Effector T cells and pharmaceutical compositions containing the same

Abstract

To provide cells that are competitive against cancer cells without starving or becoming exhausted even in glucose-depleted environments. To provide cells that are effective in metabolic (e.g., low glucose, high lactate, high fatty acid) and immunological tumor environments (e.g., chemokine profiles that induce immunosuppressive immune cells, expression of checkpoint molecules, and infiltration of suppressor cells that negatively regulate anti-tumor immune responses, such as inhibitory cytokines and Treg cells) in which normal T cells or conventional genetically modified cells are inhibited from infiltrating and, even if they infiltrate, are unable to exert long-term effector function due to starvation or exhaustion. [Solution] T cells with effector function, which have been modified to express Foxp3 (including mutant and partially deleted forms) and / or have enhanced Foxp3 expression.

Description

[Technical Field]

[0001] The present disclosure provides a method for the treatment of effector T cells (T eff ), especially metabolically altered effector T cells (T eff ), and pharmaceutical compositions comprising such cells. [Background technology]

[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. Upon TCR stimulation, T cells increase Ca 2+ The intracellular concentration of calcineurin increases, activating calcineurin. Activation of calcineurin dephosphorylates the transcription factor NFAT, which then translocates into the nucleus and interacts with other transcription factors, activating them by promoting the transcription of genes such as the IL-2 gene.

[0004] On the other hand, cancer cells consume and deplete glucose in large quantities, which means that T cells require glucose as an energy source. eff When tumor-specific T cells of this type infiltrate tumors, they receive signals via TCR, but the cells' Ca 2+ This decreases the concentration of NFAT and reduces its nuclear translocation, 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 anti-tumor 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. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides T cells with effector function (T eff ), especially effector T cells with altered metabolic capacity (T eff ) and functioning in a tumor environment without starvation or exhaustion, and a pharmaceutical composition containing such cells.

[0008] Thus, the present disclosure provides: (Item 1) Foxp3, its mutants, or any part thereof are (exogenously) expressed, and / or (endogenous) Foxp3 expression is enhanced; and Possess immune effector functions, T cells (FOXP3-T cells). (Item 2) The FOXP3-T cells described in any one of the above items, wherein the FOXP3-T cells have been modified to reduce or eliminate the function and / or expression of a factor (hereinafter referred to as a "negative regulatory factor") that negatively regulates the immune effector function induced by FOXP3. (Item 3) The FOXP3-T cell according to any one of the preceding items, wherein the negative regulatory factor comprises a Treg cell-like immunosuppressive factor, for example, an inhibitory cytokine such as TGFβ or IL-10, or an inhibitory molecule such as CTLA-4. (Item 4) The FOXP3-T cell according to any one of the preceding items, wherein the Foxp3 has been modified to confer immune effector function to the Foxp3-T cell or to maintain or enhance the immune effector function of the T cell. (Item 5) (Abandonment of inhibitory function + effector) The Foxp3 is a factor that negatively regulates immune effector function (negative regulator) so that the expression and / or function of the factor is reduced or eliminated. The FOXP3-T cell according to any one of the preceding items, wherein the T cell has been modified to impart an effector function to the T cell or to maintain or enhance the effector function of the T cell. (Item 6) The FOXP3-T cell according to any one of the preceding items, wherein the Foxp3-T cell is a cell with modified metabolic capacity. (Item 7) The FOXP3-T cells according to any one of the preceding items, wherein the Foxp3-T cells are cells with altered chemokine receptor expression and / or altered migration, infiltration, and survival in the local environment. (Item 8) The FOXP3-T cell according to any one of the above items, wherein the local environment is the environment in which the target cell is present, and when the target cell is a tumor cell, the local environment is the tumor environment or tumor microenvironment. (Item 9) A T cell (FOXP3-T cell) having effector function and modified to express Foxp3, a mutant thereof, or a part of either thereof, and / or having enhanced Foxp3 expression, wherein the FOXP3-T cell has been modified to reduce or eliminate the inhibitory function of the negative regulatory factor. (Item 10) The FOXP3-T cell according to any one of the preceding items, which has been modified so that the inhibitory function of the negative regulatory factor is reduced or eliminated, thereby maintaining or enhancing immune effector function. (Item 11) The FOXP3-T cells according to any one of the preceding items, wherein the expression of a chemokine receptor associated with migration, infiltration and survival in the local environment is enhanced in the FOXP3-T cells. (Item 12) The FOXP3-T cell according to any one of the above items, wherein the chemokine receptor includes CCR4, CCR8, etc. (Item 13) The FOXP3-T cell according to any one of the above items, which satisfies at least one predetermined condition. (Item 14) While FOXP3-T cells are effective against any tumor, they are particularly effective in tumor environments that promote Treg cell infiltration and / or suppress T cell infiltration, survival, and function. These conditions arise when the patient's tumor environment is rich in ligands (e.g., CCL17, CCL22, and CCL1) for the chemokine receptors (CCR4 and CCR8) induced by FOXP3 and / or has a nutritional environment compatible with the metabolic pattern induced by FOXP3 (e.g., low glucose, high fatty acid, and high lactate conditions). The FOXP3-T cells described in any one of the above items are effective biomarkers for diagnosing the above conditions, such as histopathologically abundant infiltration of Treg cells and / or the presence of genetic mutations in the tumor that can induce the above-mentioned tumor environment (such as EGFR mutation, ROHA mutation, or increased expression of MHC class II and self-molecules), and can be identified by tumor biopsy, gene panel testing using peripheral blood liquid biopsy, or next-generation sequencing. (Item 15) The FOXP3-T cell of any one of the preceding items, wherein the T cell comprises an exogenously inserted antigen receptor such as a chimeric antigen receptor (CAR). (Item 16) The FOXP3-T cell described in any one of the above items, wherein the CAR is expressed in the T cell. (Item 17) The FOXP3-T cell according to any one of the preceding items, in which the expression of at least one immunosuppressive gene is reduced or substantially eliminated. (Item 18) The FOXP3-T cell described in any one of the above items, wherein the immunosuppressive gene includes at least one selected from the group consisting of CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73. (Item 19) The FOXP3-T cell according to any one of the above items, which is a human effector T cell. (Item 20) A) diagnosing the local environment of a subject, wherein the diagnosis is achieved by determining the Treg status in the subject or genetic mutations in environmental factors of target cells; C) providing T cells with immune effector function and / or factors that modify the tumor environment based on the local environment; a method for improving the tumor environment or anti-tumor immune response in said subject, comprising: (Item 21) A) A step of diagnosing the local environment of a subject (test subject) (e.g., determining whether Treg cell infiltration is enhanced and / or whether T cell infiltration, survival, or function is suppressed), in which the diagnosis is achieved by determining the Treg status in the subject or genetic mutations in environmental factors of target cells (e.g., histopathologically, abundant Treg cell infiltration and / or target cells having genetic mutations that can induce the above-mentioned tumor environment (e.g., EGFR mutation, ROHA mutation, etc.)); C) providing T cells with immune effector function and / or factors that modify the tumor environment (e.g., internal infiltration, chemokines, etc.) based on the local environment; a method for improving the tumor environment or anti-tumor immune response in said subject, comprising: (Item 22) A) A step of diagnosing the immune status of a subject (subject); B) diagnosing a genetic mutation in the subject; C) providing T cells having immune effector functions according to the immune state based on the immune state and the genetic mutation, and providing factors that modify the local environment (e.g., internal infiltration, BATF, chemokines, etc.) based on the genetic mutation; a method for improving the immune status in said subject, comprising: (Item 23) A pharmaceutical comprising T cells or a cell population of said T cells having immune effector function, wherein said T cells have been modified to express Foxp3, a mutant thereof, or a part of either thereof, and / or have enhanced expression of Foxp3. (Item 24) A method for treating or preventing a disease associated with an abnormal immune status, comprising administering to a subject in need thereof an effective amount of T cells or a cell population of said T cells having immune effector function, wherein said T cells have been modified to express Foxp3, a mutant thereof, or a part of either thereof, and / or have enhanced expression of Foxp3. (Item 25) Effector T cells (T eff 1. A pharmaceutical composition comprising: a T cell comprising a human T cell line comprising a human T cell lysate; a human T cell line comprising ... (Item 26) The composition described in any one of the above items, wherein the T cells are cells with modified metabolic capacity. (Item 27) The composition of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). (Item 28) The composition described in any one of the above items, wherein the CAR is expressed in the T cell. (Item 29) A composition described in any one of the above items, in which the expression of at least one immunosuppressive gene is reduced or substantially eliminated. (Item 30) The immunosuppressive gene is selected from the group consisting of CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and and CD73. (Item 31) The composition of any one of the above items, wherein the T cells are human effector T cells. (Item A1) A cell population comprising T cells (FOXP3-T cells) that (exogenously) express Foxp3, its mutants, or any part thereof, and / or have enhanced (endogenous) Foxp3 expression and have immune effector function. (Item A2) The FOXP3-T cells suppress the function and / or expression of a factor that negatively regulates the immune effector function induced by FOXP3 (hereinafter referred to as "negative regulatory factor"). The cell population described in any one of the preceding items, which has been modified so that expression is reduced or eliminated. (Item A3) The cell population described in any one of the above items, wherein the negative regulatory factor includes a Treg cell-like immunosuppressive factor, for example, an inhibitory cytokine such as TGFβ or IL-10, or an inhibitory molecule such as CTLA-4. (Item A4) The cell population according to any one of the preceding items, wherein the Foxp3 has been modified to confer immune effector function to the Foxp3-T cells or to maintain or enhance the immune effector function of the T cells. (Item A5) (Abandonment of inhibitory function + effector) The expression and / or function of Foxp3, a factor that negatively regulates immune effector function (negative regulator), is reduced or eliminated. The cell population according to any one of the preceding items, wherein the T cells have been modified to impart an effector function to the T cells or to maintain or enhance the effector function of the T cells. (Item A6) The cell population according to any one of the preceding items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item A7) The cell population described in any one of the above items, wherein the Foxp3-T cells are cells with altered chemokine receptor expression and / or altered migration, infiltration, and survival in the local environment. (Item A8) The cell population according to any one of the preceding items, wherein the local environment is the environment in which the target cells exist, and when the target cells are tumor cells, it is the tumor environment or tumor microenvironment. (Item A9) A cell population according to any one of the preceding items, comprising T cells (FOXP3-T cells) that have effector function and have been modified to express Foxp3, a mutant thereof, or a part of either of them, and / or have enhanced Foxp3 expression, wherein the FOXP3-T cells have been modified to reduce or eliminate the inhibitory function of the negative regulatory factor. (Item A10) The cell population according to any one of the above items, wherein the cell population has been modified so that the inhibitory function of the negative regulatory factor is reduced or eliminated, thereby maintaining or enhancing immune effector function. (Item A11) The cell population described in any one of the above items, wherein the expression of a chemokine receptor in the FOXP3-T cells is enhanced, the chemokine receptor being associated with migration, infiltration, and survival in the local environment. (Item A12) The cell population according to any one of the preceding items, wherein the chemokine receptors include CCR4, CCR8, and the like. (Item A13) A cell population according to any one of the above items, which satisfies at least one predetermined condition. (Item A14) The cell population according to any one of the above items, wherein the predetermined conditions are effective against any tumor, but are particularly effective against a tumor environment in which Treg cell infiltration is enhanced and / or T cell infiltration, survival, and function are suppressed, or the cell population according to any one of the above items, wherein the predetermined conditions are generated when the patient's tumor environment is rich in ligands (CCL17, CCL22, CCL1, etc.) for the chemokine receptors (CCR4 and CCR8) induced by FOXP3, and / or has a nutritional environment compatible with the metabolic pattern induced by FOXP3 (low glucose, high fatty acid, high lactate, etc.). A cell population according to any one of the above items, wherein, in order to diagnose the above, abundant infiltration of Treg cells histopathologically and / or the presence of a gene mutation in the tumor that can induce the above tumor environment (EGFR mutation, ROHA mutation, or increased expression of MHC class II and self-molecules) are effective biomarkers, and can be identified by tumor biopsy, gene panel testing using peripheral blood liquid biopsy, or next-generation sequencing. (Item A15) The cell population described in any one of the preceding items, wherein the T cells contain an exogenously inserted antigen receptor such as a chimeric antigen receptor (CAR). (Item A16) The cell population described in any one of the above items, wherein the CAR is expressed in the T cells. (Item A17) The cell population according to any one of the preceding items, in which the expression of at least one immunosuppressive gene is reduced or substantially eliminated. (Item A18) A cell population described in any one of the above items, wherein the immunosuppressive gene includes at least one selected from the group consisting of CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73. (Item A19) The cell population described in any one of the above items, wherein the T cells are human effector T cells. (Item A20) A) A step of diagnosing the local environment of a subject, wherein the diagnosis is achieved by determining the Treg status in the subject or genetic mutations in environmental factors of target cells; C) providing a T cell or cell population according to any one of the preceding paragraphs, and / or a factor that modifies the tumor environment, based on the local environment; a method for improving the tumor environment or anti-tumor immune response in said subject, comprising: (Item A21) A) A step of diagnosing the local environment of a subject (test subject) (e.g., determining whether Treg cell infiltration is enhanced and / or whether T cell infiltration, survival, or function is suppressed), in which the diagnosis is achieved by determining the Treg status in the subject or genetic mutations in environmental factors of target cells (e.g., histopathologically, abundant Treg cell infiltration and / or target cells having genetic mutations that can induce the above-mentioned tumor environment (e.g., EGFR mutation, ROHA mutation, etc.)); C) providing a T cell or cell population according to any one of the preceding items, and / or a factor that modifies the tumor environment (e.g., internal infiltration, chemokines, etc.) based on the local environment; a method for improving the tumor environment or anti-tumor immune response in said subject, comprising: (Item A22) A) A step of diagnosing the immune status of a subject (subject); B) diagnosing a genetic mutation in the subject; C) providing the T cells or cell population according to any one of the preceding items based on the immune state and the genetic mutation, and providing a factor that modifies the local environment (e.g., internal infiltration, BATF, chemokine, etc.) based on the genetic mutation; a method for improving the immune status in said subject, comprising: (Item A23) A pharmaceutical comprising T cells or a cell population of said T cells having immune effector function, wherein the T cells have been modified to express Foxp3, a mutant thereof, or a part of either thereof, and / or the expression of Foxp3 has been enhanced. (Item A24) A method for treating or preventing a disease associated with an abnormal immune status, comprising administering to a subject in need thereof an effective amount of T cells or a cell population of said T cells having immune effector function, wherein said T cells or said cell population of T cells have been modified to express Foxp3, a mutant thereof, or a part of either thereof, and / or have enhanced expression of Foxp3. (Item A25) Effector T cells (T eff a pharmaceutical composition comprising a cell population of wherein the T cells have been modified to express Foxp3 and / or have enhanced expression of Foxp3. (Item A26) The composition according to any one of the preceding items, wherein the T cells are cells with modified metabolic capacity. (Item A27) The composition of any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). (Item A28) The composition described in any one of the above items, wherein the CAR is expressed in the T cells. (Item A29) The composition according to any one of the preceding items, in which the expression of at least one immunosuppressive gene is reduced or substantially eliminated. (Item A30) A composition described in any one of the above items, wherein the immunosuppressive genes include CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73. (Item A31) The composition according to any one of the preceding items, wherein the T cells are human effector T cells. (Item B1) A method for treating or preventing a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject an effective amount of the cell, cell population, composition, or medicament described in any one of the above items. (Item C1) A cell, cell population, composition or medicament according to any one of the preceding items for treating or preventing a disease, disorder or condition. (Item D1) Use of a cell, cell population, composition or medicament described in any one of the preceding items for the manufacture of a medicament for treating or preventing a disease, disorder or condition.

