Survivor t cells, survivor t cell population, and pharmaceutical composition

Abstract

The present disclosure provides cells that are competitive with respect to cancer cells, without experiencing starvation or exhaustion, even in glucose-depleted environments. Specifically, the present disclosure provides survivor T cells with enhanced glucose uptake capacity. The T cells are modified so as to express a glucose transporter and / or configured such that the expression of a glucose transporter is enhanced. Also provided are a cell population including such cells, a pharmaceutical composition, and a treatment method.

Description

Survivor T cells, survivor T cell populations and pharmaceutical compositions

[0001] The present disclosure relates to survivor T cells, survivor T cell populations with enhanced glucose uptake capacity, and pharmaceutical compositions comprising such cells or cell populations.

[0002] Cancer cells reprogram their metabolic systems to favor their own cell proliferation, actively utilizing glycolysis, which is inefficient at producing ATP even in the presence of oxygen, to increase glucose uptake and lactate production (Warburg effect).

[0003] In the tumor microenvironment, there are many factors that suppress the metabolic activity and function of antitumor T cells. In addition to immunosuppressive factors, metabolic competition between tumors and T cells contributes to the formation of an immunosuppressive environment. When T cells are stimulated by TCR, Ca ２＋ The intracellular concentration of calcineurin increases, activating calcineurin. Activation of calcineurin causes the dephosphorylated transcription factor NFAT to translocate into the nucleus, where it interacts with other transcription factors to promote and activate the transcription of genes such as the IL-2 gene.

[0004] On the other hand, cancer cells consume large amounts of glucose and deplete it, so T cells, which require glucose as an energy source, ｅｆｆ When tumor-specific T cells of this type infiltrate tumors, they receive signals from TCR but do not respond to the Ca ２＋ The concentration of NFAT decreases and nuclear translocation of NFAT is reduced, resulting in T cell dysfunction and suppression of cell proliferation and cytokine production.

[0005] In addition to glucose, cancer cells also consume large amounts of nutrients such as amino acids and fatty acids, which cause T cells to malfunction. Thus, in the tumor microenvironment, the metabolic mechanisms that support the active proliferation of cancer cells inhibit the antitumor effector functions of tumor-specific T cells.

[0006] Therefore, there is a need for cells that can compete with cancer cells without starving or becoming exhausted even in such an environment.

[0007] The present disclosure provides T cells with enhanced glucose uptake capacity that function without starvation or exhaustion even in a tumor environment, a cell population containing the same, and a pharmaceutical composition containing such cells.

[0008] Accordingly, the present disclosure provides the following: [Item 1] T cells with enhanced glucose uptake ability. [Item 2] The cells of Item 1, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 2A] The T cells of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 3] The T cells of any one of the above items, wherein the T cells have effector function. [Item 4] The T cells are effector T cells (T ｅｆｆ ) precursor cells. [Item 5] The T cell of any one of the above items, wherein the CAR is expressed in the T cell. [Item 6] The T cell of any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item 7] The T cell of any one of the above items, wherein the glucose transporter is GLUT3. [Item 8] The T cell of any one of the above items, wherein the T cell is a human T cell. [Item 9] A cell population comprising T cells with enhanced glucose uptake ability, wherein the T cells have the property of having effector function when introduced into a body. [Item 10] The cell population of any one of the above items, wherein the T cells have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter. [Item 11] The cell population of any one of the above items, wherein the T cells have effector function. [Item 12] The cell population according to any one of the preceding items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 12A] The cell population according to any one of the preceding items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 13] The cell population is a T ｅｆｆ [Item 14] The cell population according to any one of the above items, comprising T ｅｆｆ[Item 14A] The cell population according to any one of the above items, comprising progenitor cells of T ｅｆｆ[Item 15] The cell population according to any one of the above items, wherein the CAR or TCR is expressed on the T cells. [Item 16] The cell population according to any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item 17] The cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item 18] The cell population according to any one of the above items, wherein the T cells are human T cells. [Item 19] A pharmaceutical composition comprising the T cells or the cell population according to any one of the above items. [Item 20] The pharmaceutical composition according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 20A] The pharmaceutical composition according to any one of the above items, which is for the cure of cancer. [Item 21] A pharmaceutical composition for preventing or treating a disease in a subject, the pharmaceutical composition comprising: (A) collecting a value of glucose uptake ability or an index related thereto from the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate manner and at an appropriate dose. [Item 21A] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 21B] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 21C] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item 21D] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer. [Item 22] The composition according to any one of the above items, wherein the glucose transporter is GLUT3.[Item 23] The T cell or cell population of any one of the above items for use as a pharmaceutical. [Item 24] The cell or cell population of any one of the above items, which is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 24A] The cell or cell population of any one of the above items, which is for the cure of cancer. [Item 25] A cell or cell population for the prevention or treatment of a disease in a subject, the cell or cell population being characterized by: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting a T cell with enhanced glucose uptake ability or a cell population comprising said T cell, which has an appropriate expression characteristic according to the index; and (C) administering the T cell with enhanced glucose uptake ability or the cell population comprising said T cell to the subject in an appropriate manner and dosage. [Item 25A] The cell or cell population of any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 25B] The cell or cell population of any one of the above items, wherein the T cell comprises a chimeric antigen receptor (CAR). [Item 25C] The cell or cell population of any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or infectious disease. [Item 25D] The cell or cell population of any one of the above items, wherein the disease comprises cancer. [Item 26] The cell or cell population of any one of the above items, wherein the glucose transporter is GLUT3. [Item 27] ​​A method for treating or preventing a subject in need thereof, comprising administering to the subject an effective amount of the T cell or cell population of any one of the above items. [Item 28] The method of any one of the above items, wherein the treatment or prevention in the subject is for the treatment or prevention of cancer, an autoimmune disease, allergy, or infectious disease. [Item 28A] The method of any one of the above items, wherein the treatment or prevention in the subject is for the cure of cancer.[Item 29] A method for preventing or treating a disease in a subject, the method comprising: (A) collecting a value of glucose uptake ability or an index related thereto from the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate dosage regimen. [Item 29A] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item 29B] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 29C] The method of any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or an infectious disease. [Item 29D] The method of any one of the above items, wherein the disease comprises cancer. [Item 30] The method of any one of the above items, wherein the glucose transporter is GLUT3. [Item 31] Use for producing a medicament comprising the T cells or cell population described in any one of the above items, wherein the cell or cell population comprises the T cells or cell population described in any one of the above items. [Item 32] The use described in any one of the above items, wherein the medicament is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item 32A] The use described in any one of the above items, wherein the medicament is for the cure of cancer. [Item 33] The use described in any one of the above items, wherein the medicament is for the prevention or treatment of a disease in a subject, the medicament comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population comprising the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population comprising the T cells to the subject in an appropriate manner and dosage. [Item 33A] The use of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR).[Item 33B] The use of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item 33C] The use of any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or an infectious disease. [Item 33D] The use of any one of the above items, wherein the disease comprises cancer. [Item 34] The use of any one of the above items, wherein the glucose transporter is GLUT3. The present disclosure also provides the following: [Item B0] Effector T cells that have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM]. [Item B1] Effector T cells comprising a chimeric antigen receptor (CAR) that have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM]. [Item B2] The T cells described in the above items, wherein the CAR is expressed in the T cells. [Item B3] The T cells described in any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item B4] Effector T cells comprising a chimeric antigen receptor [CAR] that have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM], and the glucose transporter is GLUT3. [Item B5] Effector T cells into which the GLUT3 gene has been introduced. [Item B6] The T cells according to any one of the above items, which are human T cells.[Item B7A] A cell population comprising effector T cells that have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM]. [Item B8] The cell population according to any one of the above items, wherein the CAR is expressed in the T cells. [Item B7] A cell population comprising effector T cells that comprise a chimeric antigen receptor [CAR] and have been modified to express a glucose transporter and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM]. [Item B8] The cell population according to any one of the above items, wherein the CAR is expressed in the T cells. [Item B9] The cell population according to any one of the above items, wherein the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4. [Item B10] A cell population comprising effector T cells that have been modified to express a glucose transporter comprising a chimeric antigen receptor [CAR] and / or have enhanced expression of a glucose transporter, wherein the T cells exhibit effector function under low glucose conditions [0.5 mM] at least equivalent to that under normal glucose conditions [10 mM], and the glucose transporter is GLUT3. [Item B11] A cell population comprising effector T cells into which a GLUT3 gene has been introduced. [Item B12] The cell population according to any one of the above items, wherein the T cells are human T cells. [Item B13] A pharmaceutical composition comprising the T cells according to any one of the above items or the cell population according to any one of the above items. [Item B14] The pharmaceutical composition according to any one of the above items, which is for the treatment or prevention of cancer, autoimmune diseases, allergies, or infectious diseases. [Item B15] The pharmaceutical composition according to any one of the above items, which is for the cure of cancer.[Item B16] A pharmaceutical composition for preventing or treating a disease in a subject, the pharmaceutical composition comprising: (A) collecting a value of glucose uptake ability or an index related thereto from the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population containing the T cells to the subject in an appropriate manner and at an appropriate dose. [Item B17] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B18] The pharmaceutical composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item B19] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, an allergy, or an infectious disease. [Item B20] The pharmaceutical composition according to any one of the above items, wherein the disease comprises cancer. [Item B21] The composition according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B22] The T cell according to any one of the above items or the cell population according to any one of the above items for use as a pharmaceutical. [Item B23] The cell or cell population according to any one of the above items, for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item B24] The cell or cell population according to any one of the above items, for the cure of cancer. [Item B25] A cell or cell population for the prevention or treatment of a disease in a subject, the cell or cell population being characterized by: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population comprising said T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or the cell population comprising said T cells to the subject in an appropriate manner and dosage.[Item B26] The cell or cell population according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B27] The cell or cell population according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item B28] The cell or cell population according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or infectious disease. [Item B29] The cell or cell population according to any one of the above items, wherein the disease comprises cancer. [Item B30] The cell or cell population according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B31] A method for treating or preventing a subject in need thereof, comprising administering to the subject an effective amount of the T cell or cell population according to any one of the above items. [Item B32] The method according to any one of the above items, wherein the treatment or prevention in the subject is for the treatment or prevention of cancer, an autoimmune disease, allergy, or infectious disease. [Item B33] The method of any one of the above items, wherein the treatment or prevention of the subject is for the purpose of curing cancer. [Item B34] A method for the prevention or treatment of a disease in a subject, the method comprising: (A) collecting a value of glucose uptake ability or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake ability or a cell population containing the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake ability or a cell population containing the T cells to the subject in an appropriate dosage and administration. [Item B35] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B36] The method of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item B37] The method of any one of the above items, wherein the disease includes cancer, an autoimmune disease, an allergy, or an infectious disease. [Item B38] The method according to any one of the above items, wherein the disease includes cancer.[Item B39] The method according to any one of the above items, wherein the glucose transporter is GLUT3. [Item B40] Use of the T cells according to any one of the above items or the cell population according to any one of the above items for manufacturing a medicament comprising the cells or cell population. [Item B41] The use according to any one of the above items, wherein the medicament is for the treatment or prevention of cancer, autoimmune disease, allergy, or infectious disease. [Item B42] The use according to any one of the above items, wherein the medicament is for the cure of cancer. [Item B43] The use according to any one of the above items, wherein the medicament is for the prevention or treatment of a disease in a subject, the medicament being characterized by: (A) collecting a value of glucose uptake capacity or an index related thereto in the subject; (B) selecting T cells with enhanced glucose uptake capacity or a cell population comprising the T cells, which have an appropriate expression characteristic according to the index; and (C) administering the T cells with enhanced glucose uptake capacity or the cell population comprising the T cells to the subject in an appropriate manner and dosage. [Item B44] The use according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). [Item B45] The use according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). [Item B46] The use according to any one of the above items, wherein the disease comprises cancer, an autoimmune disease, allergy, or infectious disease. [Item B47] The use according to any one of the above items, wherein the disease comprises cancer. [Item B48] The use according to any one of the above items, wherein the glucose transporter is GLUT3.

[0009] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated, and further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0010] Note that features and significant actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings.

[0011] The present disclosure makes it possible to provide T cells and / or related cell populations with enhanced glucose uptake capacity, and by using such cells and / or related cell populations, it is possible to provide chimeric antigen receptor (CAR) T cells and / or related cell populations that function in a tumor environment without starvation or exhaustion.

[0012] The cells disclosed herein can be referred to as metabolically enhanced T cells (survivor T cells) or related cell populations, which utilize the mechanism by which tumor cells survive in the tumor environment. The cells and related cell populations disclosed herein are capable of maintaining effective cytotoxic activity and cytokine production in low-glucose environments, such as tumor microenvironments, which could not be achieved with conventional immune checkpoint inhibitor therapy, and can exert a high antitumor effect in vivo. Furthermore, by using a transient glucose transporter expression system that expresses glucose transporters in response to increased glucose demand, it is possible to avoid hyperdifferentiation and exhaustion of T cells due to excessive activation and induce memory phenotype T cells that can maintain their effects for a long period of time.

