the culture. 6. To further investigate the long-term amplification of stem cell-like memory T cells and the retention of stem cell-like characteristics by mannose, T cells cultured up to day 12 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody. The following day, the cells were transferred to a new cell culture plate and cultured for another 24 days. Next, T cells cultured up to day 24 were continuously stimulated for 24 hours with a final concentration of 1 μg / ml human CD3 antibody, transferred to a new cell culture plate, and cultured until day 36 (CE36 & ME36). Flow cytometry was used to detect the proportion of stem cell-like memory T cells in the culture and the expression level of the transcription factor TCF-1, which controls T cell differentiation. 7. Flow cytometry revealed that lymphocyte populations mainly composed of the above-mentioned stem cell-like memory T cells were obtained on days 12 and 36 of culture. As a result, as shown in Figure 7, compared to the control group, prolonged exposure to mannose significantly increased the expression of the transcription factor TCF-1, which controls the differentiation of stem cell-like memory T cells. Includes.

[0113] <Example 7: Detection of in vivo antitumor function of mannose-inducible mouse stem cell-like memory T cells> This embodiment is, 1. In vivo antitumor experiments on mannose-induced stem cell-like memory T cells in mice were performed using 4-8 week old C57BL6 / N mice. To demonstrate that memory stem cell-like T cells obtained by mannose treatment have superior antitumor function and persistence compared to conventionally treated T cells, the present invention includes a step intended to investigate the in vivo antitumor function of mannose-induced stem cell-like memory T cells using an OT-IT cell model. 2. First, put 2 x 10 on the C57BL6 / N mouse. 5 B16-OVA cells were subcutaneously inoculated into the axilla of the right hind limb, and the tumor volume was measured using calipers after the tumor cells had grown subcutaneously in the mouse for approximately 8-10 days. The tumor volume was 75 mm². 3 ~150mm 3 Once this was reached, 4 × 10⁶ C57BL6 / N tumor-bearing mice were given. 6 The process involved intravenously injecting mannose-treated or untreated OT-IT cells into the tail vein, measuring tumor volume every two days, and on approximately day 14 (specific operating time varied depending on the experimental results), collecting spleen and tumor tissue from C57BL6 / N mice. The mouse tumor tissue was then pulverized, subjected to Percoll density gradient centrifugation, and analyzed after flow cytometry staining. 3. As a result, as shown in Figure 8, first, when the tumor growth volume was statistically analyzed, the tumor growth rate of mice in the group of mice treated with mannose-treated T cells was significantly slower compared to the control group. Furthermore, flow cytometry staining revealed that the infiltration rate 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 a stronger and more persistent antitumor effect in the adoptive mouse model. Includes.

[0114] <Example 8: Detection of in vivo antitumor function of human stem cell-like memory T cells induced by long-term mannose treatment> This embodiment is, 1. In vivo antitumor experiments on mannose-induced stem cell-like memory T cells in mice were performed using 8-10 week old NSG mice. To demonstrate that memory stem cell-like T cells obtained by mannose treatment have superior antitumor function and persistence compared to conventionally treated T cells, the present invention includes a step intended to investigate the in vivo antitumor function process of stem cell-like memory T cells treated with mannose in vitro over a long period using a 1G4 TCR T cell model. 2. First, set the NSG mouse to 3x10 6 HepG2-NYESO tumor cells were subcutaneously inoculated into the axilla of the right hind limb. After the tumor cells had grown subcutaneously in the mouse for approximately 8-10 days, the tumor volume was measured using calipers. The tumor volume was 75 mm². 3 ~150mm 3 Once this is reached, 6 × 10⁶ mice with NSG tumors are given. 6 The process involved intravenously injecting mannose-treated or untreated 1G4 TCR T(CE12 / ME12 / CE36 / ME36) cells into the tail vein, measuring tumor volume every two days, collecting tumor tissue from NSG mice on approximately day 14 (specific operating time varied depending on the experimental results), pulverizing the mouse tumor tissue, performing Percoll density gradient centrifugation, and analyzing the tissue after flow cytometry staining. 3. As a result, as shown in Figure 9, when the tumor growth volume was statistically analyzed, it was found that the tumor growth rate of mice in the experimental group of adoptive mice treated with mannose for a long period was significantly slower compared to the control group. This indicates that mannose-treated 1G4 TCR T cells exhibit a stronger antitumor effect in the adoptive mouse model. Includes.

[0115] It should be understood that the above embodiments are illustrative and not intended to encompass all possible embodiments included in the claims. Furthermore, various modifications and changes can be made based on the above embodiments without departing from the scope of this disclosure. Similarly, various technical features of the above embodiments can be arbitrarily combined to form additional embodiments of the invention that may not be expressly described. Therefore, the above embodiments are merely illustrative of some embodiments of the invention and do not limit the scope of protection of the invention.

Claims

1. A method for inducing and amplifying stem cell-like memory T cells in vitro, S1: A step to activate and amplify T cells, S2: A step of culturing T cells by adding mannose to the culture medium during or after activation. A method for inducing and amplifying stem cell-like memory T cells in vitro, characterized by including the following:

2. Step S1 includes coating a cell culture plate with an antibody, and then activating and amplifying T cells in a cytokine-containing medium. Preferably, the antibodies are CD3 antibodies and CD28 antibodies. Preferably, the cytokine is selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21, and is preferably IL-2, the method according to claim 1.

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

4. The method according to any one of claims 1 to 2, characterized in that the concentration of mannose is 2.0 to 100.0 mM, preferably 5.0 to 50.0 mM.

5. S3: The process further includes the step of changing the culture medium to one containing cytokines and mannose to obtain a lymphocyte population mainly composed of stem cell-like memory T cells, Preferably, the cytokine is selected from one or more of the group consisting of IL-2, IL-7, IL-15, IL-17, and IL-21, and is preferably IL-2. Preferably, the time for changing the culture medium is 1 to 3 days, preferably 2 days. Preferably, the lymphocyte population comprises CD4 T cells, CD8 T cells, γδ T cells, TILs, TCR-T cells, CAR-T cells, and STAR-T cells, according to any one of claims 1 to 4.

6. S4: This further includes a step of stimulating the T cells obtained in step S3 with an antibody and culturing them further. Preferably, the culture is carried out for 12 days. Preferably, step S4 is performed cyclically two or more times. Preferably, the antibody is a CD3 antibody and / or a CD28 antibody, and preferably a CD3 antibody. Preferably, the concentration of the antibody is 0.5 to 2 μg / mL, and preferably 1 μg / mL, the method according to any one of claims 1 to 4.

7. The method according to any one of claims 1 to 6, characterized in that the species origin of the T cells includes human-derived T cells or mouse-derived T cells.

8. A lymphocyte population prepared by the method described in any one of claims 1 to 7.

9. The lymphocyte population described in claim 8 is included, Preferably, further comprising pharmaceutically acceptable excipients, Preferably, a pharmaceutical composition characterized by further comprising additional therapeutic agents.

10. The use of a lymphocyte population prepared by the method of any one of claims 1 to 7, the lymphocyte population according to claim 8, or the composition according to claim 9 in the preparation of a pharmaceutical for adoptive immunotherapy, Preferably, the adoptive immunotherapy is an antitumor immunotherapy, an anti-infective immunotherapy, and an autoimmune disease therapy, as used.

11. Use of mannose in the induction and amplification of stem cell-like memory T cells in vitro.

Images