[0009] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. 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 apparent to those skilled in the art by referring to the following description of the preferred embodiments of the invention and the drawings. [Effects of the Invention]

[0011] The present disclosure provides a method for producing metabolically altered effector T cells (T eff ) can be provided, and by using such cells, it is possible to provide chimeric antigen receptor (CAR) T cells that function in the tumor environment without starvation or exhaustion.

[0012] The cells disclosed herein can be said to be enhanced T cells, in which the mechanisms by which regulatory T cells infiltrate, survive, and function in the tumor environment are applied to non-regulatory T cells. By using Foxp3 as a target, it is possible to simultaneously induce factors necessary for T cells to infiltrate tumors and continue to act for a long time, such as changes in chemokine expression profiles and metabolic patterns. In addition, by knocking out / knockdown the inhibitory molecules of regulatory T cells, which are by-products, it is possible to confer only anti-tumor effects to T cells, and induce high anti-tumor activity not seen in conventional engineered T cells. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 illustrates the concept of the present disclosure. Representatively, we demonstrate the use of FOXP3 to induce factors useful for T cell effector function in non-Treg cells, thereby enhancing antitumor efficacy. The efficacy of CAR-T cell therapy in solid tumors is limited. This is because the unique immune microenvironment formed in solid tumors creates a hypoxic, low-glucose, and high-lactate environment, causing exhaustion and apoptosis in CAR-T cells, which rely on glycolysis for metabolism, leading to dysfunction. On the other hand, regulatory T cells (Tregs), unlike CAR-T cells, efficiently infiltrate and activate in the tumor microenvironment. This is because they are not dependent on glucose alone but can overcome metabolic checkpoints using fatty acids and lactate present in the tumor microenvironment as nutrient sources. Furthermore, enhanced infiltration function due to increased expression of chemokine receptors such as CCR4 and CCR8 allows them to maintain infiltration and activation in the hostile tumor microenvironment. In this disclosure, by expressing FOXP3, the master regulator of Tregs, in T cells, CAR-T cells are endowed with the diverse functions of FOXP3, such as metabolic reprogramming and chemokine receptor expression, thereby enhancing effector function and enhancing anti-tumor effects. [Figure 2]Figure 2 shows T cells expressing FOXP3 (FOXP3-T cells). By expressing FOXP3, the master regulator of regulatory T cells (Treg cells), these cells are obtained with factors that negatively regulate effector functions (negative regulators) and factors that positively regulate effector functions (positive regulators) that are inherent in parental T cells (unmodified T cells) and / or induced by FOXP3. The former are typified by the expression of Treg-like inhibitory cytokines and inhibitory molecules, while the latter are typified by metabolic reprogramming, tumor invasion and survival, cytotoxic activity, and cytokine production. [Figure 3] Figure 3 shows FOXP3-T cells in which negative regulatory factors are suppressed or abrogated, while positive regulatory factors are maintained or enhanced. As shown, to selectively obtain effector functions important for anti-tumor immune responses, negative regulatory factors of effector function are suppressed or abrogated using gene editing (e.g., CRISPR / Cas9) or RNA interference (e.g., shRNA) (A). Alternatively, FOXP3 derivatives (mutant FOXP3 or partially deleted FOXP3) that are modified to selectively maintain or enhance positive regulatory factors without inducing negative regulatory factors are introduced (B). These methods allow the establishment of T cells with enhanced anti-tumor activity (including genetically modified T cells such as CAR-T cells). [Figure 4] Figure 4 shows the results of directly introducing factors that enhance effector function into T cells. As shown in Figure 4, factors that positively regulate effector function, the expression of which is controlled by the transcription factor FOXP3 (here, the metabolic transporter genes CD36 and MCT1, and the chemokine receptor CCR4) were introduced into T cells individually or in combination, resulting in increased expression of each factor. However, when two or three factors were introduced simultaneously, the expression efficiency of each factor gradually decreased, suggesting that this method is not feasible for the cumulative gene introduction of numerous factors that enhance effector function controlled by FOXP3. [Figure 5]Figure 5 shows the expression of immunosuppressive molecules induced by wild-type FOXP3 transfection and their downregulation using RNA interference. Comparing the phenotypes of cCAR and wild-type FOXP3-transfected CAR-T (WT), we confirmed increased expression of Treg-derived immunosuppressive molecules such as CTLA4 and CD25. Furthermore, we confirmed that simultaneous downregulation of immunosuppressive molecules such as CTLA4 and TGFβ1 could be achieved using the shRNA system. This demonstrates the increased expression of factors (immunosuppressive molecules) that negatively regulate effector function as a side effect of wild-type FOXP3 transfection and the suppression or abrogation of these factors using RNA interference. Comparing the phenotypes of conventional CAR-T cells (normal CAR-T cells, cCAR-T cells, without FOXP3-related manipulation) and wild-type FOXP3-transfected CAR-T cells (WT), we confirmed increased expression of Treg-like immunosuppressive molecules such as CTLA4 and CD25, which can negatively regulate FOXP3-induced effector function. We confirmed that simultaneous downregulation of immunosuppressive molecules such as CTLA4 and TGFβ1 could be achieved using shRNA-based RNA interference. [Figure 6]Figure 6 shows the phenotype of wild-type FOXP3-transduced CAR-T cells. The phenotypes of conventional CAR-T cells (CAR-T cells without FOXP3 transduced, abbreviated as cCAR) and wild-type FOXP3-transduced CAR-T (CAR-FOXP3, abbreviated as WT) were compared. FCM analysis revealed increased FOXP3 expression in WT cells (a), an increased fraction of memory T cells (responsible for long-term antitumor activity) (b), decreased proportions of exhaustion markers PD-1+ and TIM-3+ cells (c) and TOX expression (d), and increased expression of chemokine receptors (CCR4 and CCR8) that induce intratumoral T cell migration (e). Furthermore, decreased expression of the glucose transporter GLUT1 and decreased glucose uptake (2-NBDG) were observed in WT cells, suggesting impaired glycolytic function. On the other hand, increased CPT1A expression and increased fatty acid uptake (BODIPY-FL) suggested enhanced fatty acid oxidation (f). Analysis using an extracellular flux analyzer revealed increased oxidative phosphorylation (OCR) activity in a low-glucose environment (Glu 0.5mM RPMI) (g). Furthermore, suppression of fatty acid oxidation by adding etomoxir reduced the maximum OCR only in WT, suggesting that the increased oxidative phosphorylation activity in WT under a low-glucose environment may be dependent on fatty acid oxidation (h). GSEA using scRNAseq also revealed increased expression of genes involved in oxidative phosphorylation and fatty acid oxidation in WT compared to cCAR (i). [Figure 7]Figure 7 shows an example of a CAR construct incorporating FOXP3 (CAR-FOXP3 construct). Based on a second-generation anti-CD19 CAR containing 4-1BB and CD3z as signaling molecules, wild-type FOXP3 was incorporated via the 2A sequence to create an all-in-one construct expressing CD19 CAR, wild-type FOXP3, and a gene transfer marker (tEGFR) (A). To suppress negative regulatory factors for effector function, constructs were constructed combining RNA interference, the CRISPR / Cas9 system (B), and a dominant-negative TGFβR (C). CAR-FOXP3 constructs were also constructed incorporating mutant FOXP3 (D) or partially truncated FOXP3 (E), which suppress negative regulatory factors and selectively maintain or enhance positive regulatory factors for effector function. The CD19 scFv can be replaced with other scFvs (anti-mesothelin, anti-ROR1, anti-EGFR RvIII, etc.). [Figure 8] Figure 8 shows the increase in chemokine receptor expression due to FOXP3 transfection. As shown, FCM was used to compare the expression of CCR4, CCR5, CCR8, and CXCR3 in CD19CART cells (CART) and FOXP3-transfected CD19CART cells (CART-FOXP3). Compared to 19CART (CART), 19CART-FOXP3 (CART-FOXP3) showed increased expression of the above chemokine receptors (CCR4, CCR5, CCR8, and CXCR3). [Figure 9] Figure 9 shows the cytokine production ability of FOXP3-transfected cells. The cytokine production ability of CART-FOXP3 cells was evaluated by intracellular staining. CART-FOXP3 cells exhibited cytokine production equal to or greater than that of conventional CD19CART cells. [Figure 10]Figure 10 shows the antitumor effect of FOXP3-expressing CAR-T using an NSG mouse model. (a) The human leukemia cell line Nalm-6 was infused into NSG mice, and the antitumor effects of untrunduced T cells (UTD), cCAR, and WT were confirmed. WT showed a reduction in tumor volume compared to UTD, but showed earlier tumor recurrence compared to cCAR. (b) The human pancreatic cancer cell line Aspc-1 (tumor volume 20-150 mm3) was infused into NSG mice, and the antitumor effects of UTD, cCAR, and WT were evaluated. cCAR significantly suppressed tumor growth compared to WT and UTD, while the antitumor effect of WT showed no significant difference from UTD. [Figure 11] The identification of highly efficient sh-CTLA4 and sh-TGFb is shown in Figure 11. As shown, in the present disclosure, multiple shRNAs were constructed (see Tables 2 and 3), and highly efficient shRNAs were identified for each. [Figure 12] Figure 12 shows the identification of highly efficient sg-CTLA4 and sg-TGFb. We constructed multiple shRNAs (see Tables 3 and 4) and successfully eliminated CTLA-4 and TGFb completely using CRISPR-Cas9. [Figure 13] Figure 13 shows the enhanced proliferation of CART cells by suppressing CTLA-4 and TGF-β. CTLA-4 and TGF-β were knocked down in CD19CART-Foxp3 cells using shRNA. CD19CART-Foxp3 cells lacking both molecules exhibited greater proliferation than normal CART cells. [Figure 14] Figure 14 shows the blockade of TGFb signaling by dominant-negative TGFb receptor (dnTGFbR). Introducing dnTGFRbR into CD19CART-Foxp3 cells blocked TGFb signaling and increased IFN-γ production. [Figure 15]Figure 15 shows the structure of FOXP3 and an example of the mutation site. (a) The three-dimensional