[0013] Figure 1 shows an example of the structure of a metabolically engineered anti-EGFRvIII CAR (3C10CAR_GLUT3) lentiviral vector plasmid. A single-chain fragment (scFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII, which is expressed in glioblastoma (GBM), etc., was constructed and linked to CD8 hinge, CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BBICD, and CD3z to construct a CAR. A high-affinity glucose transporter (GLUT3) was encoded by the CAR via the P2A sequence. The figure shows an example of the same 3C10CAR_GLUT3 incorporated into a third-generation lentiviral vector plasmid containing the EF-1a promoter. Figure 2 shows enhanced T cell surface GLUT3 expression and glucose uptake. The upper panel shows the structures of the conventional CAR (3C10 CAR) and the metabolically engineered CAR (GLUT3 CAR). The lower left shows images of CAR and GLUT3 expression on T cells for 3C10 CAR and GLUT3 CAR under a fluorescence microscope. The lower middle shows GLUT3 expression quantified by fluorescence intensity. The lower right shows the results of quantifying intracellular glucose uptake using the 2-NBDG method. The conventional CAR (3C10 CAR) and the metabolically engineered CAR (GLUT3 CAR) were transfected into T cells derived from healthy donors to obtain CAR-T cells. Fluorescence microscopy confirmed homogeneous GLUT3 expression on the GLUT3 CAR-T cell membrane. Furthermore, GLUT3 CAR-T cells exhibited enhanced glucose uptake compared to T cells and conventional CAR-T cells (3C10 CAR-T cells). Figure 3 shows results demonstrating T cell expansion efficiency equivalent to that of conventional CAR-T cells. The X axis represents the number of days after transduction, and the Y axis represents LOG2 (FC). Peripheral blood mononuclear cells (PBMCs) containing T cells were stimulated with anti-CD3 / 28 beads, followed by CAR gene transduction, and T cell proliferation efficiency was analyzed. Under both normal glucose conditions (10 mM) and low glucose conditions (0.5 mM), GLUT3 CAR-T cells had establishment efficiency equivalent to that of conventional CAR-T cells (3C10 CAR-T cells). Figure 4 shows results demonstrating enhanced T cell glycolytic capacity due to GLUT3 expression.The graph on the left shows the glycolytic capacity of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu conc. 10 mM) and low glucose conditions (Glu conc. 0.5 mM). The graph below shows the glycolytic capacity of 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (Glu conc. 10 mM). The left panel on the right shows glycolysis values ​​observed after the addition of glucose, and the right panel shows glycolytic capacity values ​​observed after the addition of oligomycine. The metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Fluxanalyzer. The GLUT3 CAR had enhanced glycolytic activity compared to the 3C10 CAR in both low-glucose and normal-glucose environments. Figure 5 shows results demonstrating that GLUT3 CAR-T cells have a competitive advantage under low glucose conditions. The left panel shows the experimental procedure. The middle panel shows the cell count ratios of 3C10 CAR-T cells and GLUT3 CAR-T cells on days 0 and 3. The right panel shows the ratio of 3C10 CAR-T cells to GLUT3 CAR-T cells on day 3. 3C10 CAR-T cells (unlabeled) and GLUT3 CAR-T cells (CSFE-labeled) were co-cultured under normal glucose and low glucose conditions. Both cells showed comparable survival under normal glucose conditions, but significant survival of GLUT3 CAR-T cells was observed under low glucose conditions. Figure 6 shows results demonstrating the enhanced cytokine production ability of GLUT3 CAR-T cells. The left panel shows intracellular IFNg and IL-2 analysis of CD8-positive cells at the top, and the amount of intracellular IFNg and IL-2 at the bottom of CD4-positive cells. The right panel shows the percentage of cells positive for IFNγ, IL-2, and TNF-α, respectively. After antigen stimulation of 3C10 CAR-T cells and GLUT3 CAR-T cells, the percentage of cytokine-producing cells was analyzed by FCM. Significant increases in the expression of cytokines (IFN-g, IL-2, TNF-α), which are important for T cell maintenance and activation and for the exertion of antitumor activity, were observed. Figure 7 shows results demonstrating that the function of GLUT3 CAR-T cells is maintained even under low glucose conditions.The panels, starting from the left, show the percentage of cells producing IFNγ, IL-2, and TNF-α under normal glucose conditions (10 mM) and low glucose conditions (0.5 mM), respectively. Cytokine production capacity under normal glucose and low glucose was compared. Conventional CAR-T cells (3C10 CAR-T cells) showed an extreme decrease in function under low glucose. On the other hand, GLUT3 CAR-T cells showed high cytokine production capacity under normal glucose, and even under low glucose, they exhibited function comparable to the cytokine production capacity of 3C10 CAR-T cells under normal glucose. Figure 8 shows the results demonstrating the enhanced cytotoxic activity of GLUT3 CAR-T cells. The left panel shows the results of the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against the U87d cell line at a glucose concentration of 10 mM, and the right panel shows the results of the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against the U87d cell line at a glucose concentration of 0.5 mM. Cytotoxic activity was analyzed using an EGFRvIII-expressing cell line as a target. Cytotoxic activity was analyzed under low glucose and normal glucose conditions, and under both conditions, GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells. Figure 9 shows the results showing the properties of GLUT3 CAR that confer T cell memory phenotype. The left panel shows the expression of CD45 and CD27 before (preco-culture) and after (co-culture) co-culture with the U87d tumor cell line, and the right panel shows, from left to right, the percentage of CD45RA-positive and CD27-negative fractions before and after co-culture. The memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. A stronger tendency for differentiation into effector T cells was observed in GLUT3 CAR-T cells compared with 3C10 CAR. Figure 10 shows the suppression of inhibitory molecule expression in GLUT3 CAR-T cells. From left to right, data for PD-1, LAG3, and TIM3 are shown. The expression of inhibitory molecules under stimulation with EGFRvIII antigen-positive cells was compared. In GLUT3 CAR-T cells, suppression of PD-1, LAG3, and TIM3 expression was observed. Figure 11 shows mRNA analysis (evaluation of metabolism, exhaustion, activation, and differentiation) of GLUT3 CAR-T cells.The upper left panel shows, from left to right, PDCD1 expression, LAG3 expression, and TIM3 expression. The upper right panel shows, from left to right, enrichment plots related to lactate metabolism and glucose metabolism. The lower panel shows factors with increased expression based on gene ontology. Detailed analysis of 3C10 CAR-T and GLUT3 CAR-T cells was performed using mRNA expression. Similar to FCM, GLUT3 CAR-T cells showed a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors. These factors are associated with enhanced T cell effector functions, such as activation and cytokine production. Figure 12 demonstrates the generality of this gene, demonstrating that similar effects can be achieved not only with the 3C10 CAR but also with the CD19 CAR. The left panel shows an experiment using the 3C10 CAR, and the right panel shows an experiment using the CD19 CAR. In each panel, the upper left shows intracellular IL-2, IFNg, and TNF-a analysis of mock-transfected T cells, 3C10 CAR-T cells, and GLUT3 CAR-T cells, the upper right shows the percentage of IFNg-, IL-2-, and TNFa-positive cells (from left) for 3C10 CAR-T cells and GLUT3 CAR-T cells under normal glucose conditions (10 mM), and the lower left shows the percentage of IFNg-, IL-2-, and TNFa-positive cells (from left) for 3C10 CAR-T cells and GLUT3 CAR-T cells under low glucose conditions (0.5 mM). The lower right shows the cytotoxic activity of 3C10 CAR-T cells and GLUT3 CAR-T cells against (from left) U87d and U251MGd cell lines under low glucose conditions (0.5 mM). We confirmed that the same effect as the 3C10 CAR was obtained when GLUT3 was loaded onto the CD19 CAR. It was also shown to be effective against pancreatic cancer cells in which CD19 was expressed in a pancreatic cancer cell line. Figure 13 shows that rapid tumor eradication was achieved in an intracranial xenograft model. The upper left panel shows a simple scheme of the experiment. Briefly, 2.5 x 10 cells were intracranially injected into immunodeficient NSG mice. ４ U87d tumor cell line was transplanted, and 5 days later, 1x10 ６This is a model in which CAR T cells are administered via the tail vein and tumor burden is quantified over time by bioluminescence imaging (BLI). The middle left panel shows tumor imaging images on days 0 and 11. The graph on the bottom left shows luminescence intensity (reflecting tumor burden) over time. The middle-middle panel shows Kaplan-Meier curves for each treatment group. Below the middle panel, tumor imaging images on days 34, 38, and 42 in tumor-rechallenged mice are shown. The right column shows peripheral blood CD3 ＋ Figure 14 shows the design of the GLUT3 expression system. The symbols in the diagram represent the EF-1 promoter (EF-1a), anti-EGFRvIII CAR (EGFRvIII CAR), P2A sequence (P2A), and high-affinity glucose transporter (GLUT3), respectively. Stbl-GLUT3 is identical to GLUT3 CAR. Figure 15 shows the antitumor effects of mock T cells and GLUT3 CAR-T cells evaluated in a mouse model. The experimental schematic is shown at the top. The Kaplan-Meier curve after treatment is shown at the top left. The transition of tumor burden over time, quantified by bioluminescence imaging (BLI), is shown at the bottom. Mock T cells did not suppress tumors, while GLUT3 CAR-T cells achieved complete remission (CR) in four mice. Figure 16 shows a tumor rechallenge experiment (sudden death of mice in the GLUT3 CAR-T cell group). The tumor burden and survival after GLUT3 CAR-T cell therapy are shown (tumor imaging images on day 1 (after challenge), day 5 of rechallenge, and day 12 of rechallenge are shown). Tumors (U87d) were re-implanted into the cured mice from the experiment shown in the previous figure, and rejection ability was evaluated (a "surrogate for memory formation"). 1x10 ５After re-implantation of tumor cells, one of four mice in the GLUT3 CAR-T group achieved a CR. However, three of the four mice died (non-tumor amplification death). Figure 17 shows the time course of CAR T cells demonstrating enhanced long-term survival after stimulation under the above conditions. This figure shows the in vitro (CAR) T cell stimulation and culture test plan. Non-CAR-transfected T cells (UTD), 3C10 CAR-T cells, and GLUT3 CAR-T cells were evaluated. Groups were divided into unstimulated and stimulated groups (stimulated with U87d cell line or EGFRvIII magnetic beads). Furthermore, each group was cultured under glucose-free conditions (0 mM), low glucose conditions (0.5 mM), and normal glucose conditions (10 mM) (27 groups in total). The analyses shown in the schedule on the right were performed over time for each group. Under low glucose conditions, normal CAR-T 3C10 cells do not exhibit effective cytokine production, while GLUT3 CAR-T cells exhibit effective cytokine production. However, excessive glucose uptake in GLUT3 CAR-T cells leads to terminal differentiation into CCR7-negative effector memory T cells (Tem) or terminally differentiated RA-positive T cells (Temra) over time (day 3 or day 7), loss of stemness, and apoptosis. When CAR-T cells (established with normal glucose) are suddenly placed under low glucose conditions of 0 mM or 0.5 mM (day 0), the highly glucose-dependent GLUT3 CAR-T cells are prone to apoptosis. Figure 18 shows enhanced long-term survival in vivo. The above antitumor effects, survival, and adverse events are examined using a system similar to the mouse described above. In this figure, the U87d tumor cell line was implanted intracranially into immunodeficient NSG mice. Five days later, mock-transduced T cells (UTD), 3C10 CAR-T cells, and GLUT3 CAR-T cells were administered via the tail vein, and the antitumor effect and survival were analyzed using bioluminescence imaging. In Experiment 1, the established CAR-T cells were used as is; in Experiment 2, they were administered after exposure to a high-glucose environment; in Experiment 3, tumors were implanted into the liver, which has been reported to have a low-glucose, high-lactic acid environment; and in Experiment 4, T cell engraftment and tumor infiltration were evaluated. In Experiment 1, GLUT3 CAR-T cells demonstrated superior antitumor activity.In Experiment 2, glucose exposure led to overactivation of GLUT3 CAR-T cells, resulting in apoptosis and hyperdifferentiation, attenuating antitumor activity. In rechallenge, tumors were rejected using GLUT3 CAR-T cells. In Experiment 4, superior CAR-T cell engraftment and tumor T cell infiltration were observed in GLUT3 CAR-T cells. Experiment 3 was an exploratory experiment using a liver tumor model in which immunosuppression due to a low glucose, high lactate environment has been suggested, and is expected to yield results similar to those in Experiment 2. Figure 19 shows data (in vitro data) demonstrating enhanced long-term survival after stimulation. From the left, cell death results for GLUT3 CAR-T cells under 0 mM, 0.5 mM, and 10 mM conditions are shown. Cell death in each CAR-T cell type was examined using Annexin V and 7-AAD. In unstimulated GLUT3 CAR-T cells, apoptosis and cell death rates were comparable under no or low glucose conditions (0 mM, 0.5 mM) compared with normal glucose conditions (10 mM). Figure 20 shows that low glucose conditions impair CAR-T cell function. (a-c) Interstitial fluid was collected by low-speed centrifugation from surgical or biopsy specimens from patients with glioblastoma (GBM), non-small cell lung cancer, and colon cancer (n = 5 each), and glucose concentrations were examined. (a) Experimental scheme. (b) Glucose concentrations in interstitial fluid and paired serum samples from GBM specimens. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ***, P < 0.001. (c) Glucose concentrations in interstitial fluid from GBM, non-small cell lung cancer (lung cancer), and colorectal cancer (colorectal cancer) specimens. Data are shown as mean ± SEM. Statistical analysis was performed by one-way analysis of variance; *, P<0.05. (d, e) Mock-transduced T cells and conventional EGFRvIII CAR-T cells were prepared from PBMCs from healthy volunteers (n=5).(d) Cytokine production in mock-transformed T cells (Mock T) and conv EGFRvIII CAR-T cells (conv CAR-T) upon stimulation with EGFRvIII-expressing tumor cells (U-87 MGΔ) under low glucose (0.5 mM) or high glucose (10 mM) conditions was examined by intracellular cytokine staining. Representative contour plots (left) and summaries of the frequencies of cytokine-producing cells (IFN-γ, IL-2, and TNFα) are shown (right). Data are presented as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01. (e) Cytokine (IFN-γ, IL-2, TNFα) production by conv EGFRvIII CAR-T cells cocultured with U-87 MGΔ cells for 16 hours under low glucose (0.5 mM) or high glucose (10 mM) conditions was measured by ELISA. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; **, P<0.01; ****, P<0.0001. (f) Gene expression in EGFRvIII CAR-T cells 24 hours after stimulation with EGFRvIII beads under low glucose (0.5 mM) or high glucose (10 mM) conditions. Pathways enriched under low glucose conditions compared to high glucose conditions are shown according to the Molecular Signatures Database. (g) Gene expression of selected genes is shown in a heatmap. Gene expression was normalized by row. All experiments were performed at least twice. Figure 21 shows that GLUT3 overexpression enhances the metabolic fitness of CAR-T cells, increasing cytotoxicity and cytokine production under low glucose conditions. (a) Constructs of conventional EGFRvIII CAR (conv CAR, top) and EGFRvIII CAR stably expressing GLUT3 (GLUT3 CAR, bottom). (b) Immunofluorescence staining of conv EGFRvIII CAR-T cells (conv CAR-T) and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T). The white dotted circle indicates T cells not transduced with CAR. Katyusha (red), 4,6-diamino-2-phenylindole (DAPI: blue), GLUT3 (green). (c) Expression levels of GLUT1 (left) and GLUT3 (right) in CAR-T cells.(d) Glucose uptake by CAR-T cells measured by glucose analog 2-NBDG uptake assay. A representative histogram (left) and a summary of mean fluorescence intensity (MFI) (right) are shown. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ns, not significant; ****, P<0.0001. (e, f) Glycolytic capacity (e) and metabolic phenotype (f) of CAR-T cells under low glucose (0.5 mM) conditions as determined by flux analyzer. Data are presented as mean ± SD. (g, h) CAR-T cells were prepared from PBMCs of healthy individuals (n=5). (g) Cytokine production in EGFRvIII-expressing tumor cells (U-87 MGΔ) stimulated under low glucose (0.5 mM) conditions was examined by intracellular cytokine staining. Representative contour plots (left) and summaries of the frequencies of cytokine-producing cells (IFN-γ, IL-2, and TNFα) are shown (right). Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; *, P<0.05; **, P<0.01; ****, P<0.0001. (h) Cytokine production (IFN-γ, IL-2, and TNFα) by CAR-T cells cocultured with U-87 MGΔ cells under low glucose (0.5 mM) conditions for 16 hours was measured by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ****, P<0.0001. (i) The cytotoxicity of CAR-T cells under low glucose (0.5 mM) conditions was measured using a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87 MGΔ-Luc) were cocultured for 24 hours at the indicated effector-to-target (E:T) ratio, and specific lysis was calculated based on luciferase activity. Data are shown as mean ± SEM. Statistical analysis was performed by Student's t-test; **, P<0.01. (j, k) Expression of exhaustion markers (PD-1 and Tim-3) by CAR-T cells. PD-1. ＋ CAR-T cells (j) and PD-1 ＋ Tim-3 ＋A summary of the frequency of the CAR-T cell (k) population is shown. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; ****, P<0.0001. Figure 22 shows that low glucose conditions impair the function of CAR-T cells. (a) Volcano plot showing differentially expressed genes in conv EGFRvIII CAR-T cells cultured under low glucose (0.5 mM) conditions compared to high glucose (10 mM) conditions. The X-axis is log ２ (fold change) (log ２ FC), and the Y axis is -log １０ (adjusted p value) [(-log １０ (padj)). The dotted line represents log ２ (fold change) cutoff value, x = ±1.4 and -log １０The cutoff value for the adjusted p-value, y=0.1, is shown. The top 5 and bottom 5 genes are highlighted in green and purple, respectively. Figure 23 shows that GLUT3 overexpression enhances the metabolic fitness of CAR-T cells. (a) Expression of GLUT1 (left) and GLUT3 (right) in conv EGFRvIII CAR-T cells (conv CAR-T), GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T), U-87 MGΔ cells, and U-251 MGΔ cells. Representative histograms are shown. (b) Glycolytic parameters of CAR-T cells cultured under low glucose (0.5 mM) conditions were measured by a glycolytic stress test. Data are shown as mean ± SD. Statistical analysis was performed by Student's t-test; ns, not significant; ***, P<0.001. (c, d) Competition assay of CAR-T cells under low glucose (0.5 mM) or high glucose (10 mM) conditions (72-hour culture). (c) Experimental schema (n=3). (d) Representative contour plot (left) and summary of the proportion of each CAR-T cell population (right). Data are shown as mean ± SEM. Statistical analysis was performed by Student's t-test; ***, P<0.001. Figure 24 shows that stable expression of GLUT3 improves CAR-T cell function under low glucose conditions. (a-c) Cytokine production by mock T cells (Mock T), conv EGFRvIII CAR-T cells (conv CAR-T), and GLUT3 EGFRvIII CAR-T cells (GLUT3 CAR-T) (n=5) upon stimulation with EGFRvIII-expressing tumor cells (U-87 MGΔ) under high glucose (10 mM) conditions was examined by intracellular staining. (a) CD3 ＋ Summary of the frequency of cytokine (IFN-γ, IL-2, TNFα) producing CAR-T cells. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; ***, P<0.001. (b) Representative contour plot (left) and cytokine (IFN-γ, IL-2, TNFα) producing CD8 ＋Summary of CAR-T cell frequencies (right). Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; ***, P<0.001. (c) Representative contour plots (left) and cytokine (IFN-γ, IL-2, TNFα)-producing CD4 ＋Summary of CAR-T cell frequency (right). Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; **, P<0.01. (d, e) The cytotoxicity of CAR-T cells against U-87 MGΔ cells and U-251 MGΔ cells under low glucose (0.5 mM) or high glucose (10 mM) conditions was determined by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (U-87 MGΔ-Luc and U-251 MGΔ-Luc) were cocultured at the indicated effector-to-target (E:T) ratio for 24 hours, and specific lysis was calculated based on luciferase activity. (d) Cytotoxicity of CAR-T cells against U-251 MGΔ-Luc cells under low glucose (0.5 mM) conditions. Data are presented as mean ± SEM. Statistical analysis by Student's t-test; *, P<0.05; **, P<0.01. (e) Cytotoxicity of CAR-T cells against U-87 MGΔ-Luc cells or U-251 MGΔ-Luc cells under high glucose (10 mM) conditions. Data are shown as mean ± SEM. Statistical analysis by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. (F, G) Expression of exhaustion markers (PD-1, f; PD-1 and Tim-3, g) by the indicated CAR-T cell populations. Representative contour plots are shown. Figure 25 shows that stable expression of GLUT3 improves CAR-T cell function independently of the type of scFv. (a, b) Mock T cells (Mock T), conv CD19 CAR-T cells (conv CD19 CAR-T), and GLUT3 CD19 CAR-T cells (GLUT3 CD19 CAR-T) were prepared from PBMCs of healthy volunteers (n=5). (a) Cytokine production by the indicated CAR-T cells after stimulation with CD19-expressing tumor cells (NALM6) under low glucose (0.5 mM) conditions, as determined by intracellular cytokine staining. Representative contour plots (left) and a summary of the frequency of cytokine (IFN-γ, IL-2, TNFα)-producing cells (right) are shown. Data are presented as mean ± SEM.Statistical analysis by Student's t-test: *, P<0.05; ***, P<0.001; ***, P<0.0001. (b) Cytokine (IFN-γ, IL-2, TNFα) production by conv CD19 CAR-T cells and GLUT3 CD19 CAR-T cells cocultured with NALM6 cells for 16 hours under low glucose (0.5 mM) conditions was measured by ELISA. Data are shown as mean ± SEM. Statistical analysis by Student's t-test: *, P<0.05; ***, P<0.001; ***, P<0.0001. (c) The cytotoxicity of conv CD19 CAR-T cells and GLUT3 CD19 CAR-T cells against NALM6- and CD19-expressing AsPC-1 cells under low glucose (0.5 mM) conditions was determined by a luciferase-based assay. CAR-T cells and luciferase-expressing target cells (NALM6-Luc and CD19-expressing AsPC-1-Luc) were cocultured at the indicated effector-to-target (E:T) ratio for 24 hours, and specific lysis was assessed based on luciferase activity. Data are presented as mean ± SEM. Statistical analysis was performed by Student's t-test; ns, not significant; *, P<0.05; **, P<0.01.