structure of FOXP3 predicted using Alphafold3 is shown. FOXP3 forms a dimeric leucine zipper structure and is suggested to bind to RUNX1 and NFAT1 via the forkhead region. Through this complex formation, FOXP3 is thought to induce transcriptional suppression of inflammatory cytokines and increased expression of immunosuppressive molecules. (b) and (c) FOXP3 structure and mutation site. Introducing FOXP3 mutations from the leucine zipper region to the forkhead region into CAR T cells inhibits complex formation with RUNX1 and NFAT1. This allows CAR-T cells to acquire the tumor local survival function of regulatory T cells while reducing the immunosuppressive function induced by FOXP3, thereby improving antitumor efficacy. We also investigated the possibility of reducing immunosuppressive function by introducing FOXP3 mutations observed in IPEX syndrome, a fatal autoimmune disease associated with FOXP3 mutations. [Figure 16]Figure 16 shows the antitumor effect of mutant FOXP3-expressing CAR-T (FOXP3-T cells containing CAR) in an NSG mouse model. (a) Human pancreatic cancer cell line Aspc-1 (tumor volume 90-725 mm3) was infused into NSG mice, and the antitumor effects of untrunculous T cells (UTD), cCAR, WT, F325D, F331D, F331D, K332D, H334D, R337Q, F340D, Y342F, W348Q, M370I, A372P, R386H, R397W, E399R / E401A, and D409A mutations were confirmed. The R397W mutation demonstrated significant tumor regression compared to all other groups. The K332D, R337Q, M370I, A372P, and R386H mutations demonstrated comparable antitumor effects to those of cCAR. (b) Human pancreatic cancer cell line Aspc-1 (tumor volume 20-180 mm3) was infused into NSG mice, and the antitumor effects of UTD, cCAR, WT, K332D, R337Q, Y342F, R356E, F367L, M370I, A372P, R386H, V396E, R397W, V398E, and V408E mutations were evaluated. The cCAR, R356E, F367L, M370I, A372P, R386H, and R397W mutations demonstrated comparable antitumor effects. (c) MYC, an oncogene involved in increased glycolytic activity and cell proliferation, was overexpressed in the human pancreatic cancer cell line Aspc-1, and refractory tumors (tumor volume 180-400 mm3) were infused into NSG mice to confirm the antitumor effects of cCAR, K332D, M370I, and R397W mutations. MYC-expressing models have been reported to induce a low-glucose, high-lactate tumor microenvironment through increased glycolytic activity. In this model, the R397W mutation demonstrated greater tumor regression than all other groups. [Figure 17]Figure 17 shows an example of fatty acid metabolic activity in a high-fat environment. (a) Established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 at a CAR-T:Tumor ratio of 10:1 for 72 hours in a low-sugar, high-fat environment (0 mM glucose, 2 mM palmitic acid). (b) Changes in CAR-T cell numbers in the culture environment were evaluated in three donors. WT, R356E, F367L, A372P, and R397W mutations tended to have significantly higher viable cell numbers than cCAR. (c) CPT1A expression was evaluated by FCM in three donors at 72 hours of co-culture. CPT1A expression tended to be higher in R356E, F367L, M370I, A372P, R386H, and R397W mutations than in cCAR. (d) Glucose 0mM RPMI was supplemented with palmitic acid (0.2mM, 0.5mM, 1.0mM) and the human leukemia cell line Nalm6 was incubated with CAR-T:Tumor at a ratio of 10:1 and co-cultured for 72 hours to confirm CPT1A expression. WT, F367L, and R397W mutants showed a tendency for CPT1A expression to increase in a fatty acid concentration-dependent manner, but this tendency was not observed in cCAR. (e) Celltrace violet 2.5µM was stained before Nalm6 stimulation, and expression was evaluated 72 hours later. F367L, M370I, A372P, and R397W mutants had higher division ability than cCAR. [Figure 18]Figure 18 shows an example demonstrating the evaluation of metabolic activity in a high-fat environment. Established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 at a CAR-T:Tumor ratio of 10:1 in a low-glucose, high-fat environment (0 mM glucose, 2 mM palmitic acid) for 48 hours. After this, the CAR-T cells were transferred to RPMI with 0 mM Glu, and their oxidative phosphorylation activity (oxygen consumption rate [OCR]) and glycolytic activity (proton efflux rate [PER]) were analyzed in two donors using an extracellular flux analyzer (Tcell Metabolic Profiling Kit). A tendency for increased OCR after BAM15 stimulation was observed in Donor 2 with WT, K356E, and R397W mutations, and in Donor 3 with WT, F367L, M370I, A372P, and R386H mutations. [Figure 19] Figure 19 shows another example demonstrating oxidative phosphorylation activity in a high lactate environment. (a) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc1 at a CAR-T:Tumor ratio of 10:1 for 96 hours in a low-glucose, high-lactate environment (glucose 0 mM, lactate 40 mM). (b) Changes in CAR-T cell numbers in the culture environment were evaluated in one donor. WT, F367L, M370I A372P, and R397W mutants tended to have higher cell viability than cCAR. (c) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc-1 at a CAR-T:Tumor ratio of 10:1 in a low-sugar, high-fat environment (0 mM glucose, 40 mM lactate) for 96 hours. After this, CAR-T cells were transferred to RPMI with 0 mM Glu, and their oxidative phosphorylation activity (oxygen consumption rate [OCR]) and glycolysis activity (extracellular oxidation rate [ECAR]) were analyzed in one donor using an extracellular flux analyzer (Mito Stress test). WT, K356E, F367L, M370I, and R386H mutant CAR-T cells tended to have increased OCR compared to cCAR upon FCCP stimulation. [Figure 20]Figure 20 shows an example demonstrating the proliferation potential and phenotypic changes of CAR-T cells following repeated stimulation. (a) The established CAR-T cells were co-cultured with the pancreatic cancer cell line Aspc-1 at a 1:1 ratio, and stimulation with Aspc-1 was repeated every 96 hours. (b) Changes in CAR-T cell numbers (doubling) following repeated stimulation were evaluated. F367L and R397W mutations tended to result in higher cell proliferation than cCAR. (c) Cells were stained with 2.5 μM Celltrace violet before Aspc-1 stimulation, and expression was evaluated on day 12 (after three stimulations). WT, F367L, and A372P mutations had higher division potential than cCAR. (d) Ki-67 expression after a single stimulation with Aspc-1 was confirmed by FCM in two donors. Ki-67 expression tended to be higher in WT, K356E, F367L, M370I, A372P, R386H, and R397W mutants compared to cCAR. (e)-(f) Tim-3 and TOX expression levels and the percentage of PD-1+ and Tim-3+ cells after three rounds of Aspc-1 stimulation were confirmed by FCM in one donor. Tim-3 expression tended to be lower in WT, K356E, F367L, A372P, R386H, and R397W mutants compared to cCAR. TOX expression tended to be lower in WT, R356E, F367L, and R397W mutants compared to cCAR. Additionally, the percentage of PD-1+ and Tim-3+ cells tended to be lower in WT, K356E, F367L, A372P, R386H, and R397W mutants. [Figure 21] Figure 21 shows the changes in phenotypes in the tumor environment. After engraftment of the pancreatic cancer cell line Aspc1 in NSG mice, CAR-T cells were administered. 10 days later, tumors were excised and the phenotypes of the CAR-T cells contained within the tumors were evaluated. Regarding the percentage of PD-1+ and Tim-3+ cells, the Y342F, R386H, and R397W mutations showed a decrease in the percentage of both positive cells (a). Furthermore, TOX expression tended to decrease with the Y342F, R386H, and R397W mutations (b), while CCR8 expression tended to increase with the M370I and A372P mutations. [Figure 22]Figure 22 shows the changes in gene expression profiles as determined by RNA sequencing. RNA was extracted 12 days after CAR-T cell establishment and RNA sequencing was performed. (a) Heatmap revealed three groups: a group showing gene expression patterns similar to Th2 cells, including IL13 (A372P, R337Q, R397W), a group showing gene expression patterns similar to WT (F367L, Y342F, R386H, K332D, K356E), and a group showing gene expression patterns similar to cCAR (M370I). (b) Volcanoplots were generated comparing the gene expression patterns of WT and each FOXP3-CART mutant with cCART. The R397W mutation exhibited characteristic gene expression patterns, including elevated expression of TNFRSF8 (CD30) and BATF3, in addition to increased expression of IL13. (c) Volcanoplots were generated comparing the gene expression patterns of each FOXP3-CART mutant with WT. Compared to WT, Y342F, F367L, and R386H mutations showed decreased expression of genes involved in the suppressive function of regulatory T cells, such as MYB and CTLA4. Y342F, R386H, and R397W mutations showed decreased expression of genes involved in the dysfunction of CAR-T cells, such as ID3 and SOX4. R337Q, A372P, and R397W showed a gene expression profile similar to Th2 cells, including IL13 and IL4, and the R397W mutation in particular showed characteristic increased expression of genes such as BATF3 and TNFRSF8. [Figure 23]Figure 23 shows the evaluation of metabolic activity and cytokine profiles by GSEA analysis. GSEA analysis was performed on PI3-AKT-mTOR signaling (a), glycolysis activation (b), MYC target gene expression (c), IL-2-STAT5 signaling (d), and IFN-γ response (e) to evaluate changes in gene expression patterns relative to cCAR. WT, K332D, Y342F, F367L, and R386H mutations showed decreased expression of PI3-AKT-mTOR signaling, glycolysis, and MYC target genes. WT, K332D, Y342F, F367L, and R386H mutations also showed decreased gene expression related to IL-2-STAT5 signaling and IFN-γ response. [Figure 24] Figure 24 shows the analysis of various mutants. Gene ontology analysis. Gene ontology analysis (MF) was performed to compare changes in molecular function by RNA sequencing with cCAR. WT, K356E, and F367L mutations showed increased expression of genes related to cell invasiveness, such as chemokine receptor activity. On the other hand, R337Q, A372P, and R397W mutations showed increased expression of genes related to ATP hydrolysis activity and protein kinase activity. [Figure 25] Figure 25 shows the changes in the binding pattern of FOXP3 dimers and RUNX1 as determined by Alphafold3. Using Alphafold3, the complex structure of FOXP3 dimers and RUNX1 was predicted for each FOXP3 mutation. The following mutations altered the binding of RUNX1: F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A. The F367L mutation, in particular, abolished the binding of RUNX1 to the FOXP3 forkhead region. DETAILED DESCRIPTION OF THE INVENTION

[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, terms used in this specification should be understood to have the meaning 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 one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will prevail. te) takes precedence.

[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" refers to ±10% of the following numerical value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including 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, "immune effector function" refers to the ability to directly or indirectly damage or suppress target cells (e.g., tumor cells, virus-infected cells, antigen-positive cells, etc.). In the case of T cells, immune effector function can be determined by methods such as cytotoxicity assays, surface antigen analysis by flow cytometry, intracellular cytokine staining, secreted cytokines by ELISA, and gene expression profiling by RNA sequencing. Examples of immune effector function include preferential infiltration into inflammatory sites and sustained function in the cancer microenvironment, which is hostile to immune system effector cells (e.g., killer T cells) in conditions such as hypoxia and malnutrition. In addition to T cells, "immune effector function" can also include biological activities attributable to the Fc region of antibodies. Examples of such immune effector functions include 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 B cell activation.

[0018] As used herein, "conferring" immune effector function or the like refers to adding or enhancing that function to a specific subject. Specific examples include introducing molecules that induce an immune response into cells or tissues. Also included are methods of inducing the expression of immune-related genes using chemicals or biological reagents. Furthermore, this also applies to using gene editing technology to give immune cells a specific function. This also includes protein modification or the introduction of peptides for the purpose of regulating the immune system. Note that "conferring" is not limited to artificial methods, but also applies to cases where naturally occurring processes are utilized.

[0019] As used herein, "maintenance" of "immune effector function" and the like means stably maintaining that function in a specific subject. Specific examples include adjustments to allow immune cells to consistently exert their functions even under the influence of external environments and internal factors. It also includes methods for appropriately supplying nutrients, cytokines, or other physiologically active substances so as not to impair the homeostasis of the immune system. Furthermore, this also applies to stabilizing gene expression and controlling mechanisms that prevent protein degradation. It may also apply to therapeutic or preventive interventions to prevent the decline of adaptive and innate immune functions. Note that "maintenance" applies not only to external interventions but also to methods that support the subject's endogenous mechanisms.

[0020] As used herein, "enhancement" of "immune effector function" and the like means exerting that function at a higher level than normal in a specific subject. Specific examples include methods of increasing cytokine secretion by promoting immune cell activation. Chemical or genetic engineering techniques for enhancing the function of antigen-presenting cells are also included. Furthermore, methods of amplifying signaling between immune cells to strongly induce immune responses also fall under this category. Other examples include using drugs or treatments that stimulate the immune system to enhance the ability to eliminate pathogens. Note that "enhancement" is not limited to cases of external intervention, but also includes the enhancement of naturally occurring immune responses.

[0021] As used herein, "modification" of a gene or the like refers to a specific gene or an element related thereto. "Modification" refers to any manipulation performed with the purpose of changing the function or properties of a gene. This "modification" includes not only changes to the base sequence of the gene itself but also manipulations that affect the regulation of gene expression and the function of gene products (e.g., proteins). Specific examples of "imparting" function include introducing artificial receptors (CARs) that specifically recognize tumor cells into T cells to enhance a new tumor immune response. "Maintaining" function includes introducing stable promoters to maintain stable gene expression even during cell division or under stress. "Enhancing" function includes modifying specific amino acid sequences to enhance catalytic efficiency and improve enzyme activity. CRISPR / Cas9 is one method for achieving these modifications. This technology can be used to add functions by inserting new sequences into specific gene sequences or to edit epigenetic regulatory regions to stabilize expression. Genetic vectors are also widely used for modification, including the introduction of new genes to confer new functions and the maintenance of gene expression by designing vectors that enable long-term expression. Another common approach is promoter modification. By designing specific promoters and enhancers, it is possible to add new expression patterns or maintain stable expression. Furthermore, by using chemical induction systems, it is possible to introduce mechanisms that cause genes to be expressed only in the presence of specific chemicals, thereby enhancing expression in a controlled manner. These modification techniques are widely applied not only to manipulate gene function, but also to improve the properties of gene products and add new biological functions.

[0022] As used herein, "chemokine receptor expression" refers to the phenomenon or state in which chemokine receptors are expressed on the cell surface. Chemokine receptors are transmembrane proteins that bind to specific signaling molecules called chemokines and play an important role in regulating cell migration, migration, and function. "Chemokine receptor expression" is achieved through gene transcription, translation, and subsequent protein transport to the cell membrane. Specifically, expression of CCR5 and CXCR4 in immune cells contributes to cell migration to inflammatory sites and immune responses to pathogens. Furthermore, expression of chemokine receptors in tumor cells may be involved in tumor progression and metastasis. Methods for regulating chemokine receptor expression include the following: First, CRISPR / Cas9 can be used to manipulate chemokine receptor genes and enhance or control their expression. Second, modification of the promoter region can induce receptor expression under specific conditions. Third, technologies that control post-translational modifications of chemokine receptors and improve their transport efficiency to the cell membrane may be utilized. Because "chemokine receptor expression" significantly affects cellular functions, it is an important concept in the fields of immune response research, drug development, and tumor treatment.

[0023] As used herein, "migration into a local environment" refers to the phenomenon in which specific cells move toward a specific local environment (e.g., an inflammatory site, a tumor microenvironment, or a site of tissue damage). This migration occurs in response to chemical, physical, or biological stimuli and is an essential process for cells to migrate to an appropriate location and perform their functions. Factors that induce cell migration include secreted molecules such as chemokines and cytokines. For example, chemokines (e.g., CXCL12) secreted at inflammatory sites attract immune cells that express specific chemokine receptors (e.g., CXCR4), promoting their migration into the local environment. Physical factors, such as substrate stiffness and intercellular adhesion, can also affect cell migration. Methods for regulating "migration into a local environment" include the following: First, manipulations that enhance or control the expression of chemokine receptors to improve migration ability. Second, methods that selectively induce specific cells by controlling the concentration of chemokines or cytokines. Third, techniques that modify cell surface molecules to optimize migration efficiency may also be used. "Migration to the local environment" is a key factor in the development of inflammatory diseases and tumors. This concept plays an important role in a wide range of fields, including tumor immunotherapy and regenerative medicine, and is essential for understanding cellular functions and developing therapeutic strategies. Chemokine receptors are transmembrane proteins that bind to specific signaling molecules called chemokines and play an important role in regulating cell migration, migration, and function. There are various types of chemokine receptors, each of which exerts its function by binding to a specific chemokine. Specific examples of chemokine receptors include the CC motif chemokine receptor (CCR family), which includes CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10. The CXC motif chemokine receptor (CXCR family), which includes CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7. Furthermore, there is the C-X3-C motif chemokine receptor CX3CR1, and the XC motif chemokine receptor XCR1. Other examples of chemokine receptors include Duffy antigen receptor (DARC), CC motif chemokine receptor-like 1 (CCRL1), CC motif chemokine receptor-like 2 (CCRL2), aberrant chemokine receptor 1 (ACKR1), and aberrant chemokine receptor 2 (ACKR2). These chemokine receptors play an important role in cell migration in response to the local environment and are involved in many physiological and pathological processes, including inflammatory responses, immune surveillance, tumor progression, and regenerative medicine. In this specification, chemokine receptor expression is interpreted as a broad concept that encompasses these functions and roles.

[0024] As used herein, the term "ligand for a chemokine receptor" refers to a molecule that binds to a specific chemokine receptor and induces, regulates, or inhibits its activity. Ligands for chemokine receptors are primarily proteins called chemokines, which play important roles in regulating cell migration, migration, and immune responses. Specific examples of ligands that bind to CC motif chemokine receptors (CCR family) include: CCL17 (TARC) and CCL22 (MDC) as ligands for CCR4; CCL1 (I-309) as ligands for CCR8; CCL2 (MCP-1) as ligands for CCR2; and CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) as ligands for CCR5. CCL20 (MIP-3α) binds to CCR6, and CCL19 and CCL21 bind to CCR7, respectively. Furthermore, ligands that bind to the C-X-C motif chemokine receptors (CXCR family) include CXCL12 (SDF-1) for CXCR4, CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) for CXCR3. CX3CL1 (fractalkine), a C-X-C motif chemokine, binds to CX3CR1, and XCL1 (lymphotiactin), an XC motif chemokine, binds to XCR1. These ligands guide specific cells to the local environment through binding to chemokine receptors, contributing to inflammatory responses, immune regulation, tumor microenvironment formation, and tissue repair. Furthermore, the strength and duration of signaling depend on the concentration of the ligand and its affinity for the receptor, determining the biological role of each chemokine. The relationship between chemokine receptors and their ligands is crucial in physiological and pathological processes.

[0025] As used herein, "genetic mutations that can induce a tumor environment" refers to genetic mutations that affect the properties of cells and their surrounding environment, contributing to the formation and maintenance of the tumor microenvironment (TME). These genetic mutations affect intracellular and extracellular signal transduction, immune response evasion, angiogenesis, metabolic regulation, and cell-cell interactions, and may promote tumor progression and metastasis. For example, mutations in KRAS, PIK3CA, and BRAF cause abnormal activation of signal transduction pathways, inducing cell proliferation and the secretion of inflammatory cytokines, thereby promoting tumor formation. Furthermore, as a mutation related to immune evasion, TP53 deficiency or mutation impairs tumor suppressor function and creates an environment that evades immune surveillance. Furthermore, overexpression of CD274 (PD-L1) can also be caused. Mutations and abnormalities in the JAK / STAT pathway contribute to the creation of an immunosuppressive cellular environment. Mutations in VEGFA and HIF1A promote angiogenesis, which plays a key role in the tumor microenvironment. These mutations promote the formation of new blood vessels that supply oxygen and nutrients to tumors, supporting tumor growth. Genetic mutations associated with altered tumor metabolism, such as IDH1 / IDH2 mutations, contribute to the formation of an immunosuppressive tumor environment through the accumulation of abnormal metabolites. Similarly, abnormalities in SDH (succinate dehydrogenase) and FH (fumarate hydratase) induce a metabolic state favorable for tumorigenesis. Furthermore, genetic mutations that promote extracellular matrix (ECM) remodeling, such as MMP (matrix metalloproteinase)-related genes, promote ECM degradation, facilitating tumor cell invasion and metastasis. Furthermore, abnormalities in the NF-κB pathway induce the secretion of inflammatory cytokines (e.g., IL-6 and CXCL8), inducing carcinogenic inflammation. Epigenetic abnormalities are also important in shaping the tumor environment. Abnormalities in DNMT3A cause abnormal DNA methylation, suppressing the expression of tumor suppressor genes. Mutations in EZH2 alter chromatin modifications, promoting tumor progression and immune evasion. These genetic mutations affect a wide range of processes in the tumor microenvironment, creating favorable conditions for tumor growth and progression. Understanding these mutations is crucial for developing new approaches to tumor therapy and tumor control strategies.

[0026] As used herein, the term "immunosuppressive genes" refers to genes encoding molecules that suppress or regulate immune responses. These genes play important roles in suppressing excessive immune system responses and maintaining immune homeostasis. They are also involved in physiological and pathological processes, such as suppressing the tumor microenvironment, autoimmune diseases, and graft rejection. Specifically, immunosuppressive genes include the following: CTLA-4, a molecule that directly suppresses T cell activation and plays an important role in regulating the immune response; TGF-β, a cytokine that promotes the formation of an immunosuppressive environment and suppresses the proliferation and activity of immune cells; CD25 (IL-2Rα) is involved in regulating T cell proliferation and activity; OX40 and GITR contribute to the maintenance of regulatory T cells (Tregs) with immunosuppressive functions; ICOS is involved in maintaining regulatory T cell function and balancing the immune response; CD80 (B7-1) and CD86 (B7-2) are costimulatory molecules that induce immunosuppression through interaction with CTLA-4. TGF-β receptors (TGFβR) suppress immune cell activity through TGF-β signaling. IDO (indoleamine 2,3-dioxygenase) is a molecule that promotes immunosuppression through tryptophan metabolism. IL-10 and IL-35 function as anti-inflammatory cytokines, creating an immunosuppressive environment. CD39 and CD73 also degrade ATP to produce adenosine, resulting in immunosuppressive effects. These genes play a particularly important role in tumor immune evasion and the creation of an immunosuppressive environment. Understanding the function of immunosuppressive genes and techniques for modulating their action are crucial for cancer immunotherapy, autoimmune disease treatment, and transplantation medicine. The development of therapeutic strategies targeting these genes will lay the foundation for new medical technologies based on immune regulation.