[0014] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0015] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0016] As used herein, "about" means ±10% of the numerical value that follows. For example, "about 20" includes "18 to 22." Numerical ranges include all values ​​between and at the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0017] As used herein, the term "T cells" is used in the broad sense used in the art and refers to lymphocytes produced in the bone marrow that have migrated to the thymus and matured. T cells may be CD45-positive and CD3-positive cells among normal fractions of peripheral blood and bone marrow-derived mononuclear cells. T cells used may be, but are not limited to, T cells isolated from a donor, particularly a human donor. Examples of T cells and cells derived therefrom include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained indefinitely under cell culture conditions. It is known that there are multiple types of T cells based on their functions, including effector memory T cells (T ｅｆｆ Examples of peripheral T cells include T cells (Tnv), naive T cells (Tscm), stem cell memory T cells (Tscm), central memory T cells (Tcm), and terminally differentiated RA-positive T cells (Temra). As used herein, "peripheral T cells" refers to T cells present outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues. As used herein, the term "peripheral T cells" refers to a cell population that contains peripheral T cells, and does not require that the T cells be isolated. In addition to T cells, cell fractions containing various lymphocytes, such as peripheral blood mononuclear cells (PBMC), may also be used.

[0018] As used herein, a "cell population" refers to a population containing two or more cells, and may be, for example, a collection of cells gathered in a planar manner, or a cell mass formed by cells adhering to each other in a three-dimensional manner. Furthermore, a "cell population" may be formed by a single type of cell, or may contain multiple types of cells. When referring to a cell population of T cells of the present disclosure, it is sufficient that the cell population contains at least one cell that can have effector function in the location where it is to function (typically in the body), such as Tnv, Tscm, Tcm, or Tempra.

[0019] As used herein, "flow cytometry" refers to a technique for measuring the number of cells, solids, and other biological particles suspended in a liquid, as well as their individual physical, chemical, and biological properties. A device using this technique is called a "flow cytometer." In this disclosure, the "positive" and "negative" status of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) is determined by flow cytometry, as commonly used in the art. More specifically, in flow cytometry, cells are lined up and flowed, and the number of cells is counted using spectroscopic techniques. For example, target cells are counted by irradiating cells labeled with fluorescent or luminescent enzymes with laser light, and the resulting fluorescent or luminescent signals are detected by a detector such as a photodiode. Furthermore, the detection results from the detector can be input into a computer, and a two-dimensional plot can be generated and displayed. This allows for easy understanding of the presence and number of target cells.

[0020] As used herein, the term "effector function" refers to biological activities such as cytotoxic activity, cytokine production, and division / proliferation that are caused by signals mediated by TCR or CAR in T cells, and to biological activities caused by the Fc region of an antibody in other immune cells. Effector functions include, for example, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells. In the present disclosure, whether or not a T cell has an effector function can be determined by confirming that the T cell has cytotoxic activity, cytokine production, and division / proliferation using the techniques exemplified in the Examples and the like.

[0021] As used herein, the term "effector cell" or "effector T cell" refers to a T cell having an effector function and exerting cytotoxic effects on target cells, and is a T ｅｆｆ As used herein, whether a cell is an "effector" (cell) can be determined by methods such as cytotoxicity assays, surface antigen analysis by flow cytometry, and intracellular cytokine staining. Effector T cells are immune cells that perform effector functions, such as mediating antibody-dependent cellular cytotoxicity (ADCC). Effector T cells include, for example, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils, and can be isolated from natural tissues such as blood.

[0022] As used herein, "effector T cells (T ｅｆｆ"Effector T cell precursors" refer to cells that are not effector cells but acquire effector function by migrating to a location where they should function (e.g., within the body) or by other stimuli. Such cells include lymphoid precursors, Tnv, Tscm, Tcm, and Tempra. Effector T cell precursors are activated early in the immune response and mature into effector T cells through proliferation and differentiation. However, some precursors have the potential to differentiate into memory T cells. These precursors have specific gene expression patterns and metabolic states that contribute to the prolongation of memory function. Several molecular and metabolic characteristics contribute to this prolongation of memory function. First, effector T cell precursors have a unique metabolic program. These cells rely predominantly on oxidative phosphorylation rather than glycolysis, which is characteristic of effector T cells. This metabolic pathway is energy efficient and allows for long-term cell survival. This metabolic characteristic is a key factor supporting the long-term maintenance of memory T cell function. Next, the role of transcription factors is also important. Effector T cell precursors express transcription factors such as T-bet and Eomes, which promote differentiation into memory T cells and enable their long-term survival and function. Furthermore, these precursor cells, expressing surface markers such as CD62L and CCR7, have the ability to migrate to secondary lymphoid tissues. This homing function allows precursor cells to receive necessary survival signals in secondary lymphoid tissues, contributing to the maintenance of memory function. Furthermore, suppression of antigenic stimulation also contributes to the extension of memory function. Some precursor cells are suppressed from full differentiation into effector T cells by release from antigenic stimulation, instead retaining the properties of memory T cells. This mechanism suppresses excessive effector responses while maintaining memory function. Additionally, the cytokine environment surrounding the cells also plays an important role. In particular, cytokines such as IL-7 and IL-15 provide the survival signals necessary for the maintenance of memory T cells, which significantly contributes to the extension of memory function.These factors allow effector T cell precursors to survive longer than normal effector T cells and retain memory functions that enable rapid and powerful secondary immune responses. This property is thought to play an important role not only in immune defense against infectious diseases but also in cancer immunotherapy and vaccine development. Thus, understanding the properties of effector T cell precursors is of great significance for both basic immunological research and clinical applications.

[0023] The maintenance of memory T cell phenotype in effector T cell precursors refers to the phenomenon in which some precursor cells retain memory T cell-specific phenotypes (e.g., surface markers, metabolic state, transcription factor expression, etc.) during the differentiation process into effector T cells. These precursor cells survive even after the end of an immune response and have the ability to provide long-term immunological memory. This phenomenon is thought to be an important mechanism for T cells to balance the process of exerting effector function with memory function, which allows long-term survival and rapid secondary immune responses. The characteristics of this phenomenon are explained below. First, effector T cell precursors maintain surface markers specific to memory T cells. These include homing molecules such as CD62L and CCR7, and the expression of these molecules allows precursor cells to maintain their ability to migrate to secondary lymphoid tissues. Second, these precursor cells also maintain a characteristic metabolic state. Specifically, oxidative phosphorylation predominates over glycolysis, maintaining an energy-efficient metabolic program that enables long-term survival. This metabolic characteristic is important for maintaining memory T cell function. Furthermore, progenitor cells maintain a characteristic balance in the expression of transcription factors. Appropriate expression of transcription factors such as T-bet and Eomes ensures both differentiation into effector T cells and maintenance of the memory T cell phenotype. Furthermore, progenitor cells are responsive to cytokine signals and can receive survival signals such as IL-7 and IL-15. This responsiveness allows progenitor cells to survive for long periods while retaining memory T cell characteristics. Finally, these progenitor cells are capable of antigen-independent maintenance. In other words, memory T cell progenitors retain their phenotype even in the absence of antigen and maintain a state of readiness for a rapid immune response. Due to these characteristics, the maintenance of the memory T cell phenotype by effector T cell progenitors plays a crucial role in the flexibility of the immune system and the establishment of long-term memory immunity. These characteristics are thought to be of great significance in infectious disease prevention, cancer immunotherapy, and vaccine design.

[0024] As used herein, the term "antigen receptor" refers to any molecule that specifically binds to a target antigen and transmits a signal into a cell that expresses the receptor. In the present disclosure, an antigen receptor is one that can be introduced into a T cell and may be a natural or artificial molecule, and includes, for example, a T cell receptor (TCR) / B cell receptor (BCR) as well as a chimeric antigen receptor (CAR).

[0025] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered receptor that can confer antigen specificity to cells (e.g., immune cells). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. A CAR is an engineered receptor that transfers antigen specificity to immune system cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, or a combination thereof, NK cells, macrophages, etc.). CARs may include, for example, an antigen-specific targeting region, an extracellular domain, a transmembrane domain, a costimulatory domain, and / or an intracellular signaling domain. They may also include bispecific CARs that use multiple (usually two) types of antigen-specific targeting regions. Preferably, the CAR of the present disclosure includes at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0026] As used herein, "T cell receptor (TCR)" refers to a receptor present on T cells. TCRs are heterodimeric receptor molecules consisting of two TCR polypeptide chains. There are two types: αβ TCRs, which are expressed by normal T cells, and γδ TCRs, which have specialized functions. α and β chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transduce intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens proliferated within cells following viral infection and cancer antigens derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Furthermore, antigens derived from foreign microorganisms are taken up by antigen-presenting cells by endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules. Regarding αβ-type TCRs, it is said that the specificity is expressed by the unique combination of the α and β gene products.

[0027] As used herein, the term "glucose uptake ability" refers to the ability of a cell to take up glucose per unit time. The uptake of glucose into a cell can be evaluated by a technique such as the 2-NBDG uptake assay.

[0028] As used herein, "enhanced glucose uptake ability" refers to improving glucose uptake ability by any method. Improvement of glucose uptake ability can be achieved by modifying immune cells (e.g., T cells) to express one or more glucose transporters and / or by modifying them to enhance glucose transporter expression. Such genetically modified immune cells are predicted to exhibit high glucose uptake, for example, in a low-glucose environment (e.g., a tumor microenvironment). Therefore, immune cells co-expressing one or more glucose transporters and a chimeric receptor polypeptide may exhibit superior physiological activities (e.g., in a tumor microenvironment, such as low-glucose conditions, optionally in the presence of a therapeutic antibody), such as cell proliferation, activation (e.g., increased cytokine production, e.g., IL-2 or IFNγ production), cytotoxicity, and / or in vivo anti-tumor activity.

[0029] As used herein, the term "glucose transporter" (GLUT) refers to a transporter that transports glucose (grape sugar) by facilitated diffusion. Molecular species of GLUT, GLUT1 to GLUT14, have been reported, and among them, for example, GLUT1 is expressed in a wide range of tissues and is responsible for constant baseline glucose transport, GLUT2 is activated when the sugar concentration increases and enhances glucose uptake, GLUT3 plays a central role in the central nervous system and exerts transport function even at low glucose concentrations, and GLUT4 is activated in response to insulin stimulation.

[0030] As used herein, the term "tumor microenvironment" is used interchangeably with "tumor environment." The tumor microenvironment (TME) refers to the local biological environment surrounding tumor tissue and encompasses a variety of factors involved in tumor progression, growth, metastasis, drug resistance, and other processes. This environment includes not only tumor cells but also non-tumor cells such as immune cells (e.g., T cells and macrophages), fibroblasts, vascular endothelial cells, and adipocytes. Humoral factors such as extracellular matrix (ECM), cytokines, chemokines, and growth factors are also important components. Furthermore, the tumor microenvironment is influenced by physical factors such as oxygen concentration, pH, and nutritional status. Changes in intercellular interactions and physical factors in the tumor microenvironment are believed to play important roles in tumor malignancy and therapeutic responsiveness. In particular, immunosuppressive microenvironments have attracted attention as factors that reduce the efficacy of immunotherapy. In the present invention, the "tumor microenvironment" refers to the collection of biological and physical factors, including these elements and their interactions, and is an important target in tumor treatment and diagnosis.