[0027] As used herein, "enhanced infiltration of Treg cells and / or suppressed infiltration, survival, and function of T cells" refers to a state in which regulatory T cells (Tregs) selectively infiltrate specific local environments, such as tumor microenvironments and inflammatory sites, resulting in enhanced immunosuppressive properties while hindering the infiltration of effector T cells (T cells) and inhibiting their survival and function.

[0028] Treg cells are responsible for negatively controlling immune responses, and their infiltration into the tumor microenvironment is enhanced by the induction of immunosuppressive chemokines (e.g., CCL22, CCL28). This infiltration strengthens immunosuppression in the local environment, preventing tumor cells from evading the immune system and slowing tumor progression. The proliferation of effector T cells (e.g., CD8+ T cells and Th1 cells) plays an important role in eliminating tumors and infected cells. However, suppression of their infiltration, survival, and function significantly reduces the immune response in the local environment. Suppression of T cell infiltration, survival, and function is often caused by the following factors: First, immunosuppressive factors (e.g., TGF-β and IL-10) secreted by tumor cells and Treg cells inhibit T cell migration and activity in the local environment. Second, metabolic conditions in the tumor microenvironment, such as low glucose and high lactate, limit T cell energy supply and impair their function. Furthermore, overexpression of immune checkpoint molecules, such as PD-L1 and CTLA-4, suppresses T cell proliferation and cytotoxic activity. A specific example is the selective infiltration of Treg cells into inflammatory sites and the tumor microenvironment, physically inhibiting the infiltration of effector T cells. Furthermore, even when T cells reach the tumor microenvironment, inhibitory signals from tumor cells and Treg cells induce apoptosis of T cells or significantly reduce their cytotoxic activity and cytokine secretion. This situation not only plays an important role in immune evasion and progression of tumors, but is also associated with the development of chronic inflammatory diseases and autoimmune diseases. By targeting these immunosuppressive mechanisms, the present invention provides a new strategy for cancer immunotherapy and the treatment of immune-related diseases.

[0029] As used herein, the term "factors that modify the tumor environment" refers to factors that affect the structure and function of the tumor microenvironment (TME) and contribute to the regulation of tumor progression and immune responses. These factors are often secreted by tumor cells, immune cells, fibroblasts, vascular endothelial cells, and the extracellular matrix (ECM), and play an important role in promoting tumor survival, growth, and metastasis. Factors that modify the tumor environment include internal infiltration factors, which promote the infiltration of Treg cells and macrophages into the tumor microenvironment. Specifically, the chemokines CCL22 and CCL28 induce the infiltration of Treg cells and enhance the immunosuppressive environment in tumors. Furthermore, colony-stimulating factor 1 (CSF-1) promotes macrophage infiltration and promotes tumor progression. Chemokines regulate the migration of tumor cells and immune cells and are important for modifying the tumor environment. For example, CXCL12 (SDF-1) promotes tumor cell proliferation and angiogenesis, while CXCL8 (IL-8) attracts neutrophils and macrophages to tumors, creating an inflammatory environment. These chemokines also promote tumor cell migration and metastasis. Furthermore, immunosuppressive cytokines are also important factors in modifying the tumor microenvironment. TGF-β and IL-10 suppress effector T cell activity while promoting the expansion of immunosuppressive cells such as Treg cells and M2 macrophages. This allows tumors to better evade immune attack. Metabolic factors also contribute to modifying the tumor microenvironment. Lactic acid, produced by tumor cells through aerobic glycolysis, creates a local acidic environment and reduces effector T cell function. In addition, hypoxic conditions increase the expression of HIF-1α (hypoxia-inducible factor 1α), promoting angiogenesis and tumor cell adaptation. VEGF (vascular endothelial growth factor) is another angiogenic factor. VEGF helps supply nutrients and oxygen to tumor cells by forming new blood vessels, but heterogeneous vascular structures result in the impediment of immune cell infiltration. Furthermore, matrix metalloproteinases (MMPs), which are extracellular matrix (ECM) remodeling factors, facilitate tumor cell invasion and metastasis through ECM degradation.Finally, exosomes and extracellular vesicles secreted by tumor cells and immune cells contain chemokines, cytokines, and microRNAs, which contribute to modifying the tumor environment. These factors interact to shape the tumor microenvironment, creating favorable conditions for tumor growth and progression. Furthermore, the development of therapeutics targeting these factors offers new possibilities for tumor treatment.

[0030] As used herein, the term "immune status of a subject" refers to the overall state of the subject's immune system at that time. This immune status includes the composition of immune cells, the degree of activation, the secretion pattern of cytokines, and the suppression or enhancement of immune responses. The immune status of a subject reflects the dynamic balance of the immune system, including immune function, immune response, and immune activation. The immune status of a subject includes not only normal immune function in healthy individuals but also abnormal immune function during disease progression or treatment. For example, in tumor patients, the immune status is characterized by the proportion of immunosuppressive cells (e.g., regulatory T cells (Tregs) and M2-type macrophages) in the tumor microenvironment and the degree of suppression of effector T cell function. On the other hand, in patients with autoimmune diseases, abnormal activation of autoreactive T cells and B cells is an important factor in the immune status. Specifically, the following factors can be used as indicators of the immune status: First, the composition of immune cells includes the proportion and distribution of lymphocytes, macrophages, neutrophils, monocytes, and dendritic cells in the blood and tissues, as well as the balance of T cell subsets (e.g., CD4+ T cells, CD8+ T cells, and Treg cells). Second, the activation state of immune cells is important, including the expression of cell surface markers (e.g., PD-1, CTLA-4), activation markers (e.g., CD69, HLA-DR), and effector functions (e.g., cytokine secretion, cytotoxic activity). Third, the secretion levels of inflammatory cytokines (e.g., IL-6, TNF-α, IFN-γ) and immunosuppressive cytokines (e.g., TGF-β, IL-10) are also important indicators for assessing immune status. Furthermore, the presence and activity of antibodies against specific pathogens or antigens, as well as the function of memory T cells and memory B cells, are also important indicators of immune memory. In tumor patients and chronic inflammatory conditions, the proportion and function of immunosuppressive cells (e.g., Treg cells, myeloid-derived suppressor cells (MDSCs)) are important factors in the immune status. These factors can be measured using methods such as blood and tissue sample analysis, flow cytometry, ELISA, real-time PCR, and immunofluorescence staining. Appropriate assessment of immune status plays a crucial role in understanding disease, developing treatment strategies, and confirming the effectiveness of novel therapies.

[0031] As used herein, "diagnosing a genetic mutation" means detecting, identifying, and analyzing the presence or absence of a genetic mutation, such as a change in base sequence, deletion, insertion, or duplication, in a specific gene of a subject (examinee). This diagnosis is performed for the purposes of risk assessment, progression monitoring, treatment selection, and prognosis prediction for diseases associated with genetic mutations. Gene mutations are diagnosed using a variety of methods. The first is sequence analysis. Next-generation sequencing (NGS) and Sanger sequencing directly analyze the base sequence of a target gene, enabling highly accurate identification of mutations. Polymerase chain reaction (PCR) is also commonly used to amplify specific gene regions and rapidly detect the presence or absence of mutations. Real-time PCR (qPCR) and allele-specific PCR can diagnose specific gene mutations with high sensitivity. Hybridization techniques, such as DNA microarrays and Southern blotting, detect mutations using probes complementary to specific gene sequences. This method is effective for comprehensively evaluating large-scale genomic mutations. A new diagnostic technique, the CRISPR-Cas system, can rapidly and accurately detect specific gene mutations. This method utilizes the target recognition capabilities of CRISPR and is highly accurate and efficient. In addition, genetic mutations can be indirectly diagnosed by measuring the expression and function of abnormal proteins caused by genetic mutations. These techniques include Western blotting and mass spectrometry. Diagnosing genetic mutations plays an extremely important role in the early detection of cancer, hereditary diseases, infectious diseases, and other diseases, as well as in determining treatment strategies. For example, diagnosing mutations in tumor-related genes such as TP53, KRAS, and PIK3CA in cancer makes it possible to evaluate tumor characteristics and sensitivity to therapeutic drugs. In addition, diagnosing mutations in BRCA1 and BRCA2 can predict the risk of developing breast cancer and ovarian cancer.

[0032] As used herein, immune status refers to the current activity and state of an individual's immune system. In the present invention, T cells with immune effector function are selectively provided according to this immune status. These T cells regulate immune responses and achieve specific therapeutic goals. Gene mutations refer to structural or functional changes in DNA sequences. The present invention provides factors that modify the local environment based on these gene mutations. These factors include internal infiltration, BATF (Basic Leucine Zipper ATF-like Transcription Factor), and chemokines, and play a role in regulating the local environment, such as the tumor environment and inflammatory sites.

[0033] As used herein, the term "nutritional environment compatible with FOXP3-induced metabolic patterns" refers to the nutritional conditions that are optimal for cells to adopt a specific metabolic pattern as a result of gene expression regulation by FOXP3 (Forkhead box P3). This nutritional environment plays an important role in the survival and function of regulatory T cells (Tregs) regulated by FOXP3, and examples of such nutritional environments include low glucose, high fatty acid, and high lactate conditions. FOXP3 is a transcription factor essential for Treg differentiation and function, and regulates multiple cellular processes, including energy metabolism. Tregs have metabolic properties distinct from those of normal immune cells, being less dependent on glycolysis and instead more dependent on fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). This allows them to secure energy supplies even in low glucose environments, while efficiently utilizing fatty acids as an energy source under high fatty acid conditions. Furthermore, high lactate conditions are known to promote Treg differentiation and suppressive function. Lactate is often abundant in tumor microenvironments and inflammatory sites as a by-product of cellular metabolism. In this environment, Tregs can utilize lactate for metabolism, maintaining their suppressive function without competing with other immune cells. Specifically, FOXP3 regulates the expression of metabolic genes (e.g., CPT1A, ACADL) to promote fatty acid oxidation. Furthermore, by suppressing the mTOR pathway, activation of glycolysis is suppressed, shifting metabolism to fatty acid oxidation. This metabolic adaptation allows Tregs to secure a sustainable energy supply and exert their immunosuppressive function even under nutrient deficiency. A "nutritional environment compatible with the FOXP3-induced metabolic pattern" is crucial for the pathogenesis of tumors and inflammatory diseases. In particular, the characteristic metabolic state of low glucose and high lactate in the tumor microenvironment enhances the dominance of Tregs and contributes to the formation of an immunosuppressive environment. These findings provide important guidance for the development of novel therapeutic strategies targeting the metabolic properties of Tregs.

[0034] As used herein, "invasion into the local environment" refers to the physical penetration of specific cells into tissues or microenvironments. This "invasion" refers to the process by which cells penetrate or degrade the surrounding intercellular matrix or tissue structure to reach a specific local environment (e.g., an inflammatory site, tumor tissue, or injury site). Cell motility, adhesion, and matrix-degrading ability are key factors in invasion. For example, enzymes that degrade the extracellular matrix (e.g., matrix metalloproteinases (MMPs)) play an important role in tumor cell invasion. Furthermore, when immune cells infiltrate an inflammatory site, chemokines and cytokines induce migration, and matrix-degrading enzymes enable their infiltration into local tissues. Methods for regulating "invasion into the local environment" include the following: First, regulating the expression of extracellular matrix-degrading enzymes can enhance or suppress invasive ability. Second, controlling the expression and activity of chemokine receptors can enhance the efficiency of specific cells invading the target local environment. Third, genetic modification to enhance cell motility and methods to regulate the expression of adhesion molecules are used. Thus, "local invasion" is a concept that plays an important role in many physiological and pathological processes, such as tumor progression and metastasis, induction of immune responses, and promotion of tissue repair. Understanding and modulating its mechanisms is important for developing therapeutic strategies.

[0035] As used herein, "survival in a local environment" refers to the ability of a particular cell to survive under stressful or adverse conditions in a particular local environment (e.g., an inflammatory site, a tumor microenvironment, tissue under hypoxic conditions, an injured site, etc.). This "survival" refers to the ability of a cell to continue to function as it is. Local survival is an essential element for maintaining a stable immune system and contributing to physiological or pathological processes while adapting to the environment. Factors that contribute to survival in the local environment include ensuring nutrient supply, regulating the oxidative stress response, increasing the expression of anti-apoptotic molecules, and the action of immunosuppressive factors. For example, tumor cells survive under unfavorable conditions such as hypoxia and nutrient deprivation by upregulating the expression of hypoxia-inducing factor (HIF-1α). Furthermore, immune cells, under the influence of local cytokines, evade the apoptotic pathway and contribute to inflammation and repair. Methods for promoting or regulating "survival in the local environment" include the following: First, enhancing the expression of anti-apoptotic molecules (e.g., Bcl-2 family proteins) to avoid cell death. Second, modulating the expression of antioxidant factors and related transcription factors (e.g., Nrf2) to control the oxidative stress response. Third, techniques for supporting survival in the local environment include modifying molecules involved in the extracellular matrix and nutrient supply. Thus, "survival in the local environment" is a central element in physiological and pathological processes such as tumor progression, immune responses, and tissue repair, and elucidating and controlling its mechanisms is of great significance for the development of novel therapeutic approaches.

[0036] As used herein, "(immune) effector cells" or "(immune) effector T cells" refer to immune cells or T cells that exert cytotoxic effects on target cells. Whether a cell is an "effector" can be determined by methods such as cytotoxicity assays, surface antigen analysis by flow cytometry, and intracellular cytokine staining. Effector cells are immune cells that perform effector functions, such as mediating antibody-dependent cellular cytotoxicity (ADCC). Effector 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.

[0037] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered receptor that can confer antigen specificity to a cell (e.g., an immune cell). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. Preferably, the CAR of the present disclosure comprises at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0038] As used herein, "Foxp3," "FOXP3," and "foxp3" (also referred to as Scurfin) are used interchangeably and refer to gene names such as NP_001107849 (protein) or NM_001114377 (mRNA), regardless of species. They are transcription factors that control immune responses and are primarily expressed in regulatory T cells (Tregs). In this specification, control sequences for mutants and the like are designated NM_014009.4 (mRNA) and NP_054728.2 (protein) (SEQ ID NO: 27). Foxp3 possesses a forkhead DNA-binding domain and plays a role in regulating the differentiation, maintenance, and function of Tregs. It plays an important role in maintaining immune homeostasis and suppressing autoimmune diseases, and its abnormal expression is associated with autoimmune diseases, allergic diseases, or tumor progression. As used herein, Foxp3 refers to the protein encoded by the human FOXP3 gene, as well as proteins synthesized based on a sequence identical or substantially identical to the nucleotide sequence, and also encompasses its physiologically active derivatives and mutants. When it is necessary to clearly distinguish between species, it will be referred to as mouse Foxp3 or human FOXP3.

[0039] As used herein, a "variant" of a gene (protein or nucleic acid molecule) refers to a protein or nucleic acid molecule derived from that gene (protein or nucleic acid molecule) that contains one or more amino acid or nucleotide substitutions, deletions, insertions, or additions, provided that these changes maintain some or all of the biological function of the FOXP3 protein.

[0040] The case of FOXP3 will be explained. FOXP3 is a transcription factor that plays an important role in the expression and function of regulatory T cells (Tregs). The "FOXP3 mutant" referred to herein includes, but is not limited to, the following specific modifications: 1) amino acid substitution: for example, a natural mutation (e.g., Phe367Ser) or an artificial mutation (e.g., Arg48Ala); 2) amino acid deletion: for example, a deletion of 1 to 10 amino acids in the Exon 7 region; 3) amino acid insertion: for example, an insertion of 1 to 5 amino acids in the Forkhead domain; and 4) modification: for example, the addition of a phosphorylation site or a glycosylation modification, or any combination thereof. Examples of such modifications include the following: Examples include Phe367Ser mutant: an example in which Phe367 is substituted with Ser, enhancing the suppressive ability of regulatory T cells; Exon 2 deletion mutant: an example in which the Exon 2 region is deleted, resulting in reduced Treg expression but partial maintenance of regulatory immune responses; and Arg48Ala mutant: a mutant in which Arg48 in the Forkhead domain is substituted with Ala, resulting in reduced DNA binding ability. It should be understood that the scope of the present specification regarding "FOXP3 mutants" is intended to clearly identify the subject matter of the invention through specific examples of changes in amino acid sequence and descriptions based on their biological functions. Furthermore, by providing specific examples of the biological functions of the mutants, it is understood that those skilled in the art can easily carry out the invention.

[0041] As used herein, a "portion" of a gene (protein or nucleic acid molecule) refers to a partial element or attribute that constitutes the whole of the target function or structure, and includes a partial structure within the scope necessary to achieve a specific function or characteristic. A "portion" may also be defined by the length of a specific amino acid sequence, ranging from 10 amino acids to the full length (e.g., 431 amino acids, the entire length of FOXP3). Specifically, sequences containing 20, 50, 100, 200, or 300 amino acids, or the full length, in increments of 10 amino acids, are considered. The term "portion" is used to clarify elements relevant to the scope of the invention from a structural or functional perspective. As used herein, the term "portion" refers to, for example, the following: functional portion: a portion of the FOXP3 protein that is responsible for a specific functional property, such as DNA binding ability, transcriptional repression ability, or nuclear transport ability; structural portion: a specific region of the FOXP3 protein, such as the Forkhead domain, Exon 2 region, or transcription start site; and sequence portion: a specific amino acid sequence or motif (e.g., the sequence around Phe367).

[0042] In an exemplary embodiment, examples of portions of a gene (protein or nucleic acid molecule) include a 10-amino acid portion: a 10-amino acid sequence within the Forkhead domain of the FOXP3 protein; a 20-amino acid portion: a 20-amino acid sequence contained in the Exon 2 region; a 50-amino acid portion: a region contributing to the transcriptional repression ability of FOXP3; a 100-amino acid portion: a range including the entire Forkhead domain of the FOXP3 protein; a 200-amino acid portion: a sequence including the entire Exon 7 region; and a full-length (396 amino acids in humans): the entire FOXP3 protein. The definition and description of "portion" herein are provided to avoid ambiguity in the scope of the invention and to enable those skilled in the art to easily understand and implement the invention based on specific examples and literature. Furthermore, the meaning of "portion" is clearly stated structurally, functionally, and sequentially, thereby enabling the subject matter of the invention to be identified.

[0043] As used herein, "metabolic capacity" refers to the ability to consume energy to maintain and / or activate T cell function. Each T cell activates a subtype-specific metabolic mechanism, and impairment of this mechanism leads to a decrease in the survival rate and function of T cells. For example, the metabolic capacity of peripheral blood CD4+ and CD8+ effector T cells (T eff In tumors, glycolysis is promoted by mTOR signaling, whereas in regulatory T cells (Tregs), AMPK signaling promotes fatty acid oxidation followed by mitochondrial oxidative phosphorylation. The tumor microenvironment is low glucose and hypoxic, and T cells infiltrating the tumor site are prone to hyperglycemia. T cells are also in this environment. Therefore, the metabolic capacity of T cells can be defined as their ability to adapt to the environment using various molecular mechanisms. Metabolic capacity can be assessed by methods such as flux analyzer analysis, glucose uptake analysis (2-NBDG uptake assay), and expression analysis of metabolic factors by flow cytometry or RNA sequencing.

[0044] As used herein, the terms "negative regulator of (immune) effector function" or "negative regulator" are used interchangeably and refer to molecules, genes, or proteins that play a role in suppressing, inhibiting, or attenuating specific immune effector functions (e.g., immune response, signal transduction, cell proliferation, or metabolic activity) in cells or tissues. These factors may take the form of transcription factors, signal molecules, enzymes, or non-coding RNAs.

[0045] As used herein, the term "local environment" refers to the environment in which target cells reside, and refers to the surrounding environment of target cells, a complex environment that includes physical, chemical, and biological factors. When target cells are tumor cells, it is sometimes referred to as the tumor environment or tumor microenvironment. The characteristics of the "local environment" vary depending on the type of target cell. For example, when target cells are tumor cells, the local environment is referred to as the tumor environment or tumor microenvironment. The tumor microenvironment includes stromal cells, immune cells, vascular endothelial cells, and extracellular matrix surrounding tumor cells. Oxygen concentration, pH, nutritional status, and secreted factors (e.g., cytokines and chemokines) are also important factors that characterize the tumor microenvironment. The tumor microenvironment has a significant impact on tumor progression, metastasis, and therapeutic response. For example, the presence of immunosuppressive cell groups (e.g., regulatory T cells and tumor-associated macrophages) in the tumor microenvironment suppresses the immune response against tumors. Meanwhile, even in normal tissues, the local environment is involved in maintaining tissue homeostasis, regeneration, and pathological changes. Thus, the term "local environment" as used herein is to be interpreted as including the surrounding conditions and factors in which the target cells reside and which influence their biological behavior.

[0046] As used herein, "inhibitory function" refers to the action of attenuating, inhibiting, or stopping a specific physiological or biochemical process or the inherent function of a cell or tissue, and a factor with such a function is called an "inhibitory factor." This "inhibitory function" is achieved by a factor, structure, or pathway that functions at the molecular, cellular, or system level. For example, immunosuppressive function refers to the action of reducing the activation, proliferation, or production of effector molecules of immune cells. Furthermore, signal transduction inhibitory function can refer to the function of suppressing the transmission of signal molecules or the activity of receptors.

[0047] As used herein, specific examples of molecules with "inhibitory function" include inhibitory cytokines (e.g., IL-10, TGF-β), inhibitory receptors (e.g., PD-1, CTLA-4), or genes or regulatory factors that control their expression. Furthermore, factors with "inhibitory function" may act as non-coding RNA or specific epigenetic modifiers. In the present invention, manipulation of factors with "inhibitory function" may reactivate or enhance the function of specific cells or tissues.

[0048] As used herein, "abandonment of inhibitory function" refers to the reduction or elimination of the inhibitory effect on a specific physiological or biochemical process (hereinafter referred to as "inhibitory function"), and is used herein to mean the same as "reduction or elimination" of "inhibitory function." This "abandonment of inhibitory function" or "reduction or elimination of inhibitory function" occurs at the molecular, cellular, or tissue level and refers to a state in which the function of the target inhibitor is completely undetectable.

[0049] The mechanism that causes the "abandonment of inhibitory function" or "decrease or disappearance of inhibitory function" of inhibitors The methods include: Gene knockout: The gene encoding the targeted inhibitor is disrupted, completely stopping production of the factor. RNA interference (RNAi): Inhibition of factor expression by degrading or silencing the mRNA of the inhibitor. Antibody neutralization: Antibodies that specifically bind to inhibitors inhibit their activity. Small molecule inhibitor: A molecule that acts at the active site of an inhibitor, causing it to lose its function. "Abandonment of inhibitory function" refers to the complete loss of the inhibitory function, rather than simply a reduction in the inhibitory function. For example, in the case of an immunosuppressive factor, it refers to the complete inactivation of the factor, thereby releasing the suppression of the immune response. In the case of a signal transduction inhibitor, it refers to the complete removal of the inhibitory effect on the signal pathway, allowing the signal to be transmitted normally.

[0050] Specific examples of factors with inhibitory functions include inhibitory receptors such as PD-1 and CTLA-4, and inhibitory cytokines such as IL-10 and TGF-β. Abolishing the inhibitory functions of these factors is expected to result in, for example, activation of immune responses and improvement of certain disease states.

[0051] It should be noted that the definitions of "abandonment of inhibitory function" and "decrease or disappearance of inhibitory function" in this specification are not intended to limit the scope of the claims, but are intended to illustrate specific examples of the invention.

[0052] As used herein, the term "negative regulator" refers to a factor that negatively regulates immune effector functions induced by Foxp3. These factors play a role in suppressing or regulating immune responses, particularly in the immunosuppressive mechanism mediated by regulatory T cells (Tregs). Negative regulators prevent excessive immune responses and autoimmune reactions by suppressing cytokine production, cell activation, and the expression of effector functions. This includes molecules whose expression is directly or indirectly regulated by Foxp3, including those that function as secreted factors, cell surface molecules, and signaling molecules. Negative regulators can contribute to immunosuppression in the tumor microenvironment and may contribute to tumor progression and resistance to immunotherapy. On the other hand, these factors may also be useful target molecules from the perspectives of transplantation immunology and the treatment of autoimmune diseases. As used herein, the term "negative regulator" refers to a general group of molecules that negatively regulate immune effector functions, encompassing their physiological and pathological roles. Negative regulatory factors include Treg cell-like immunosuppressive factors, for example, inhibitory cytokines such as TGFβ and IL-10, and inhibitory molecules such as CTLA-4.