[0031] As used herein, the term "tumor microenvironmental conditions" refers to the tumor microenvironment or equivalent conditions. Therefore, "tumor microenvironmental conditions" refers to a broad concept encompassing not only the conditions constituting the tumor microenvironment but also similar or equivalent environmental conditions to which tumors are exposed within the body. Specifically, it encompasses not only the local biological environment surrounding tumor tissue, but also endogenous and exogenous factors that affect tumors when they reside within the body. This environment includes interactions between tumor cells and non-neoplastic cells (e.g., immune cells, fibroblasts, vascular endothelial cells, etc.), humoral factors such as the extracellular matrix (ECM), cytokines, chemokines, and growth factors, and physical factors such as oxygen concentration, pH, nutritional status, and physical pressure. Furthermore, "tumor microenvironmental conditions" also encompasses situations in which tumors interact with a broader physiological environment within the body, and is therefore not limited to the local environment surrounding the tumor. In the present invention, "tumor microenvironmental conditions" refers to a comprehensive range of environmental conditions that tumors encounter within the body, including factors that affect tumor progression and therapeutic response.

[0032] As used herein, "modification" broadly refers to any structural, functional, or expression-related change made to a gene (including nucleic acids and their corresponding proteins), including naturally occurring or artificial modifications. Specifically, this term includes sequence changes due to nucleotide substitution, deletion, insertion, addition, or a combination thereof; changes to the amino acid sequence or properties of the encoded protein; and chemical modifications (e.g., glycosylation, phosphorylation, acetylation, etc.) aimed at adding or improving functionality. "Modification" also includes exogenous gene introduction (e.g., transfection or viral vector-mediated gene introduction), transformation, knockout or knock-in of specific genes using genome editing technology, and suppression of gene expression (e.g., suppression using RNA interference or CRISPR technology). Furthermore, epigenetic modifications (e.g., DNA methylation, histone modifications, etc.) and modifications of expression regulatory elements are also included. As used herein, "modification" refers comprehensively to any form of change that affects the structure, expression, function, or biological behavior of a gene, including not only endogenous changes but also changes introduced or manipulated by exogenous means.

[0033] As used herein, the term "disease" is broadly interpreted to refer to a state of mental or physical discomfort or inconvenience in humans or animals, and refers to any condition that cannot be considered a healthy state and is not specifically defined, such as illness, disability, various symptoms, etc. Diseases that may be the subject of the present disclosure include, but are not limited to, diseases in which an immune response may be associated, such as cancer, autoimmune diseases, allergies, and infectious diseases.

[0034] As used herein, a subject having an "immune response" to a certain component or substance means that some kind of immune reaction occurs against the component or substance. The component or substance can be identified by observing changes in various immune cells or increases or decreases in immune-related substances (e.g., cytokines) in the subject or biological components (e.g., cells) derived from the subject. The response can be determined by objective indicators or by subjective judgment based on the experience of a physician or other professional.

[0035] As used herein, whether a subject "has immunological memory" of a certain component or substance can be evaluated by measuring whether the component or substance (i) enhances cytokine production in an antigen-dependent manner in memory CD4-positive T cells or has a proliferation-promoting effect in the subject or in a biological component (e.g., cell, etc.) derived from the subject, (ii) alters the expression of a surface antigen on memory regulatory T cells, (iii) changes the ratio of Treg to Th1, (iv) induces IFN-γ production from T-bet-positive Th1 cells, (v) alters the ability to produce IFN-γ, (vi) alters the ability to produce IL-2, and (vii) alters the ability to produce TNF-α, or (viii) has an antibody specific to the component or substance in the blood, and confirming that at least one of these results is positive.

[0036] As used herein, the term "infectious disease" refers to any infectious disease, and encompasses any type of infectious disease, such as viral infections (including any viral form, such as single-stranded or double-stranded DNA viruses and RNA viruses), bacterial infections, protozoan infections, and mycoplasma infections. Examples of such infectious diseases include tuberculosis, coronavirus, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonella infection, pathogenic E. coli, toxoplasmosis, Zika virus, herpesvirus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza, MERS, rabies, and diphtheria.

[0037] As used herein, the term "immune abnormality" refers to any disease, disorder, or condition that is caused or suspected to be caused at least in part by an abnormality in the immune system. It refers to a condition in which an abnormality in immunity occurs due to some cause, resulting in susceptibility to infectious diseases or allergic reactions. Immune abnormalities include, but are not limited to, allergies, autoimmune diseases, etc. When the immune response is directed against a self-antigen, it is generally referred to as an autoimmune disease, and when directed against an external antigen, it is referred to as an allergy. A strong immune response can be said to result in an autoimmune disease state against self-antigens and an allergic state against non-self antigens, while a weak immune response can result in a cancer state against self-antigens and an infectious disease state against non-self antigens.

[0038] As used herein, the term "autoimmune disease" refers to an excessive immune response to a specific self-antigen. Autoimmune diseases are diseases caused by a breakdown of immune tolerance, in which the immune system, which is responsible for recognizing and eliminating foreign substances, overreacts and attacks the body's own normal cells and tissues, resulting in symptoms.Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus erythematosus: SLE (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leakage of leukocytes; central nervous system CNS (Clinical System) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP), autoimmune uveitis, or autoimmune thrombocytopenia.

[0039] As used herein, "allergy" refers to an excessive immune response to a specific non-self antigen, and is a disease in which an immune response occurs to an "allergen." An "allergen" refers to an antigen that can react with the antibody of a subject with an allergic disease, and includes allergens derived from the pollen of trees (acacia, alder, velvet beech, beech, birch, maple, mountain cedar, red cedar, cottonwood, cypress, American elm, autumn elm, Douglas fir, rubber tree, eucalyptus tree, Chinese hackberry, hickory, American linden, sugar maple, mesquite, paper mulberry, and kona). Allergens derived from plant pollen (cotton, larkspur, longgrass, bromegrass, corn, broadleaf fescue, sorghum, oat, orchard grass, ricegrass, morning glory, ryegrass, rice, morning glory, timothy grass, water hyacinth, pigweed, etc.), and allergens derived from plant pollen (cotton, larkspur, longgrass, bromegrass, corn, broadleaf fescue, sorghum, oat, orchard grass, rice bran, morning glory, timothy grass, pigweed, corn, Allergens derived from insects (silkworms, mites, honeybees, wasps, ants, cockroaches, etc.), allergens derived from bacteria (alternaria, aspergillus, botulinum, candida, cephalosporin, etc.), allergens derived from insects (silkworms, mites, honeybees, hornets ... Examples of allergies include, but are not limited to, allergens derived from animal hair (dog, cat, bird, etc.), allergenic proteins derived from house dust, and allergens derived from food (OVA, etc.). Representative diseases of "allergy" include atopic dermatitis, allergic rhinitis (hay fever, etc.), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergies, drug allergies, and urticaria.

[0040] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0041] In one aspect, the present disclosure provides immune cells such as T cells with enhanced glucose uptake ability, cell populations containing such cells, pharmaceutical compositions containing such cells or cell populations, and other related technologies.

[0042] In one aspect of the present disclosure, T cells with enhanced glucose uptake ability are provided. T cells modified and / or enhanced to express a glucose transporter have unexpectedly been shown to be significantly effective against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases when applied to CAR-T cells, TCR-T cells, etc. (see Figure 16). The cells of the present disclosure are advantageously those with enhanced glucose uptake ability. Furthermore, the cells of the present disclosure preferably have effector functions after being introduced into the place where they are to function, particularly into the body. Such cells include T ｅｆｆ Examples include, but are not limited to, T ｅｆｆ The progenitor cells may be lymphoid progenitor cells, such as lymphoid progenitor cells, Tnv, Tscm, and Temra.

[0043] In one embodiment, the present disclosure may relate to effector T cells comprising a chimeric antigen receptor (CAR), modified to express a glucose transporter, and / or with enhanced glucose transporter expression. In another embodiment, the present disclosure may relate to a cell population comprising effector T cells comprising a chimeric antigen receptor (CAR), modified to express a glucose transporter, and / or with enhanced glucose transporter expression. The present disclosure provides "effector T cells," which have not been provided by conventional techniques. In this embodiment, effector T cells refer to immune cells (e.g., T cells) that exert cytotoxic effects on target cells (as described elsewhere herein). As described elsewhere herein, effector function includes cytotoxic activity, cytokine production, and division / proliferation resulting from signals mediated by TCR or CAR, but one important function of effector T cells is to exert cytotoxic effects on target cells.

[0044] On the other hand, the prior art has not provided evidence that effector T cells can exert cytotoxic effects on target cells. Rather than suggesting this, the prior art has not demonstrated cure through rechallenge or other experiments, and in many cases, nodules remain, indicating lack of cure. While various cancer treatments are known, most of these treatments have only reduced tumors, failing to eradicate them. This is because they lack the cytotoxic effect on target cells that is presently demonstrated. In the prior art, tumor suppression is sometimes observed through the effects of cytokines, etc., and most anticancer drugs only have this indirect effect. In the prior art, unlike the present disclosure, tumor suppression in in vivo models does not necessarily result in cytotoxic effects on target cells. Rather, tumors remain, and the tumors lack the cytotoxic effect on target cells. In this respect, the present disclosure demonstrates effects distinct from those of the prior art.

[0045] More specifically, in an embodiment of the present disclosure, chimeric antigen receptor T (CAR-T) cell therapy has not been shown to be effective against solid cancers; this disclosure also solves this problem. Here, the inventors have discovered that CAR-T cell dysfunction in solid cancers is caused by glucose deficiency in the tumor microenvironment (TME), and that metabolic supplementation significantly improves the antitumor effect of CAR-T cells. Massive glucose consumption by cancer cells reduces glucose levels in the TME of solid cancers, resulting in CAR-T cell impairment. In a specific embodiment of the present disclosure, they have found that expressing GLUTs, which are glucose transporters, in CAR-T cells restores cytokine production and killing activity, or improves effector function. This disclosure provides a treatment for various solid cancers that have not been cured even with combination therapy utilizing chemotherapeutic agents and radiation. Certain solid cancers possess a metabolically challenging TME, and CD8 ＋ The function of effector T cells, including T cells, is impaired by adoptive transfer of CAR-T cells into the TME. This disclosure is particularly effective in cases where neural cells, such as neurons and glial cells, require glucose as an energy source. Furthermore, certain aggressive cancer cells consume more glucose than normal cells due to aerobic glycolysis (known as the Warburg effect), a characteristic metabolism of cancer cells, leading to glucose depletion in the TME. On the other hand, naive T cells shift their energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to meet their increased energy demand. Therefore, intense metabolic competition via glucose occurs between activated T cells and cancer cells in the TME. While cancer cells can survive and continuously proliferate by taking up sufficient glucose within the TME, activated T cells lose their function due to the failure of metabolic competition. Equal or greater expression of GLUT by CAR-T cells that exogenously express GLUT or have enhanced endogenous expression may enable complete competition with cancer cells.

[0046] Specifically, preferred, non-limiting embodiments of the present disclosure are as follows: Despite the clinical success of chimeric antigen receptor T (CAR-T) cell therapy in hematological malignancies, its application to refractory solid tumors, including glioblastoma (GBM), has not yet been successful, and clinical trials of CAR-T cells for solid tumors have failed to demonstrate efficacy. The present disclosure also addresses this issue. Here, the inventors have discovered that the dysfunction of CAR-T cells in GBM is due to glucose deficiency in the tumor microenvironment (TME), and that metabolic supplementation significantly improves the antitumor efficacy of CAR-T cells. Massive glucose consumption by cancer cells reduces glucose levels in the TME of GBM, resulting in CAR-T cell impairment. In a preferred embodiment of the present disclosure, stable expression of the high-affinity glucose transporter GLUT3 in CAR-T cells restored cytokine production and killing activity. Glioblastoma (GBM) is a devastating malignant brain tumor with an annual incidence rate of 3.19 per 100,000 people. The advent of combination therapy consisting of temozolomide and radiation in 2005 led to the development of oncolytic field therapy (Novo TTF) for primary GBM, improving the prognosis of GBM patients in clinical practice. However, the 5-year overall survival rate for GBM remains below 20%. Given the clinical success of chimeric antigen receptor T-cell (CAR-T cell) therapy in hematological malignancies, several preclinical and clinical trials of CAR-T cell therapy for GBM are being actively investigated. While some patients treated in clinical trials have shown promising results, such as CAR-T cell infiltration into the tumor microenvironment (TME) and reduction in tumor burden, most patients do not respond to CAR-T cell therapy. GBM harbors a metabolically challenging TME, resulting in CD8 ＋The function of effector T cells, including T cells, is impaired by adoptive transfer of CAR-T cells into the TME. In the brain, neural cells, such as neurons and glial cells, require glucose as an energy source, and the brain consumes 25% of the body's glucose-derived energy. Furthermore, aggressive GBM cells consume three times more glucose than normal neurons due to aerobic glycolysis (known as the Warburg effect), a metabolic pathway characteristic of cancer cells. This leads to glucose depletion in the GBM TME. On the other hand, naive T cells shift their energy production from oxidative phosphorylation and fatty acid oxidation to aerobic glycolysis upon activation to meet their increased energy demand. Therefore, intense metabolic competition via glucose occurs between activated T cells and cancer cells in the GBM TME. In a preferred embodiment, GLUT3, encoded by SLC2A3 and physiologically expressed in neurons and glial cells, has a five-fold higher affinity for glucose than GLUT1, the major glucose transporter expressed in T cells. As a result, GLUT3-expressing GBM cells can survive and continue to proliferate while taking up sufficient glucose within the TME, while activated T cells lose their function due to failure of metabolic competition. In this preferred embodiment, given the significant glucose uptake by GBM cells via high-affinity GLUT3, equal or greater expression of GLUT3 by CAR-T cells may enable them to fully compete with GBM cells. In one preferred embodiment of the present disclosure, a construct is provided in which SLC2A3 is linked to a CAR to induce GLUT3 expression in CAR-T cells.

[0047] (Cell Population) In one aspect of the present disclosure, a cell population is provided that includes immune cells such as T cells with enhanced glucose uptake capacity. At least a portion of the T cells included in this cell population have been modified and / or enhanced to express a glucose transporter, and unexpectedly, when applied to CAR-T cells, TCR-T cells, etc., it has been shown that these cells are significantly effective against diseases such as cancer, autoimmune diseases, allergies, and infectious diseases (see Figure 16, etc.). The cell population of the present disclosure is advantageous in that it has enhanced glucose uptake capacity. Furthermore, the cell population of the present disclosure preferably includes cells that have effector function after being introduced into the place where they are to function, particularly into the body. Such cells include T ｅｆｆ Examples include, but are not limited to, T ｅｆｆ The progenitor cells may be lymphoid progenitor cells, such as lymphoid progenitor cells, Tnv, Tscm, and Temra.

[0048] In one embodiment of the present disclosure, a cell population comprising the T cells described above is advantageous in that the cell population comprises about 10% or more T cells having a characteristic of the present disclosure (e.g., enhanced or expression of GLUT, effector function, or both). In one embodiment, the cell population of the present disclosure can have a percentage of T cells having a characteristic of the present disclosure of about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0049] In one embodiment, the cell population of the present disclosure comprises T cells that have the property of having effector function when introduced into the body.

[0050] In one embodiment, the cell population of the present disclosure, the T cells comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR), preferably a chimeric antigen receptor (CAR).

[0051] In one embodiment, in the cell population of the present disclosure, the T cells are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter.

[0052] In one embodiment, in the cell population of the present disclosure, the T cells have effector function.

[0053] In one embodiment, the cell population of the present disclosure is T ｅｆｆ , and / or T ｅｆｆ Preferably, the cell population (or T ｅｆｆ The progenitor cells of the lymphoid lineage include at least one of lymphoid progenitor cells, Tnv, Tscm, Tcm, and Tempr.

[0054] In one embodiment, in the cell population of the present disclosure, the CAR is expressed by the T cells.

[0055] In one embodiment, in the cell population of the present disclosure, the glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, preferably GLUT3.

[0056] In one embodiment, in the cell population of the present disclosure, the T cells are human T cells.

[0057] In one embodiment, the cell population of the present disclosure is provided as a medicament or pharmaceutical composition. Its use may be for the prevention or treatment of cancer, for the cure of cancer, or for the prevention of metastasis or recurrence of cancer. In a specific embodiment, the cell population of the present disclosure may be used to prevent or treat cancer so that it does not recur.

[0058] <Glucose transporter> Cancer cells reprogram their metabolic system to favor their own cell proliferation, actively utilizing the glycolytic pathway, which is less efficient in producing ATP even in the presence of oxygen, to increase glucose uptake and lactate production (Warburg effect). Therefore, cancer cells consume and deplete large amounts of glucose, and T cells, which require glucose as an energy source, are depleted. ｅｆｆWhen tumor-specific T cells of this type infiltrate tumors, they receive signals from TCR but do not respond to the Ca ２＋ The concentration drops, leading to starvation and exhaustion.

[0059] Thus, in one embodiment of the present disclosure, T cells and cell populations are provided that are genetically engineered to express glucose transporters to promote aerobic glycolysis and increase the competitiveness and / or fitness of immune cells in a glucose-depleted tumor microenvironment (TME).

[0060] In one embodiment, glucose transporters include GLUT1, GLUT2, GLUT3, and GLUT4, and among them, the glucose transporter with the highest affinity for glucose is GLUT3. Thus, in a preferred embodiment, the glucose transporter utilized in the cells and cell populations of the present disclosure is GLUT3.

[0061] GLUT3 was first identified in the mouse brain and was initially defined as a neuronal glucose transporter. GLUT3 was subsequently shown to be expressed in other glucose-requiring cells, such as mouse sperm, which provide the energy needed for movement, and blastocysts, which are important for post-implantation development. Furthermore, GLUT3 is expressed in immune cells, such as lymphocytes, monocytes, macrophages, and platelets, where it is normally stored in intracellular vesicles and translocates to the cell surface upon activation to maintain metabolic switches. More recently, it has been shown to be expressed in T cells, particularly CD8 ＋ Since GLUT3 is highly expressed during differentiation and activation, it has been suggested that T cells depend not only on GLUT1 but also on GLUT3 for glucose uptake.

[0062] T ｅｆｆ When glucose transporters such as GLUT3 are activated in the liver, glucose uptake is enhanced, resulting in enhanced effector functions such as cytotoxic activity and cytokine production.

[0063] In one embodiment of the present disclosure, the T cells or T cells included in the cell population of the present disclosure may include effector cells.

[0064] In one embodiment of the present disclosure, the T cells and T cells included in the cell populations of the present disclosure may advantageously be human T cells.

[0065] <Chimeric Antigen Receptor (CAR)> In another aspect of the present disclosure, there is provided a T cell or cell population comprising such a cell with enhanced glucose uptake ability, wherein the T cell comprises a chimeric antigen receptor (CAR) and the T cell has been modified to express a glucose transporter and / or the expression of the glucose transporter has been enhanced. The CAR contained in the T cell or T cells contained in the cell population of the present disclosure may be contained as a protein, or may comprise a nucleic acid molecule expressing the CAR, as long as it can function as a CAR. In a representative embodiment, the CAR of the present disclosure may be expressed in the T cell.

[0066] The CARs disclosed herein comprise at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0067] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing the antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling domain via a transmembrane domain. CARs feature MHC-independent antigen binding properties of monoclonal antibodies to redirect T cell specificity and reactivity toward selected targets. MHC-independent antigen recognition can confer the ability of CAR-expressing T cells or T cells in a cell population to recognize antigens independently of antigen processing, allowing tumor immune escape.

[0068] The intracellular T cell signaling domain of a CAR can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen. <T Cell Receptor (TCR)>

[0069] In another aspect of the present disclosure, there is provided a T cell or cell population comprising such a cell with enhanced glucose uptake ability, wherein the T cell comprises a T cell receptor (TCR) and the T cell has been modified to express a glucose transporter and / or the expression of the glucose transporter has been enhanced. The TCR contained in the T cells or T cells included in the cell population of the present disclosure may be contained as a protein, or may comprise a nucleic acid molecule expressing the TCR, as long as it is capable of functioning as a TCR. In a representative embodiment, the TCR of the present disclosure may be expressed in a T cell.

[0070] As used herein, TCR refers to a heterodimeric receptor molecule consisting of two TCR polypeptide chains. There are αβ-type TCRs expressed by normal T cells and γδ-type TCRs with specialized functions. The α- and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ chain, CD3ε chain, CD3γ chain, and CD3δ chain), transduce intracellular signals after antigen recognition, and initiate various immune responses. Endogenous antigens, such as viral antigens proliferated within cells following viral infection and cancer antigens derived from cancer cells, are presented as antigen peptides on MHC class I molecules. Furthermore, antigens derived from foreign microorganisms are taken up by antigen-presenting cells via endocytosis, processed, and then presented on MHC class II molecules. These antigens are recognized by TCRs expressed by CD8+ T cells or CD4+ T cells, respectively. It is also known that costimulatory molecules such as CD28, ICOS, and OX40 molecules are important for stimulation via TCR molecules. In the case of αβ-type TCRs, the gene products of α and β can be combined to express specificity. TCRs can be modified as appropriate and may contain a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain includes the intracellular domain of a T cell receptor, for example, the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen.

[0071] (Extracellular Domain) In one embodiment, the CAR used in the T cells or T cells contained in the cell population disclosed herein comprises an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof can be selected appropriately depending on the type and number of ligands on the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a specific disease state. Thus, examples of cell surface markers that can act as a ligand for the antigen-binding domain in the CAR of the present disclosure include tissue-specific markers, tumor-specific markers, markers associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0072] The extracellular binding domain of the CAR can be composed of a single-chain variable fragment (scFv) obtained by fusing the variable heavy and light regions of a mouse or humanized monoclonal antibody. Alternatively, scFv derived from a Fab (not from an antibody, e.g., obtained from a Fab library) can be used. The scFv can be fused to a transmembrane domain and then to an intracellular signaling domain.

[0073] In one embodiment, the antigen-binding domain portion of the CAR of the present disclosure binds to: (1) alloantigens including MHC class I and MHC class II; (2) extracellular self-antigens including TSHR (thyroid stimulating hormone receptor), DSG3 (desmoglein 3), and Cytokeratin 8; (3) foreign antigens including Gliadin and Ara h2; and (4) CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin), and MadCam1 (mucosal vascular addressing cell adhesion molecule). 1), and the like, but the antigens that can be targeted by the antigen-binding domain portion of the CAR of the present disclosure are not limited thereto.

[0074] In one embodiment, depending on the desired antigen to be targeted, the CAR of the present disclosure can be modified to include an antigen-binding domain specific for the desired antigen target. For example, if CD19 is the target antigen, an antibody against a cancer antigen such as CD19 can be used as the antigen-binding domain in the CAR. Non-limiting examples of cancer antigens include CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folate receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGE-4, and MAGE-5. A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, midkin BAGE, CASP-8, P-catenin, CA-125, CDK-1, ESO-1, gp75, MART-1, MUC-1, MUM-1, p53, PAP, PSA, PSMA, ras, trp-1, HER-2, TRP-1, TRP-2, IL13Ralpha, IL13Ralpha2, AIM-2, AIM-3, NY-ESO-1, C9orfl l2, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2, AR T1, ART4, B cycling, Grill, Cav-1, Cathepsin B, CD74, E-Cadherin, EphA2 / Eck, Fra-1 / Fosl 1, GAGE-1, ganglioside / GD2, GnT-V, pl, 6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOXIO, SOX11, Survivin, phCG, WT1, mesothelin, Melan-A, NY-BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the effector T cells disclosed herein are engineered to express one or more CARs to recognize one or more antigens.

[0075] Transmembrane Domains The CARs used in the T cells or T cells included in the cell populations disclosed herein can comprise one or more transmembrane domains fused to an extracellular domain.

[0076] In one embodiment, a linker domain derived from the extracellular domain may be connected to the transmembrane domain. The transmembrane domain may be natural or synthetic, and natural transmembrane domains may be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present disclosure may be derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0077] In one embodiment, the CAR used in the T cells or T cells contained in the cell population disclosed herein may have a spacer domain disposed between the extracellular domain and the transmembrane domain or between the intracellular domain and the transmembrane domain. The spacer domain may preferably have a sequence that promotes binding of the CAR to an antigen and enhances signal transduction into the cell.

[0078] (Intracellular Domain) The cytoplasmic signaling domain (or intracellular signaling domain) of a CAR is involved in activating at least one of the normal effector functions of an immune cell in which the CAR is expressed. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to carry out a specialized function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0079] In one embodiment, examples of intracellular signaling domains used in CARs include cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that initiate signal transduction following antigen receptor binding.

[0080] (Alloantigens, allergens and haptens associated with rejection reactions) CARs used in the T cells or T cells included in the cell populations disclosed herein can include those associated with alloantigens, allergens and haptens associated with rejection reactions.

[0081] <Medicinal Uses of T Cells or T Cells Contained in a Cell Population> In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population comprising such T cells, wherein the T cells have been modified to express a glucose transporter and / or the expression of the glucose transporter has been enhanced. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising T cells with enhanced glucose uptake ability or a cell population comprising such T cells, wherein the T cells comprise a chimeric antigen receptor (CAR), a T cell receptor (TCR), or the like, and the T cells have been modified to express a glucose transporter and / or the expression of the glucose transporter has been enhanced. In one embodiment of the present disclosure, the T cells or the T cells contained in the cell population of the present disclosure can have one or more characteristics of the T cells described above.

[0082] The cells, cell populations, etc. disclosed herein can be used in immunotherapy. Immunotherapy is considered to be effective against diseases that cause antigenic lesions (e.g., cancer, autoimmune diseases, allergies, infectious diseases, etc.) and diseases in which an abnormal immune response to a specific antigen is involved in the onset or progression of the pathology. For example, the cells, etc. disclosed herein can be used to treat, cure, or prevent autoimmune diseases, allergic diseases, or graft-versus-host disease (GVHD), rejection, or graft failure during transplantation. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), Sjögren's syndrome, systemic lupus erythematosus (SLE), antiphospholipid syndrome, polymyositis / dermatomyositis, systemic sclerosis, mixed connective tissue disease, vasculitis syndrome, type I diabetes, Graves' disease, Hashimoto's disease, idiopathic Addison's disease, autoimmune hepatitis, Goodpasture's syndrome, glomerulonephritis, autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenic purpura, autoimmune neutropenia, myasthenia gravis, pemphigus, vitiligo, and idiopathic azoospermia. Examples of allergic diseases include, but are not limited to, hay fever, allergic rhinitis, bronchial asthma, and atopic dermatitis. The cells, cell populations, and the like disclosed herein can also be used to treat or prevent diseases in which an abnormal immune response to a specific antigen is involved in the onset or progression of the disease.

[0083] In certain embodiments, the present disclosure provides such immunotherapy, which includes TCR-T therapy or CAR-T therapy. As used herein, "TCR-T therapy" refers to a cell therapy that utilizes modification of T cell receptors (TCRs), and is used, for example, in cancer treatment. As used herein, "CAR-T therapy" refers to a gene and cell therapy method in which a chimeric antigen receptor (CAR) (e.g., a CAR that has been genetically engineered to overcome tumor immune evasion mechanisms) is introduced into a patient's T cells, and the T cells are expanded and cultured ex vivo and then infused back into the patient.

[0084] The cells, cell populations, etc. of the present disclosure may be appropriately combined with other cancer treatments and used as a combination therapy. Typically, they may be administered in combination with one or more additional drugs. Alternatively, the combination therapy may be combined with radiation therapy. The one or more additional drugs may be any chemotherapeutic drug or may include an immune checkpoint inhibitor. Alternatively, other cancer treatments used in the combination therapy include, but are not limited to, other cancer immunotherapies (e.g., immune checkpoint inhibitors), hyperthermia, surgical procedures, etc.

[0085] In another aspect, there is provided a therapeutic agent comprising the T cells or cell population of the present disclosure, which performs a diagnosis of a disease in a subject and selects an appropriate CAR, TCR, etc. contained in the T cells or the cell population based on the diagnosis.

[0086] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the immune cells described herein (e.g., T cells, such as effector T cells) or cell populations comprising such cells and a pharmaceutically acceptable carrier. When the immune cells express a polypeptide such as a CAR or TCR, the pharmaceutical composition may further comprise an Fc-containing therapeutic agent, such as a therapeutic antibody or an Fc-fusion protein. The Fc-containing therapeutic agent can bind to a target antigen, such as an immune cell specific for a tumor antigen, a pathogen antigen, or an autoantigen. The pathogen antigen may be a bacterial antigen, a viral antigen, or a fungal antigen.

[0087] In one embodiment, the Fc-containing therapeutic agent is selected from the group consisting of adalimumab, adotrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, brentuximab, canakinumab, cetuximab, certolizumab, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibritumab, The therapeutic antibody may be, but is not limited to, mab, infliximab, ipilimumab, labetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tositumomab, ustekinumab, mogamulizumab, and vedolizumab.

[0088] Additionally, the present disclosure provides a kit comprising: (i) a first pharmaceutical composition comprising any T cell described herein or a cell population comprising such a T cell and a pharmaceutically acceptable carrier; and (ii) an Fc-containing therapeutic agent described herein and a pharmaceutically acceptable carrier.

[0089] In another aspect of the present disclosure, there is provided a method for inhibiting cells expressing a target antigen in a subject (e.g., reducing the number of such cells, inhibiting cell proliferation, and / or suppressing the activity of cells), comprising administering to the subject a cell, cell population, and / or pharmaceutical composition of the present disclosure. In one embodiment, at least a portion of the cells expressing the target antigen may be in a low-glucose environment.

[0090] In one embodiment, the subject treated by the method of the present disclosure may be a human patient suffering from cancer, such as carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Exemplary target cancers include, but are not limited to, B-cell derived cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B-cell derived cancers include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0091] In addition to treating a target disease or disorder, such as cancer or an infectious disorder, use of the T cells or cell populations of the present disclosure for the manufacture of a medicament for a medical treatment of interest is also within the scope of the present disclosure.

[0092] In one embodiment, a pharmaceutical composition comprising effector T cells comprising a chimeric antigen receptor (CAR) of the present disclosure can be used in cell therapy. In cell therapy, T cells comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR) of the present disclosure, or a cell population comprising such T cells, can be infused into a subject in need thereof as a pharmaceutical composition or as a formulation of a therapeutically effective cell population expressing a CAR or T cell receptor (TCR) of the present disclosure. The infused T cells or a cell population comprising such T cells in a subject can treat a target disease or disorder, such as cancer or an infectious disorder, in the subject. The subject may be the same subject from which the cells were obtained (autologous cell therapy), or the cells may be derived from another subject of the same species (allogeneic cell therapy).

[0093] In one embodiment, T cells comprising the CAR or (TCR) of the present disclosure or a cell population comprising such T cells can be formulated for administration to a subject using techniques known to those skilled in the art. In one embodiment, a formulation comprising therapeutically effective T cells comprising the CAR or (TCR) of the present disclosure or a cell population comprising such T cells can include a pharmaceutically acceptable excipient (carrier or diluent). The excipients included in the formulation have different purposes depending on, for example, the properties of the antigen-binding domain of the CAR of the present disclosure. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants.