[0053] Furthermore, "abolishment of expression of a negative regulatory factor (a factor that negatively regulates FOXP3-induced immune effector function)" or "reduced or eliminated expression of a negative regulatory factor" refers to a state in which the transcription or translation, or function and / or expression, of the factor is reduced or eliminated, resulting in a state in which the factor is substantially reduced or substantially undetectable in a cell or tissue. Also, "reduced expression of a negative regulatory factor" or "reduced or eliminated expression of a negative regulatory factor" means that the transcription, translation, or functional expression level of the factor is significantly reduced compared to the normal or baseline state.

[0054] As used herein, specific examples of "factors that negatively regulate (immune) effector functions" include immunosuppressive cytokines (e.g., IL-10, TGF-β), inhibitory receptors (e.g., PD-1, CTLA-4), or genes that regulate their expression, as well as IL-2 receptor (CD25), adenosine-related ectoenzymes (e.g., CD39, CD73), metabolic enzymes (IDO), etc. In addition, regulatory factors that regulate the expression of the relevant factors (e.g., specific microRNAs and transcription factors) are also included. Molecules of interest in the present invention Examples of these include inhibitory molecules / receptors, cytokines, and other molecules such as: 1. Inhibitory molecules and receptors Examples of inhibitory molecules or receptors include, but are not limited to, the following: CTLA-4, OX40, GITR, TGFβ receptor (TGFβR), IDO, CD25 (IL-2 receptor α chain), ICOS, CD80 (B7-1), CD86 (B7-2). 2. Cytokines Examples of cytokines include, but are not limited to, the following: TGF-β, IL-10, and IL-35. 3. Other molecules Examples of other molecules include, but are not limited to, the following: CD39, CD73. Negative regulators of the tumor microenvironment also include: IDO (indoleamine 2,3-dioxygenase): A tryptophan-metabolizing enzyme that contributes to the induction of Tregs and the suppression of effector T cells. Galectin-9: Expressed in tumor environments and induces apoptosis of T cells. These molecules may play important roles in regulating immune responses, creating an immunosuppressive environment, or in diseases, and are applicable as subjects of the present invention.

[0055] "Abandoning" or "reducing" the expression of these factors can be achieved by methods such as gene knockout, RNA interference (RNAi), CRISPR / Cas9 technology, neutralization with antibodies, or application of small molecule inhibitors.

[0056] As used herein, "immune effector function" means that when a function is suppressed by a negative regulatory factor, the effector function is expected to be activated, promoted, or restored by reducing or eliminating the expression of the factor. These definitions in this specification are merely examples and do not limit the interpretation of the claims.

[0057] As used herein, "altered metabolic capacity" refers to a state or structure in which metabolic capacity has been altered by any means, meaning that at least a partial or complete loss of function or an enhancement of function is observed. It is understood that the modified cells of the present disclosure may also have altered target capabilities and thus constitute part of the present disclosure. This refers to an altered state or structure of the molecules or cells of the present invention. The metabolic capacity of T cells can be modified in many ways, including chemically, structurally, and functionally. The metabolic capacity of T cells can also be modified by introducing nucleic acids. T cells with altered metabolic capacity can exhibit enhanced metabolic capacity, for example, in a low-glucose environment (e.g., a tumor microenvironment). Therefore, T cells with altered metabolic capacity can 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.

[0058] As used herein, the term "immunosuppressive gene" refers to a gene that expresses a molecule that suppresses the function of the immune system, and is capable of directly or indirectly suppressing the immune system or downregulating the immune system, and is a negative regulatory factor (negative regulatory factor). Examples of immunosuppressive genes include CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73.

[0059] As used herein, unless otherwise specified, the term "substantially" means that the value may vary within any amount that contributes to measurement errors that may occur in the embodiment. Therefore, "substantially eliminated" means, for example, that the expression has been eliminated to the extent that the effects of the present disclosure are exerted.

[0060] As used herein, "modification" in relation to genes (nucleic acids, proteins, etc.) refers to inserting a base sequence into intracellular DNA, deleting a portion of the base sequence of intracellular DNA, or a combination thereof.

[0061] As used herein, "modifying to express" Foxp3 or the like refers to modifying a cell marker that was not substantially expressed (usually below the detection limit) so that its expression is observed (or is present to a functional extent). As used herein, "enhanced" expression of Foxp3 or other markers refers to increasing the expression level of such cellular markers, preferably modifying their expression so as to enhance their function. This can be achieved by introducing the FoxP3 gene into the exogenous gene or by using the CRISPR-dCas Activation system.

[0062] As used herein, the term "T cells" refers to lymphocytes produced in the bone marrow that migrate to the thymus and mature. T cells can be CD45-positive and CD3-positive cells among the normal fraction of peripheral blood and bone marrow-derived mononuclear cells.

[0063] (Preferred embodiment) Preferred embodiments of the present disclosure are 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.

[0064] (FOXP3-T cells) In one aspect, the present disclosure provides T cells (FOXP3-T cells) that have been modified to express Foxp3, a mutant thereof, or a portion of either thereof, and / or have enhanced Foxp3 expression and immune effector function, as well as pharmaceutical compositions comprising the same and other related technologies.

[0065] In one aspect of the present disclosure, T cells (FOXP3-T cells) that have been modified to express Foxp3, its mutants, or portions thereof, and / or have enhanced Foxp3 expression and immune effector function, have been established. It was surprising that, unlike conventional predictions, the introduction of Foxp3, a master regulator of regulatory T cells (Tregs), can induce Treg-like chemokine expression patterns and metabolic patterns while maintaining the cytotoxic activity of Tregs as an immune effector, rather than as suppressor cells. Furthermore, the present disclosure is notable in that it establishes effector T cells with enhanced antitumor activity by suppressing the expression of T cell suppressor factors, a by-product of T cell development.

[0066] In one embodiment, the present disclosure provides T cells (FOXP3-T cells) that (exogenously) express Foxp3, a mutant thereof, or a portion of either thereof, or that have enhanced (endogenous) Foxp3 expression and have immune effector function.

[0067] The FOXP3-T cells of the present disclosure are capable of inhibiting FOXP3-induced immune effector functions. The gene is modified so that the function and / or expression of a factor that negatively regulates the gene (also referred to as a "negative regulatory factor") is reduced or eliminated.

[0068] In one embodiment, negative regulatory factors may include, but are not limited to, Treg cell-like immunosuppressive factors, for example, inhibitory cytokines such as TGFβ and IL-10, and inhibitory molecules such as CTLA-4.

[0069] The Foxp3 used in the present disclosure is modified to confer effector function to the Foxp3-T cells, or to maintain or enhance the effector function of the T cells.

[0070] Foxp3 as used in the present disclosure is a protein that is expressed in T cells such that the expression and / or function of a factor that negatively regulates immune effector function (negative regulatory factor) is reduced or eliminated. The T cells are modified to confer effector function or to maintain or enhance the effector function of the T cells.

[0071] In one embodiment, the Foxp3-T cells of the present disclosure are metabolically altered cells.

[0072] In one embodiment, the Foxp3-T cells of the present disclosure are cells with altered chemokine receptor expression and / or altered migration, infiltration, and survival in the local environment.

[0073] As used herein, the local environment refers to the environment in which the target cells exist, and when the target cells are tumor cells, it may be referred to as the tumor environment or tumor microenvironment.

[0074] In one specific embodiment, the present disclosure provides a T cell (FOXP3-T cell) that has immune effector function and has been modified to express Foxp3, a mutant thereof, or any portion thereof, and / or has enhanced expression of Foxp3, wherein the FOXP3-T cell has been modified to reduce or eliminate the suppressive function of the negative regulator.

[0075] In one embodiment, the suppression function of the negative regulatory factor used in the present disclosure is reduced or eliminated, resulting in the maintenance or enhancement of immune effector function.

[0076] In one embodiment, FOXP3 T cells exhibit increased expression of chemokine receptors associated with migration, invasion, and survival in the local environment, including, but not limited to, CCR4 and CCR8.

[0077] In another embodiment, the present disclosure provides FOXP3-T cells that satisfy at least one predetermined condition. While FOXP3-T cells are effective against any tumor, they are particularly effective against tumor environments that promote Treg cell infiltration and / or suppress T cell infiltration, survival, and function. These conditions occur when the patient's tumor environment is rich in ligands (e.g., CCL17, CCL22, CCL1) for the FOXP3-induced chemokine receptors (CCR4 and CCR8) and / or has a nutritional environment compatible with the metabolic pattern induced by FOXP3 (e.g., low glucose, high fatty acid, high lactate). To diagnose the above, the presence of abundant Treg cell infiltration on histopathology and / or genetic mutations in the tumor that can induce the above tumor environment (such as EGFR mutations, ROHA mutations, or increased expression of MHC class II and self-molecules) are effective biomarkers, and can be identified by tumor biopsy, gene panel testing using peripheral blood liquid biopsy, or next-generation sequencing.

[0078] In one embodiment, the FOXP3-T cells of the present disclosure comprise an exogenously inserted antigen receptor, such as a chimeric antigen receptor (CAR).

[0079] In one embodiment, a CAR of the present disclosure is expressed in a FOXP3- T cell of the present disclosure.

[0080] In one embodiment, the FOXP3-T cells of the present disclosure are cells in which the expression of at least one immunosuppressive gene is reduced or substantially eliminated.

[0081] In one embodiment, the immunosuppressive gene used may be selected from CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73.

[0082] In one embodiment, the cells of the present disclosure are human effector T cells.

[0083] (Application of FOXP3-T cells) In another aspect, the present disclosure provides a method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) diagnosing the local environment of the subject (e.g., determining whether Treg cell infiltration is enhanced and / or T cell infiltration, survival, or function is suppressed), where the diagnosis is achieved by determining the Treg status or genetic mutations of environmental factors of target cells in the subject (e.g., histopathologically finding abundant Treg cell infiltration and / or target cells having genetic mutations that can induce the tumor environment (e.g., EGFR mutation, ROHA mutation)); and C) providing T cells with immune effector function and / or factors that modify the tumor environment (e.g., internal infiltration, chemokines, etc.) based on the local environment. This method may employ any or a combination of the features described in (FOXP3-T cells).

[0084] In another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: A method for improving the immune status of a subject is provided, comprising the steps of: A) diagnosing the immune status of the subject; B) diagnosing a genetic mutation in the subject; and C) providing T cells with immune effector function corresponding to the immune status based on the immune status and the genetic mutation, and providing a factor that modifies the local environment (e.g., internal infiltration, BATF, chemokines, etc.) based on the genetic mutation. This method may employ any or a combination of the features described in (FOXP3-T cells).

[0085] In another aspect, the present disclosure provides a pharmaceutical composition comprising a T cell having immune effector function, the T cell having been modified to express Foxp3, a mutant thereof, or a part thereof, and / or having enhanced Foxp3 expression. The pharmaceutical composition may include any of the features described above in (FOXP3-T cells), or a combination thereof.

[0086] In another aspect, the present disclosure provides a method for treating or preventing a disease associated with an abnormal immune status, comprising administering to a subject in need thereof an effective amount of T cells having immune effector function, wherein the T cells have been modified to express Foxp3, a mutant thereof, or a portion of either thereof, and / or have enhanced expression of Foxp3. Embodiments of this method include any of the characteristics or A combination thereof may be employed.

[0087] In another aspect, the present disclosure provides a method for producing effector T cells (T eff (FOXP3-T cells) is a pharmaceutical composition comprising the T cells modified to express Foxp3 and / or to enhance Foxp3 expression. This composition may have any of the features or combinations thereof described in (FOXP3-T cells).

[0088] In one embodiment, the T cells of the present disclosure are metabolically altered cells.

[0089] In one embodiment, the T cells of this disclosure comprise a chimeric antigen receptor (CAR).

[0090] In one embodiment, the CAR of the present disclosure is expressed in the T cell.