[0094] In this specification, when a gene name and its product are written in all capital letters, contrary to the usual usage, it may refer to both the gene and the protein. For example, the FOXP3 gene and the FOXP3 protein may be used interchangeably, and the term FoxP3 refers to both the concept and entity (whole) of the gene or protein.

[0095] A formulation containing a therapeutically effective T cell comprising a CAR of the present disclosure, or a cell population comprising such a T cell, can be administered to a subject using methods and techniques known to those skilled in the art. Exemplary methods include, but are not limited to, intravenous injection. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarticular, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluid) administration.

[0096] (General Techniques) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press;Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). These are described in PCR Applications: Protocols for Functional Genomics, Academic Press, a special edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and the relevant portions (possibly in their entirety) of which are incorporated herein by reference.

[0097] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) and the like can be used. Other examples include Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.; M. et al. (1996). These are described in Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0098] As used herein, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." When "within the range of" two values ​​is specified herein, the range includes the two values ​​themselves. References cited herein, such as scientific literature, patents, patent applications, etc., are incorporated herein by reference in their entirety to the same extent as if each were specifically set forth.

[0099] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0100] In this example, various functions of T cells with enhanced glucose uptake were investigated. The reagents used were specifically those listed in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0101] Example 1 Methods and Materials Construction of GLUT3 3C10-CAR Construct and Viral Vector Preparation An SLC2A3 sequence (GLUT3) with a P2A sequence located immediately above it was inserted into an anti-EGFRvIII CAR backbone plasmid (3C10 CAR-Katushka2S) having the following structure: CD8 leader sequence-anti-EGFRvIII single-chain variable region (scFv)-CD8 hinge-CD8 transmembrane domain (TM)-CD28 intracellular domain (ICD)-4-1BB ICD-CD3z-P2A-Katushka2S, to construct a 3C10 CAR-GLUT3-Katushka2S lentiviral vector ( FIG. 1 ). The lentiviral vector was transfected into 293T cells together with packaging vectors psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259), and lentivirus was obtained from the supernatant.

[0102] Gene transfer Peripheral blood mononuclear cells from healthy donors were separated from heparinized whole blood by the Ficoll method (Ficoll-Paque PLUS, GE Healthcare), and then stimulated with anti-CD3 / 28 beads (Dynabeads T-Activator CD3 / CD28, Veritas). On day 1 after stimulation, CAR gene transfer was performed at an MOI of 4. The cells were cultured in the presence of 30 U / ml of IL-2 and either cryopreserved from day 8 to 12 or directly subjected to various evaluations.

[0103] Immunostaining 1×10 ６ GLUT3 3C10 CAR-T cells were fixed with 4% PFA and permeabilized with methanol. Blocking was performed with 3% BSA for 1 hour, followed by incubation with anti-GLUT3 antibody (ab15311) for 2 hours. After washing with 0.05% PBST, the cells were incubated with anti-rabbit IgG antibody (Alexa Flour 488) for 1 hour. After washing with 0.05% PBST, nuclei were stained with DAPI. After staining, the cells were attached to a slide glass using Cytospin (Thermo Fisher Scientific), mounted in VECTASHIELD (VECTOR LABORATORIES), and observed using a Keyence XZ-800 microscope.

[0104] Phenotype Analysis (FCM) The antibodies used in FCM analysis are shown in Table 1.

[0105]

[0106] The cells were washed with 4% FBS-PBS. After staining for dead cells and surface staining and washing, data was acquired using a FACSSymphony A3 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0107] In vitro CAR-T cell survival and function analysis (Figure 17) evaluated the long-term survival, memory formation, and functional maintenance of each CAR-T cell. CAR-T cells were stimulated at a 1:1 ratio with stimulatory factors (medium as a negative control, EGFRvIII stimulatory beads, U87d cell line). After 24 hours, the stimulatory factors were removed using magnetic beads or by sorting, and CAR-T cells were divided into 0 mM, 0.5 mM, and 10 mM groups and continued to be cultured. Cell counts were performed before stimulation and on days 1, 3, and 7 after stimulation. Cell survival and cell death were evaluated by analyzing memory phenotypes such as Annexin V / 7AAD, CD27, CCR4, and CD45RA, as well as inhibitory factors such as PD-1, TIM3, and LAG3 (Figure 19), and other markers (transcription factors, activation / senescence markers). In addition, RNA is extracted from each sample and detailed analysis such as RNA sequencing is performed. After this series of analyses, a portion of the day 7 cells are stimulated again in the same group, and cell survival and function analysis after repeated stimulation is similarly performed.

[0108] Cytokine analysis (FCM) CAR T cells 1 x 10 ５ cells and EGFRvIII-expressing U87 cell line (U87Δ) 5 × 10 ５ The cells were co-cultured in 1 ml of medium for 12 hours, and then cultured for 6 hours in the presence of 5 μg / ml Monensin (BD 554724, BD Bioscience) to stimulate the CAR T cells. The medium used was glucose-free RPMI 1640 (Wako) supplemented with 10% dialyzed FBS (Cytiva), with the glucose concentration adjusted to 10 mM and 0.5 mM with a glucose solution (Gibco). After fixation with Fixation / Permeabilization Diluent (Invitrogen), intracellular cytokines were stained with the antibodies shown in Table 1. Data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0109] Cytotoxicity analysis: 2 × 10 cells / well in a 96-well plate ５Target cells (luciferase-expressing U87Δ cells) were seeded and co-cultured with CAR-T cells at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1. After 16 hours, live cells were luminescently stimulated with VivoGlo Luciferin (Promega), and luciferase activity was measured using Cytation (Promega), and cytotoxic activity was calculated.

[0110] Metabolic analysis The metabolic activity of the cells was measured using an XFe24 Cell Flux Analyzer (Bioscience) and an XF Glycolysis Stress Kit (Agilent Technologies) according to the manufacturer's protocol. On the day before the analysis, the 24-well flat-bottom plate for analysis was coated with poly-D-lysine (0.1 mg / ml) and the sensor cartridge was hydrated with CO. ２ On the day of analysis, the cells for analysis were washed with analysis medium (Seahorse XF RPMI medium, L-glutamine 2 mM), and then plated at 2 × 10 cells on a poly-D-lysine-coated analysis plate. ５ The cells were seeded at the appropriate number and ２ The sample was left to stand at 37°C for 60 minutes. The sensor cartridge port was filled with glucose at a final concentration of 10 mM or 0.5 mM, oligomycin at a final concentration of 1 μM, and FCCP at a final concentration of 10 μM, and 2-DG at a final concentration of 50 mM. Analysis was then performed using a flux analyzer, and metabolic function was evaluated from OCR and ECAR.

[0111] Glucose uptake capacity evaluation 2 x 10 ４ GLUT3 CAR-T cells were seeded onto a 96-well plate and cultured for 5 hours in 200 μl of glucose-free RPMI 1640 (Wako). Five minutes before analysis, 1 μl of 2-NDBG (abcam) was added to each well. After washing, data were acquired using an LSRFortessa X20 and analyzed using FACSDiva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0112] Cranial tumor xenograft model: 2.5 x 10 luciferase-expressing U87Δ tumor cell line (U87Δ-luc) was implanted into the cranial cavity (right basal region) of NSG mice. ４ After engraftment, Mock, 3C10 CAR-T cells, GLUT3 CAR-T cells 2 x 10 ６ Cells were administered via the tail vein, and tumor burden and survival were monitored over time by intravital imaging.

[0113] (Method) Figure 1 shows the structure of metabolically engineered CAR (EGFRvIII_CAR_GLUT3). A single-chain antibody (asFv) derived from a mouse-derived antibody (clone 3C10) targeting EGFRvIII expressed in glioblastoma (GBM) was constructed, and the CAR was constructed by binding CD8 hinge, CD28 transmembrane domain (CD28TM), CD28 intracellular domain (CD28ICD), 4-1BB ICD, and CD3z. A high-affinity glucose transporter (GLUT3) was encoded under the CAR via the P2A sequence.

[0114] (Results) The results are shown in Figure 2 and subsequent figures. As shown in Figure 2, enhanced GLUT3 expression on the T cell surface and glucose uptake are demonstrated. CAR T cells were obtained by gene transfer of a conventional CAR (3C10 CAR) and a metabolically engineered CAR (GLUT3 CAR) into T cells derived from a healthy donor. Fluorescence microscopy confirmed homogeneous GLUT3 expression on the membrane of GLUT3 CAR-T cells. Furthermore, glucose uptake was enhanced in GLUT3 CAR-T cells compared to T cells and conventional CAR-T cells (3C10 CAR-T cells).

[0115] Next, Figure 3 shows the T cell expansion efficiency, which is equivalent to that of conventional CAR T cells. As shown in Figure 3, after stimulation with anti-CD3 / 28 beads, CAR was transfected and the T cell proliferation efficiency was analyzed. The GLUT3 CAR-T cells had an establishment efficiency comparable to that of conventional CAR-T cells (3C10 CAR-T cells).

[0116] Next, enhancement of T cell glycolytic capacity by GLUT3 expression is shown in Figure 4. As shown, the metabolism of GLUT3 CAR-T cells and 3C10 CAR-T cells was analyzed using a Flux analyzer. GLUT3 CAR enhanced glycolytic capacity in both low-glucose and normal-glucose environments.

[0117] Next, it was shown that GLUT3 CAR-T cells have a competitive advantage under low glucose conditions (Figure 5). As shown in Figure 5, 3C10 CAR-T cells and GLUT3 CAR-T cells were co-cultured under normal glucose and low glucose conditions. Under normal glucose conditions, both cells survived to a similar extent, but under low glucose conditions, GLUT3 CAR-T cells survived significantly longer, suggesting that they have an advantage in glucose uptake.

[0118] Next, Figure 6 shows that the cytokine production ability of GLUT3 CAR-T cells was enhanced. As shown in Figure 6, 3C10 CAR-T cells and GLUT3 CAR-T cells were stimulated with an antigen, and the percentage of cytokine-producing cells was analyzed by FCM. Significant increases in the expression of cytokines (IFN-g, IL-2, TNF-a) that are important for the maintenance and activation of T cells and the exertion of antitumor activity were observed.

[0119] Next, the function of GLUT3 CAR-T cells, which is maintained even under low glucose conditions, is shown in Figure 7. As shown, cytokine production capacity under normal glucose and low glucose conditions was compared. Conventional CAR-T cells (3C10 CAR-T cells) showed an extreme decrease in function under low glucose conditions. On the other hand, GLUT3 CAR-T cells showed high cytokine production capacity under normal glucose conditions, and even under low glucose conditions, they exhibited a function comparable to the cytokine production capacity of 3C10 CAR-T cells under normal glucose conditions.

[0120] Next, the enhanced cytotoxic activity of GLUT3 CAR-T cells is shown in Figure 8. As shown, cytotoxic activity was analyzed by targeting an EGFRvIII-expressing cell line. Cytotoxic activity was analyzed under low glucose and high glucose conditions, and under both conditions, GLUT3 CAR-T cells exhibited higher cytotoxic activity than 3C10 CAR-T cells.

[0121] Next, the characteristics of GLUT3 CAR on memory phenotype are shown in Figure 9. As shown, the memory phenotypes of 3C10 CAR-T cells and GLUT3 CAR-T cells were compared. A tendency for differentiation into effector T cells was observed in GLUT3 CAR-T cells.

[0122] Next, the suppression of inhibitory molecule expression in GLUT3 CAR-T cells is shown in Figure 10. The expression of inhibitory molecules was compared between unstimulated and stimulated with EGFRvIII antigen-positive cells. Suppression of PD-1, LAG3, and Tim3 expression was observed in GLUT3 CAR-T cells.

[0123] Next, mRNA analysis of GLUT3 CAR-T cells (evaluation of metabolism, exhaustion, activation, and differentiation) is shown in Figure 11. 3C10 CART and GLUT3 CAR-T cells were analyzed in detail using mRNA expression. As with FCM, GLUT3 CAR-T cells showed a decrease in exhaustion-related molecules and an increase in lactate metabolism and glycolysis-related factors. Overall, the data indicate an increase in T cell effector functions, such as activation and cytokine production.

[0124] Next, Figure 12 shows that similar effects can be obtained not only with the 3C10 CAR but also with the CD19 CAR, demonstrating universality. GLUT3 was loaded onto the CD19 CAR, confirming that it had the same effect as the 3C10 CAR. Furthermore, CD19 was expressed in a pancreatic cancer cell line, demonstrating its effectiveness against pancreatic cancer.

[0125] As shown in Figure 13, rapid tumor eradication was demonstrated in an intracranial xenograft model. Antitumor activity was observed in a U87Δ intracranial xenograft model. GLUT3 CAR-T cells demonstrated more rapid tumor eradication than 3C10 CAR-T cells. Furthermore, tumor rejection was confirmed by tumor challenge.

[0126] (Example 2: Enhancement of glucose transporter expression) To achieve enhanced expression of glucose transporters, a CAR construct that constitutively expresses GLUT3 was designed (same as stbl-GLUT3=GLUT3 CAR in Figure 12).

[0127] The function and antitumor effect of the CAR-introduced cells shown in Figure 12 will be analyzed.

[0128] The antitumor effect of GLUT3 CAR-T cells was evaluated in a similar mouse model as shown in Figure 15. While no CR was obtained with mock T cells or 3C10 CAR-T cells, GLUT3 CAR-T cells produced CR in four mice.

[0129] Figure 16 shows the results of tumor challenge. Specifically, it shows the sudden death of mice in the GLUT3 CAR-T cell group. Tumors (U87d) were re-implanted into cured mice from the previous experiment, and rejection ability was evaluated (a "surrogate for memory formation").

[0130] Figure 17 shows that long-term survival is enhanced after various types of stimulation. Each CAR-T cell was observed over time under each condition. As a result, under low glucose conditions, normal CAR-T (3C10) did not exhibit effective cytokine production, whereas GLUT3 CAR-T exhibited effective cytokine production. However, upon activation by excessive glucose uptake, GLUT3 CAR-T differentiated over time (day 3 or day 7) into CCR7-negative Tem or Temra (terminal differentiation, loss of stemness) and underwent apoptosis.

[0131] Furthermore, when CAR-T cells (established with normal glucose) are suddenly (on day 0) placed under low glucose conditions of 0 mM or 0.5 mM, GLUT3 CAR-T, which are highly activated and highly glucose-dependent, are prone to apoptosis.

[0132] Figure 18 shows that long-term survival after stimulation is enhanced. The antitumor effects, survival, and adverse events were examined using the same mouse system as described above. In Experiment 1, GLUT3 CAR-T showed equivalent antitumor activity. In Experiment 2, glucose exposure caused GLUT3 CAR-T hyperactivation, leading to apoptosis and hyperdifferentiation, resulting in the loss of antitumor activity. In rechallenge, GLUT3 CAR-T cells rejected the tumor. In Experiment 4, superior CAR-T cell engraftment and tumor T cell infiltration were observed in GLUT3 CAR-T, in proportion to the tumor effect. Furthermore, Experiment 3 was conducted in a liver tumor model in which immunosuppression due to a low-glucose, high-lactic acid environment has been suggested, and it is expected that similar results to Experiment 2 will be obtained.

[0133] Figure 19 shows data (in vitro data) demonstrating enhanced long-term survival after stimulation. At each time point on the left, staining data for 7-AAD and Annexin V, which reflect cell death and apoptosis, are shown. Cell death in each CAR-T cell type was examined using Annexin V and 7-AAD. Under unstimulated conditions, differences in glucose concentration were observed, but no significant differences were observed in CAR-T cells.

[0134] (Example 3: Example of CAR-T) Hereinafter, a further demonstrative example of CAR-T will be shown.

[0135] (Materials and Methods) (Cell Lines) U-87 MG and U-251 MG GBM cell lines were purchased from the American Type Culture Collection (ATCC) and the Japanese Collection of Research Bioresources Cell Bank, respectively, and cultured in D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. EGFRvIII-packaged lentiviral vectors were introduced into U-87 MG and U-251 MG cells, and the transduced cell lines were designated U-87 MGΔ and U-251 MGΔ, respectively. SUP-T1, NALM6, and AsPC-1 cells were purchased from ATCC and cultured in RPMI 1640 (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% FBS and 1% penicillin / streptomycin. A CD19 truncated vector was transfected into NALM6 cells to obtain the CD19-expressing AsPC1 cell line.

[0136] (Flow cytometry assay) Flow cytometry assay was performed as previously described (40. Kumagai S, Togashi Y, Kamada T, et al (2020) The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies. Nat Immunol; 21(11): 1346-58.; 41. Kumagai S, Koyama S, Itahashi K, et al (2022) Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments. Cancer Cell; 40(2):201-18. e9. ;42. Tada Y, Togashi Y, Kotani D, et al (2018) Targeting VEGFR2 with Ramucirumab strongly impacts effector / activated regulation T cells and CD8+ T cells in the tumor microenvironment. J Immunother Cancer; 6(1): 106. 1 and 43. Tanegashima T, Togashi Y, Azuma K, et al. (2019) Immune Suppression by PD-L2 Against Spontaneous and Treatment-Related Antitumor Immunity. Clin Cancer Res; 25(15): 4808-19. Briefly, cells were washed twice with FACS buffer and subjected to Fc blocking using Fc receptor blocking solution (BioLegend, San Diego, CA) for 10 minutes at 4°C in the dark. Cells were washed twice with FACS buffer, and antibodies targeting cell surface molecules were added and incubated for 20 minutes at 4°C in the dark.Cells were then washed twice with FACS buffer. For intracellular antigen staining, cells were incubated in fixation / permeabilization solution (BD Biosciences, San Jose, CA) for 1 hour at room temperature and then washed twice with wash buffer (BD Biosciences) according to the manufacturer's instructions. Antibodies targeting intracellular antigens were added and incubated for 20 minutes at 4°C in the dark, followed by two washes. For apoptosis assays, cells were washed once with FACS buffer and once with Annexin binding buffer. Cells were then stained with Annexin V and 7-AAD for 15 minutes at room temperature. After washing, flow cytometry (FCM) analysis was performed using an LSRFortessa X-20 cytometer (BD Biosciences) and analyzed with FlowJo ver. 10 software (BD Biosciences). The staining solution was prepared according to the manufacturer's instructions.

[0137] (Cytokine Staining) Intracellular cytokine staining was performed as previously described (40. Kumagai S, et al (2020) At Immunol; 21(11): 1346-58.; 41. Kumagai S, et al (2022) Cancer Cell; 40(2): 201-18.e9.; 42. Tada Y, et al (2018) J Immunother Cancer; 6(1): 106.1 and 43. Tanegashima T, et al (2019) Clin Cancer Res; 25(15): 4808-19.). Monensin was added to the medium for the last 5 hours of 6-hour T cell stimulation to maintain intracellular cytokines. After staining the cell surface markers, Cytofix / Cytoperm reagent (BD Biosciences) was added and incubated in the dark at 4°C for 20 minutes. The cells were then washed twice with washing solution, and antibodies targeting cytokines were added. The cells were incubated in the dark at 4°C for 20 minutes. After washing, FCM analysis was performed using an LSRFortessa X-20 cytometer (BD Biosciences) and analyzed with FlowJo ver. 10 software (BD Biosciences). The antibodies used for cell staining are summarized in Table 2. Antibodies for staining were prepared according to the manufacturer's instructions.

[0138]

[0139] (Killing assay) Total 1 x 10 ５Luciferase-expressing cells (U-87 MGΔ, U-251 MGΔ, AsPC-1, and NALM6) were co-cultured with CAR-T cells at the indicated ratios in 200 μL of RPMI 1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with low glucose (0.5 mM) or high glucose (10 mM) in a flat-bottom 96-well white plate. After 24 hours of culture, Bio-Glo (Promega, Madison, WI) was added to each well, and luminescence was measured using a multiplate reader, Cytation 5 (Agilent Technologies, Santa Clara, CA). The % specific lysis was calculated using the following formula: % specific lysis = [(experimental lysis - spontaneous lysis) / (maximum lysis - spontaneous lysis)] x 100.

[0140] (Immunofluorescence staining) After washing, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with cool methanol at -20°C for 10 minutes. Cells were blocked with 3% BSA / PBS for 60 minutes at room temperature. After blocking, cells were incubated with primary antibody for 2 hours at 4°C and secondary antibody for 1 hour at 4°C. DAPI was then added and incubated at room temperature for 5 minutes. After washing, stained cells were fixed to slides using a Cytospin and analyzed using a BZ-X710 (KEYENCE, Osaka, Japan).

[0141] Immunohistochemical (IHC) staining: IHC staining was performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded specimens. Antigen removal was performed using a steamer with citrate buffer (pH 6.0), followed by deparaffinization and rehydration. Slides were incubated with primary antibodies for 16 hours and HRP-conjugated secondary antibodies for 1 hour, and then developed with diaminobenzidine substrate. Anti-CD3ε antibody (clone: ​​SP7, catalog number: ab16669) (Abcam, Waltham, MA) was used for primary staining. Stained slides were counterstained with hematoxylin.

[0142] Hematoxylin and eosin (HE) staining was performed according to standard protocols. Deparaffinized and hydrated slides were added to hematoxylin solution and incubated for 4 minutes. After washing, the slides were incubated in eosin solution for 2 minutes. Stained slides were scanned at 40x and 400x magnifications using a BZ-X710 (Keyence). Two pathologists independently evaluated the stained slides.

[0143] (ELISA method) Total 2.0 x 10 ５ U-87 MGΔ cells and 2.0 × 10 ５ CAR-T cells were co-cultured in 24-well plates. After 24 hours of culture, supernatants were collected and subjected to ELISA to measure cytokine concentrations. IFN-γ, IL-2, and TNFα ELISA kits (R&D Systems, Minneapolis, MN) were used according to the manufacturer's instructions. Cytokines in mouse serum were analyzed by a highly sensitive LUMINEX assay (Merck Millipore, Burlington, MA) according to the manufacturer's instructions.

[0144] (CAR-T Cell Production) Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL). T cells were isolated from PBMCs by negative selection using MojoSort (BioLegend). Isolated T cells were stimulated with anti-CD3 / 28 beads (Thermo Fisher Scientific, Waltham, MA) at a T cell:bead ratio of 1:1 (day 0). 24 hours after stimulation (day 1), T cells were transduced with CAR lentivirus at an MOI of 3 and cultured in 30 U / ml IL-2. Half of the medium was replaced 24 hours later. Five days later, the CD3 / 28 beads were removed. During the culture, half of the medium was replaced on days 4 and 10, and the T cell concentration was maintained at 0.7 × 10 ６ The developed CAR-T cells were collected on day 10 and subjected to subsequent analysis.

[0145] (Animal Model) Female NSG mice (6 weeks old) were purchased from Jackson Laboratory. Before painful procedures, an anesthetic was injected intraperitoneally to prevent pain. Using a stereotaxic frame, tumor cells were inoculated 2 mm to the right of bregma, 3 mm behind, and 3 mm deep from the brain surface. A total of 2.0 × 10 cells were inoculated in 5 mL of PBS. ４ After injecting 1.0 × 10 cells over 1 minute, the 1 mm needle was removed and left for another 1 minute before being removed. Four days after tumor inoculation, tumor growth was monitored by bioluminescence imaging (BLI) and randomization to each treatment group was performed. On day 5, 1.0 × 10 cells were injected. ６ CAR-T cells were injected via the tail vein. Tumor growth after CAR-T cell injection was monitored twice weekly by BLI. Animal care and experiments were approved by the National Cancer Center Animal Experiment Ethics Committee and conducted in accordance with the guidelines of the National Cancer Center Animal Committee.

[0146] (Glucose Concentration Measurement) Tissue samples (5 × 5 mm) were washed to remove any blood. The samples were minced and centrifuged at 4°C for 10 minutes to collect interstitial fluid from the tumor. The glucose concentration of the interstitial fluid was measured using a Multiskan GO (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0147] (Metabolic Analysis) OCR (unit: pmol / min) and ECAR (unit: mpH / min) were assessed by a Seahorse XF-24 metabolic extracellular flux analyzer (Agilent Technologies). CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, On-d GLUT3 EGFRvIII CAR-T cells) stimulated with EGFRvIII beads (ACRO Biosystems, Beijing, China) for 24 hours were resuspended in glucose-free unbuffered RPMI-1640 medium (Agilent Technologies) and plated onto poly-L-lysine (BD Bioscience)-coated Seahorse cell plates (2.0 × 10 cells per well). ５Cells were plated onto a 1000-well plate (Figure 1). Perturbation profiling of metabolic pathway utilization by CAR-T cells was achieved by adding glucose (10 mM or 0.5 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (50 mM) (all from Agilent Technologies). Experiments using the Seahorse system were performed under the following assay conditions: 3 minutes of mixing, 2 minutes of waiting, and 3 minutes of measurement. Metabolic parameter values ​​were then calculated.

[0148] RNA sequencing and subsequent analysis: CAR-T cells (conv EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells, and On-d GLUT3 EGFRvIII CAR-T cells) sorted using a BD FACSymphony S6 (BD Biosciences) were stimulated with EGFRvIII beads at a 1:1 ratio under low glucose (0.5 mM) or high glucose (10 mM) conditions, supplemented with 30 U / ml IL-2. CAR-T cells were harvested on days 3 and 7, and RNA was extracted using an RNeasy kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from the isolated RNA using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA). Next-generation sequencing (paired-end reads) of 150 bp from both ends of the RNA-seq library was performed using a Novaseq X plus system (Illumina, San Diego, CA). For expression profiling with RNA-seq data, paired-end reads were aligned to the hg38 human genome assembly using STAR (Dobin A, Gingeras TR (2015) Mapping RNA-seq Reads with STAR. Curr Protoc Bioinformatics; 51:11.4.1-.4.9). RNAseqChef is a web-based platform for systematic transcriptome analysis, gene expression analysis, principal component analysis, and pathway analysis (Etoh K, Nakao M (2023) A web-based integrative transcriptome analysis, RNAseqChef, uncovers the cell / tissue type-dependent action of sulforaphane. J Biol Chem; 299 (6): 104810.).

[0149] (Statistical Analysis) GraphPad Prism 9 (GraphPad Software, San Diego, CA) was used for statistical analysis. Relationships between groups were compared using t-tests or one-way analysis of variance. Survival rates were analyzed using the Kaplan-Meier method and statistically compared using the log-rank test. A P value of <0.05 was considered statistically significant.

[0150] (Results) (GBM has low glucose levels in the TME, which impairs CAR-T cell function.) Accumulating evidence suggests that low glucose levels in the TME are a potential barrier to CAR-T cell therapy in solid tumors, but the actual glucose levels in the TME and how low glucose conditions affect CAR-T cell function remain unclear [Peng JJ, Wang L, Li Z, et al (2023) Metabolic challenges and interventions in CAR T cell therapy. Sci Immunol;8(82):eabq3016]. We examined glucose concentrations in the interstitial fluid of surgical specimens and serum (Figure 20a). The glucose concentration in the interstitial fluid of GBM specimens was approximately 10-fold lower than that in serum [<0.5 mM (average 0.217 mM)] (Fig. 20b). While low glucose concentrations are common in various types of cancer, such as non-small cell lung cancer and colorectal cancer, the lowest glucose concentrations were found in the TME of GBM. This indicates that low glucose concentrations in the TME are a characteristic of GBM (Fig. 20c).

[0151] Next, we investigated the effects of a low-glucose environment on CAR-T cell functions, including cytokine production. CAR-T cells targeting the GBM-associated antigen EGFRvIII (conv EGFRvIII CAR-T cells) were cocultured with a human GBM cell line (U-87 MGΔ) expressing EGFRvIII under low-glucose conditions (0.5 mM), which mimic the GBM TME. Upon stimulation with U-87 MGΔ, cytokine production (IFN-γ, IL-2, and TNFα) was significantly reduced in the low-glucose environment, even after a short-term (16-hour) exposure ( Figures 20d and 20e ). To further investigate the functional changes of CAR-T cells under low-glucose conditions, CAR-T cells were stimulated under low-glucose (0.5 mM) or high-glucose (10 mM) conditions for 16 hours, and gene expression profiles were analyzed. The gene expression profile was significantly altered by exposure to low glucose conditions (Fig. 22a). Gene sets related to cell cycle, differentiation, and cytokine production were significantly decreased, whereas gene sets related to the cellular response to glucose starvation and intrinsic apoptosis were increased under low glucose conditions (Fig. 20f, g). Thus, the effector function of CAR-T cells is immediately impaired when exposed to low glucose conditions, such as the TME of GBM.

[0152] (Overexpression of GLUT3 enhances the metabolic fitness of CAR-T cells.) Among the SLC2 family of glucose transporters (GLUTs), GLUT3, encoded by SLC2A3, has the highest affinity and is characterized by being predominantly expressed in neurons and glial cells, which have a high demand for glucose [Flavahan WA, Wu Q, Hitomi M, et al (2013) Brain tumor-initiating cells adapt to restricted nutrition through preferential glucose uptake. Nat Neurosci; 16(10): 1373-82.]. Therefore, we investigated whether GLUT3 expression by CAR-T cells could enhance CAR-T cell activity by promoting glucose uptake in the low-glucose TME of GBM. SLC2A3 was fused to a conventional anti-EGFRvIII CAR construct via a self-cleaving P2A sequence, allowing for stable co-expression of CAR and GLUT3 (GLUT3 EGFRvIII CAR) (Fig. 21a). GLUT3 was expressed on the membrane of T cells transfected with GLUT3 EGFRvIII CAR (GLUT3 EGFRvIII CAR-T cells) ( Figure 21b ). The expression level was approximately twice that of the parent anti-EGFRvIII CAR-T cells (conv EGFRvIII CAR-T cells) and was similar to that of the GBM cell line U-87 MGΔ, whereas no change was observed in the expression level of GLUT1 ( Figures 21c and 23a ). The amount of glucose uptake measured by a glucose uptake assay using the glucose analog 2-NBDG was greater in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells ( Figure 21d ). Accordingly, glycolysis and glycolytic capacity of GLUT3 EGFRvIII CAR-T cells were significantly improved (Fig. 21e and Fig. 23b). Metabolic analysis further revealed that GLUT3 EGFRvIII CAR-T cells had a significantly lower oxygen consumption rate (OCR) than conv EGFRvIII CAR-T cells. ｈｉｇｈ , extracellular acidification rate (ECAR) ｈｉｇｈThis confirmed a more energetic shift in glucose uptake in the low-glucose environment (Figure 21f). In a glucose competition assay in which carboxyfluorescein succinimidyl ester (CFSE)-labeled GLUT3 EGFRvIII CAR-T cells were cocultured with conv EGFRvIII CAR-T cells at a 1:1 ratio, the number of GLUT3 EGFRvIII CAR-T cells relative to conventional CAR-T cells increased at low glucose levels (0.5 mM) but remained similar under high glucose (10 mM) conditions, suggesting that GLUT3 EGFRvIII CAR-T cells are substantially competitive for glucose uptake, particularly in low-glucose environments (Figures 23c and 23d). Therefore, stable GLUT3 expression by CAR-T cells promotes glucose uptake, making CAR-T cells competitive for glucose uptake and enhancing the metabolic fitness of CAR-T cells in limited glucose environments.

[0153] GLUT3 expression improves the metabolic fitness of CAR-T cells, prompting us to examine their effector function and safety profile. We therefore investigated the effector function of GLUT3 EGFRvIII CAR-T cells using both in vitro and in vivo assays. Compared with combo EGFRvIII CAR-T cells, GLUT3 EGFRvIII CAR-T cells significantly increased cytokine production (IFN-γ, IL-2, and TNFα) at ​​both low (0.5 mM) and high (10 mM) glucose levels (Figures 21g, h and 24a). Cytokine production was significantly enhanced by CD8 ＋ T cell subsets and CD4 ＋GLUT3 EGFRvIII CAR-T cells also showed enhanced cytotoxicity against U-87 MGΔ cells and U-251 MGΔ cells, another EGFRvIII-expressing GBM cell line, under both low- and high-glucose conditions (Fig. 21i, Fig. 24d, e). Expression of efflux-related molecules, including PD-1 and Tim-3, after antigen stimulation was higher in GLUT3 EGFRvIII CAR-T cells than in conv EGFRvIII CAR-T cells (Fig. 21j, k and Fig. 24f, g). Furthermore, enhanced effector function due to stable GLUT3 expression was also observed in CAR-T cells targeting a different antigen, CD19 (GLUT3 CD19 CAR-T cells). This indicates that the enhanced metabolic fitness due to GLUT3 expression is universal for all CARs, regardless of the single-chain variable fragment (scFv) used or the antigen targeted (Fig. 25a-c). Thus, stable GLUT3 expression allows CAR-T cells to be activated in the TME with low glucose levels, despite the presence of an exhausted phenotype.

[0154] (Discussion) Enhancing the antitumor effect by improving the metabolic fitness of CAR-T cells in the TME is a novel concept for accelerating the clinical application of CAR-T cell therapy for refractory solid tumors. Although some clinical trials of CAR-T cells for GBM have failed to demonstrate clinical benefit, immunological monitoring of these trials has revealed that abundant CAR-T cells are detected in the TME. [O'Rourke DM, Nasrallah MP, Desai A, et al (2017) A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with current glioblastoma. Sci Transl Med; 9(399). ; Bagley SJ, Logun M, Fraietta JA, et al (2024) Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in Recurrent glioblastoma: phase 1 trial interval results. Nat Med; 30(5):1320-9. Brown CE, Hibbard JC, Alizadeh D, et al. (2024) Local delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade gliooma: a phase 1 trial. Nat Med; 30(4): 1001-12. Considering the presence of CAR-T cells in the TME, antigen loss and impaired migration are excluded as causes of failure, and it is possible that specific conditions in the TME of GBM are involved in inducing CAR-T cell dysfunction.Glucose is an essential nutrient for effector T cells, as they undergo glycolysis for survival and activation [Ho PC, Bihuniak JD, Macintyre AN, et al. (2015) Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses. Cell;162(6):1217-28.] Therefore, given that a significantly low glucose environment is detected in the TME of GBM, low glucose levels in the TME of GBM must be a major metabolic stress for CAR-T cells. Indeed, conv EGFRvIII CAR-T cells are in a dysfunctional state (low cytokine production and high expression of exhaustion markers). Furthermore, PD-1 signaling disrupts PI3K / Akt / mTOR signaling, further impairing the glycolytic pathway, potentially causing effector T cells to enter a negative metabolic cycle [Chang CH, Qiu J, O'Sullivan D, et al (2015) Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell;162(6):1229-41].

[0155] Considering that nervous system cells such as neurons and glial cells, and GBM, especially GBM expressing EGFRvIII, meet their high glucose demands using the high-affinity glucose transporter GLUT3 [Vannucci SJ, Maher F, Simpson IA (1997) Glucose transporter proteins in brain: delivery of glucose to neurons and glia. Glia; 21(1): 2-21. ;Chen S, Yang L, Li Z, et al (2021) EGFR / EGFRvIII partially regulates the tumorigenesis of glioblastoma through the SOX9-GLUT3 axis. Am J Transl Res; 13(6):6055-65. ; Schmidt S, Hommel A, Gawlik V, et al. (2009) Essential role of glucose transporter GLUT3 for post-implantation embryonic development. J Endocrinol; 200(1):23-33.] High expression of GLUT3 in CAR-T cells is likely to overwhelm nutrient competition. In fact, it has been demonstrated that the GLUT3 EGFRvIII CAR construct developed by the present inventors enhanced glucose uptake and effector functions such as cytokine production and cytotoxicity in GLUT3 EGFRvIII CAR-T cells.

[0156] (Example 3: Examples using precursor cells of other effector T cells) Similar experiments are performed using other T cells. For example, unpurified peripheral T cells contain Tnv, Tcm, Tem, Temra, etc., and any fraction can be used to express CAR (e.g., constitutively expressed GLUT3 CAR).

[0157] In this example, a CAR (CAR, constitutively expressed GLUT3 CAR) is introduced into a purified and enriched T cell memory fraction. In a separate experiment, CAR-T cells enriched in the T cell memory fraction are induced under culture conditions.

[0158] Experimental procedure: ・T cells that are expected to survive longer and maintain stemness were selected from T cell sources such as peripheral blood, bone marrow, and umbilical cord blood. ｅｆｆ Tnv, Tscm, and Tcm are precursor cells of T cells, or T cells that have already differentiated and mainly exert effector functions. ｅｆｆ The T cell memory-associated surface antigen expression patterns were CD45RA and Temra fractions, respectively. ＋ CCR7 ＋ CD95 － , CD45RA ＋ CCR7 ＋ CD95 ＋ , CD45RA － CCR7 ＋ , CD45RA － CCR7 and CD45RA ＋ CCR7 － Based on this, the cells are purified and concentrated using FACS sorting or magnetic beads. Each fraction is stimulated with CD3 / 28 beads, etc., and CAR is introduced using a lentiviral vector. Alternatively, after gene transfer to bulk T cells, the type and amount of cytokines added can be adjusted (using IL-7 or IL-15, or using platelet lysate) to induce T cell proliferation. ｅｆｆ It suppresses differentiation and induces the predominant proliferation of Tnv, Tscm, and Tcm, resulting in progenitor-predominant CAR-T cells. In vitro evaluation: These cells are co-cultured with U87d tumor cell lines or EGFRvIII beads, and after 1, 3, and 7 days, CFSE staining and cell counting are used to assess proliferation, and flow cytometry is used to assess activation and exhaustion. In vivo evaluation: These cells are administered intracranially to the aforementioned U87d mice, and antitumor activity and survival are assessed by BLI.

[0159] Results: When introduced into bulk T cells or differentiated T ｅｆｆCompared to the case of transfection with TEMRA, the T cells such as Tnv, Tscm, and Tcm, which are precursor cells, ｅｆｆ CAR-T cells introduced into progenitor cells or in which Tnv Tscm Tcm is predominantly amplified are expected to have high division and proliferation capacity, long-term division, and low expression of PD1, TIM3, and LAG3, and to be resistant to exhaustion.

[0160] (Example 4: Example of TCR) A similar experiment is carried out in which a TCR is introduced instead of a CAR. ｅｆｆ TCR can also be introduced into fractions such as Tnv, Tcm, Tem, and Temra.

[0161] In this example, a TCR (constitutively expressed GLUT3 TCR) is introduced into a purified and enriched T cell memory fraction. In a separate experiment, TCR- T cells enriched in the T cell memory fraction are induced under culture conditions.

[0162] Various in vitro and in vivo evaluations shown in Examples 1 to 3 are carried out.

[0163] Example 5: Prevention, prevention of reinfection, and treatment of bacterial infection This example examines the prevention, prevention of reinfection, and / or treatment of bacterial infection.

[0164] (Method) For prevention of bacterial infections (such as bacterial conjunctivitis), T cells of the present disclosure modified with an infectious agent of the bacterial infection are administered to a subject who does not have the bacterial infection.

[0165] In preventing recurrence and / or treating bacterial infections (such as bacterial conjunctivitis), T cells of the present disclosure are administered to a subject who has had and / or has a bacterial infection.

[0166] After administration, the subject is followed up for bacterial infection by techniques routinely used in the art.

[0167] Example 6: Prevention, reinfection prevention, and treatment of parasitic infections This example examines the prevention, reinfection prevention, and / or treatment of parasitic infections.

[0168] (Method) For prevention of a parasitic infection (such as Acanthamoeba keratitis), T cells of the present disclosure modified with an infectious antigen of the parasitic infection are administered to a subject who does not have the parasitic infection.

[0169] In preventing recurrence and / or treating a parasitic infection (such as, for example, Acanthamoeba keratitis), T cells of the present disclosure are administered to a subject who has had and / or has a parasitic infection.

[0170] After administration, the subject is followed up for parasitic infection by techniques routinely used in the art.

[0171] Example 7: Prevention of infection, onset, reinfection, and treatment of viral infections In this example, the prevention of infection, onset, reinfection, and / or treatment of viral infections is verified.

[0172] (Method) For the prevention of viral infections (e.g., which may include, but are not limited to, tuberculosis, malaria, yellow fever virus, smallpox virus, vaccination, measles / rubella, polio, mumps / MUMPS, rotavirus infection, chickenpox, yellow fever, Ebola, West Nile fever, Hib infection, pneumococcal infection, pertussis, Japanese encephalitis, meningococcal infection, salmonella infection, pathogenic E. coli, toxoplasmosis, Zika virus, herpesvirus type 1, EBV / Epstein-Barr virus (herpesvirus type 4), CMV / cytomegalovirus (herpesvirus type 5), influenza (virus), MARS, rabies, diphtheria, etc.), T cells of the present disclosure modified with an infectious antigen of the viral infection are administered to a subject who does not have the viral infection.

[0173] In preventing recurrence and / or treating the viral infection, the T cells of the present disclosure are administered to a subject who has had and / or is currently suffering from a viral infection.

[0174] After administration, the subject is monitored for prognosis of viral infection by techniques commonly used in the art.

[0175] Example 8: Prevention, recurrence prevention, and treatment of allergies This example examines the prevention, recurrence prevention, and / or treatment of allergies.

[0176] (Methods) In the prevention of allergies (eg, allergic conjunctivitis), T cells of the present disclosure modified with an allergic infection-causing antigen are administered to a subject who does not have the allergy-causing antigen.

[0177] In preventing recurrence and / or treating allergies (e.g., allergic conjunctivitis), T cells of the present disclosure are administered to a subject who has had and / or has the causative antigen of the allergy.

[0178] After administration, the subject is followed up for allergy by techniques routinely used in the art.

[0179] Example 9: Prevention, relapse prevention, and treatment of autoimmune disease This example examines the prevention, relapse prevention, and / or treatment of autoimmune disease.

[0180] (Method) In the prevention of autoimmune diseases (e.g., autoimmune uveitis), T cells of the present disclosure modified with an autoimmune disease infectious cause antigen are administered to a subject who does not have the allergy-causing antigen.

[0181] In preventing recurrence and / or treating an autoimmune disease (e.g., autoimmune uveitis), the T cells of the present disclosure are administered to a subject who has had and / or has the causative antigen of the autoimmune disease.

[0182] After administration, the subject is monitored for prognosis of the autoimmune disease by techniques commonly used in the art.

[0183] (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be construed solely by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2024-001532, filed on January 9, 2024, with the Japan Patent Office, the contents of which are incorporated by reference in their entirety herein.

[0184] According to the present disclosure, it is possible to provide cells that are competitive with cancer cells without starving or exhausting them, even in glucose-depleted environments such as the tumor microenvironment (TME), thereby enabling the development of strategies to improve the efficacy of cell-based immunotherapy, and is expected to find application in the medical field.

Claims

1. T cells with enhanced glucose uptake ability.

2. The T cell according to claim 1, wherein the T cell contains a chimeric antigen receptor (CAR).

3. The T cell according to claim 1 or 2, wherein the T cell has effector function.

4. The T cell is a progenitor cell of an effector T cell (T ｅｆｆ ), and the T cell according to claim 1 or 2.

5. The T cell according to any one of claims 2 to 4, wherein the CAR is expressed in the T cell.

6. The T cell according to any one of claims 1 to 5, wherein the glucose transporter includes GLUT1, GLUT2, GLUT3, and GLUT4.

7. The T cell according to any one of claims 1 to 6, wherein the glucose transporter is GLUT3.

8. The T cell according to any one of claims 1 to 7, which is a human T cell.

9. An effector T cell that contains a chimeric antigen receptor (CAR), is modified to express a glucose transporter, and / or has enhanced expression of a glucose transporter, and the T cell exhibits at least equivalent effector function under low glucose conditions (0.5 mM) as under normal glucose conditions (10 mM).

10. The T cell according to claim 9, wherein the CAR is expressed in the T cell.

11. The T cell according to claim 9 or 10, wherein the glucose transporter includes GLUT1, GLUT2, GLUT3, and GLUT4.

12. An effector T cell into which the GLUT3 gene has been introduced.

13. The T cell according to any one of claims 9 to 12, which is a human T cell.

14. A cell population containing T cells with enhanced glucose uptake ability, and the T cells have the property of having effector function when introduced into the body.

15. The cell population according to claim 14, wherein the T cells are modified to express a glucose transporter and / or have enhanced expression of a glucose transporter.

16. The cell population according to claim 14 or 15, wherein the T cells have effector function.

17. The cell population according to any one of claims 14 to 16, wherein the T cells contain a chimeric antigen receptor (CAR).

18. The cell population is T ｅｆｆ The cell population according to any one of claims 14 to 17, comprising 19. The cell population is T ｅｆｆ The cell population according to any one of claims 14 to 18, comprising progenitor cells of 20. The cell population according to any one of claims 17 to 19, wherein the CAR is expressed in the T cell.

21. The cell population according to any one of claims 14 to 20, wherein the glucose transporter comprises GLUT1, GLUT2, GLUT3, and GLUT4.

22. The cell population according to any one of claims 14 to 21, wherein the glucose transporter is GLUT3.

23. The cell population according to any one of claims 14 to 22, wherein the T cell is a human T cell.

24. A cell population comprising effector T cells modified to express a glucose transporter and / or having enhanced expression of a glucose transporter, the effector T cells comprising a chimeric antigen receptor (CAR), wherein the T cells exhibit at least equivalent effector function under low glucose conditions (0.5 mM) as under normal glucose conditions (10 mM).

25. The cell population according to claim 24, wherein the CAR is expressed in the T cells.

26. The cell population according to claim 24 or 25, wherein the glucose transporter comprises GLUT1, GLUT2, GLUT3, and GLUT4.

27. A cell population comprising effector T cells into which the GLUT3 gene has been introduced.

28. The cell population according to any one of claims 24 to 27, wherein the T cell is a human T cell.

29. A pharmaceutical composition comprising the T cells according to any one of claims 1 to 13 or the cell population according to any one of claims 14 to 28.

30. The pharmaceutical composition according to claim 29, which is for treating or preventing cancer.

31. The pharmaceutical composition according to claim 29 or 30, which is for curing cancer.

32. The pharmaceutical composition according to any one of claims 29 to 31, wherein the cure of the cancer is confirmed by tumor eradication.

33. The pharmaceutical composition according to any one of claims 29 to 32, which is for having a long-term effect on cancer.

34. The pharmaceutical composition according to any one of claims 29 to 33, which is for preventing cancer metastasis or recurrence.

35. The pharmaceutical composition according to any one of claims 29 to 34, which is for preventing cancer recurrence.

Images