[0091] In one embodiment, the expression of at least one immunosuppressive gene of the present disclosure is reduced or substantially eliminated.

[0092] In one embodiment, the immunosuppressive gene of the present disclosure includes at least one selected from the group consisting of CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73.

[0093] In one embodiment, the T cells of this disclosure are human effector T cells.

[0094] In one embodiment, the cells of the present disclosure advantageously have an altered metabolic capacity.

[0095] <Metabolic activity modification> One advantageous embodiment will be described. Cancer cells reprogram their metabolic pathways to favor their own cell proliferation, actively utilizing glycolysis, which is inefficient in producing ATP even in the presence of oxygen, and enhancing glucose uptake and lactate production (Warburg effect). Therefore, cancer cells consume and deplete a large amount of glucose, and T cells, which require glucose as an energy source, are depleted. eff When tumor-specific T cells of this type infiltrate tumors, they receive signals via TCR, but the cells' Ca 2+ Concentrations drop, leading to starvation and exhaustion.

[0096] Thus, in one embodiment of the present disclosure, FOXP3- T cells are provided that are genetically engineered to alter metabolic capacity to promote aerobic glycolysis and enhance immune cell competitiveness and / or fitness in a glucose-depleted tumor microenvironment (TME).

[0097] In one embodiment, altering metabolic capacity can be achieved by modifying the cell to express Foxp3.

[0098] Regulatory T cells (Tregs) are involved in immunosuppression and are also important cells in tumor immunity. Tregs express the master gene Foxp3, which activates the mitochondrial electron transport chain, enhancing mitochondrial function and shifting Treg metabolism towards OXPHOS and FAO. Furthermore, Foxp3 suppresses the expression of c-Myc and inhibits glycolysis. Conversely, when the glycolysis of Tregs is enhanced, Foxp3 expression decreases depending on the activation of the mTOR pathway, and the number of Foxp3 cells in the tumor also decreases. In this way, there is a close relationship between metabolism and the master transcription factor Foxp3. Furthermore, the induction of OXPHOS by Foxp3 increases the ratio of NAD+ / NADH in the cell, converting lactate to pyruvate, and promoting the localization of tumors. Under high lactate conditions, Tregs are protected from the inhibitory effects of lactate. Thus, Tregs adapt to local environments such as the tumor microenvironment by suppressing glycolysis and enhancing OXPHOS.

[0099] In one embodiment of the present disclosure, such metabolic reprogramming function by Foxp3 can be utilized to utilize the metabolic machinery of Treg cells in the FOXP3-T cells of the present disclosure.

[0100] While normal effector T cells are dependent on glucose metabolism, as disclosed herein, T eff When modified to express Foxp3 and / or enhance Foxp3 expression in tumor cells, metabolic function is altered, allowing them to take in, metabolize, and use lactate and fatty acids in addition to glucose as a nutrient source. Furthermore, a chemokine receptor expression profile that can respond to chemokines abundant in the tumor environment can be induced. Therefore, the FOXP3-T cells (T eff ) can be modified to express Foxp3 and / or enhance Foxp3 expression, thereby gaining the advantage of being able to infiltrate into T cells and maintain antitumor activity in the metabolic environment of tumors.

[0101] In representative embodiments, the FOXP3-T cells of the present disclosure are T cells that play a central role in the immune response, playing a key role in eliminating pathogens, suppressing tumor cells, or modulating the immune system. These cells can attack and regulate specific targets through cytokine production or direct cytotoxicity.

[0102] In a representative embodiment, the FOXP3-T cells of the present disclosure are a type of T cell with effector function, and typically have the following characteristics: FOXP3 expression: These cells have been genetically modified to express or enhance the expression of the FOXP3 (Forkhead box P3) transcription factor, which is typically known as a marker for regulatory T cells (Tregs) and plays an immunosuppressive role. Purpose of modification: To allow T cells with immune effector function to express FOXP3 (FOXP3-T cells of the present disclosure), while suppressing conventional T eff This gives the cells the ability to fine-tune the immune response while suppressing their pro-inflammatory and excessive immune responses. Characterization and engineering of FOXP3-T cells Modification of FOXP3 expression: These cells are created by introducing an exogenous FOXP3 gene or enhancing endogenous expression of FOXP3. This modification can be achieved using gene editing techniques (e.g., CRISPR / Cas9) or viral vectors (e.g., retroviruses, lentiviruses). Expression of mutants or parts: In addition to the full-length FOXP3 protein, parts (e.g., functional domains) or mutants may be expressed, allowing for the emphasis on specific functions. Immunological properties of FOXP3-T cells Immunosuppressive function: FOXP3 expression allows these cells to suppress conventional T eff Unlike cells, they may have the function of suppressing excessive inflammatory responses. Differentiation flexibility: By expressing FOXP3, FOXP3-T cells retain some properties similar to conventional Treg cells, but also possess the T effIt is possible to retain cell-specific effector functions. FOXP3-T cells can be applied to a variety of medical fields, including: Autoimmune diseases: Suppressing excessive immune responses may have the potential to treat autoimmune diseases (e.g., rheumatoid arthritis, type 1 diabetes, multiple sclerosis). Transplant medicine: Used to prevent rejection in organ transplants and hematopoietic stem cell transplants. Cancer immunotherapy: conventional T eff Preventing excessive inflammatory responses while maintaining the anti-tumor activity of cells This will improve the safety and efficacy of immunotherapy. Chronic inflammatory diseases: Potential to suppress pathogenic immune responses in diseases such as Crohn's disease and ulcerative colitis.

[0103] In a representative embodiment of the present disclosure, FOXP3- T cells can be generated and evaluated by the following steps: Gene transfer: The FOXP3 gene is introduced into T cells. Selection and expansion: Cells with confirmed FOXP3 expression are selected and expanded as needed. Functional assessment: Immunosuppressive effects and retention of effector functions are assessed using indicators of cytokine production and cytotoxicity. Caution: While FOXP3 T cells have immunoregulatory functions, if not properly controlled, they may cause excessive immunosuppression and increase the risk of infection and tumors. Therefore, careful evaluation of safety and efficacy is required.

[0104] In one embodiment of the present disclosure, the FOXP3-T cells (T eff ) can have reduced or substantially absent expression of at least one immunosuppressive gene. As described above, effector T cells (T eff ) to express Foxp3 and / or enhance Foxp3 expression, thereby enhancing effector T cells (T eff) can utilize the metabolic mechanism of Treg cells. On the other hand, the function of T cells may also become similar to that of Tregs, and the effector function may also change. Therefore, in one embodiment of the present disclosure, in order to maintain the effector function while not exerting the immunosuppressive function of Treg cells, effector T cells (T eff In another embodiment, the FOXP3-T cells (T eff ), effector function can also be maintained by reducing or substantially eliminating the expression of immunosuppressive genes.

[0105] In one embodiment, the immunosuppressive gene is not particularly limited as long as it expresses a molecule that functions as an inhibitor in the immune system, and examples include CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73.

[0106] In one embodiment of the present disclosure, the FOXP3-T cells of the present disclosure can be human effector T cells.

[0107] <Chimeric Antigen Receptor (CAR)> In another aspect of the present disclosure, T cells are provided in which the FOXP3-T cells of the present disclosure are modified to contain a chimeric antigen receptor (CAR). The CAR contained in the FOXP3-T cells of the present disclosure may be contained as a protein, or may contain a nucleic acid molecule expressing the CAR, as long as it can function as a CAR. In one embodiment, the CAR of the present disclosure can be expressed in effector T cells.

[0108] 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.

[0109] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides that contain 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. AR is characterized by its ability to redirect T cell specificity and reactivity toward selected targets by utilizing the antigen-binding properties of monoclonal antibodies, independent of MHC. MHC-independent antigen recognition can empower CAR-expressing T cells to recognize antigens independently of antigen processing, allowing tumors to avoid immune escape.

[0110] 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 a 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 the efficient response of lymphocytes to antigens.

[0111] (extracellular domain) In one embodiment, the CAR used in the FOXP3-T cell of the present disclosure comprises an antigen-binding domain or a portion thereof.The antigen-binding domain or a portion thereof can be appropriately selected according to the type and number of ligands on the surface of target cells.For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a specific disease state.Therefore, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR of the present disclosure can include tissue-specific markers, tumor-specific markers, those associated with virus, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0112] 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 Fab (not from an antibody, but obtained, for example, from a Fab library) can be used. The scFv can be fused to a transmembrane domain and then to an intracellular signaling domain.

[0113] In one embodiment, the antigen-binding domain portion of the CAR of the present disclosure comprises: (1) an MHC The antigen-binding domain portion of the CAR of the present disclosure can target antigens including, but not limited 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) targeting molecules including CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin), and MadCam1 (mucosal vascular addressin cell adhesion molecule 1).

[0114] 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 to 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 A-10, MAGE-C2, MAGE-A12, CEA, tyrosinase, myokinase-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, ART1, 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, MelanA, NY-BR-1, NY-CO-58, MN(gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the effector cells disclosed herein are engineered to express one or more CARs to recognize one or more antigens.

[0115] (transmembrane domain) The CAR used in the FOXP3-T cells of the present disclosure can comprise one or more transmembrane domains fused to an extracellular domain.

[0116] In one embodiment, a linker domain derived from the extracellular domain can be connected to the transmembrane domain.The transmembrane domain can be natural or synthetic, and the natural transmembrane domain can be derived from any membrane-bound or transmembrane protein.The transmembrane region particularly used in the present disclosure can be derived from the alpha, beta or zeta chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0117] In one embodiment, the CAR used in the inducible regulatory T cells 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.

[0118] (intracellular domain) The cytoplasmic signaling domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the 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 perform 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.

[0119] In one embodiment, examples of intracellular signaling domains used in CARs include the cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that initiate signal transduction following antigen receptor binding.

[0120] (Alloantigens, allergens and haptens associated with rejection) CARs used in the effector T cells disclosed herein can include those associated with alloantigens, allergens, and haptens associated with rejection reactions.

[0121] <Medicinal uses of FOXP3-T cells containing chimeric antigen receptors (CARs)> In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising a FOXP3-T cell of the present disclosure, wherein the T cell has been modified to express Foxp3 and / or has enhanced Foxp3 expression and enhanced immune effector function. In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising a FOXP3-T cell of the present disclosure, wherein the T cell comprises a chimeric antigen receptor (CAR), and the T cell has been modified to express Foxp3 and / or has enhanced Foxp3 expression and enhanced immune effector function. In one embodiment of the present disclosure, the FOXP3-T cell of the present disclosure can have one or more characteristics of the FOXP3-T cell of the present disclosure described above.

[0122] Also provided in one aspect is a therapeutic agent comprising a FOXP3-T cell or cell population of the present disclosure, which diagnoses a subject for a disease and selects an appropriate CAR for inclusion in the effector T cell or cell population based on the diagnosis.

[0123] In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the FOXP3-T cells of the present disclosure described herein and a pharmaceutically acceptable carrier. When the immune cells express a CAR polypeptide, 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.

[0124] 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, ibrixumab, ribozyme, ribozyme, ribozyme-binding protein 1 (RIP1), ribozyme-binding protein 2 (RIP2), ribozyme-binding protein 3 (RIP3), ribozyme-binding protein 4 (RIP4), ribozyme-binding protein 5 (RIP5), ribozyme-binding protein 6 (RIP6), ribozyme-binding protein 7 (RIP7), ribozyme-binding protein 8 (RIP8), ribozyme-binding protein 9 (RIP9), ribozyme-binding protein 10 (RIP1), ribozyme-binding protein 11 (RIP1), ribozyme-binding protein 12 (RIP1), ribozyme-binding protein 13 (RIP1), ribozyme-binding protein 14 (RIP1), ribozyme-binding protein 15 (RIP1), ribozyme-binding protein 16 (RIP1), ribozyme-binding protein 17 (RIP1), ribozyme-binding protein 18 (RIP1), ribozyme-binding protein 19 (RIP1), ribozyme-binding protein 20 (RIP1), ribozyme-binding protein 21 (RIP1), ribozyme-binding protein 22 (RIP1), ribo The therapeutic antibody may be, but is not limited to, momab, infliximab, ipilimumab, labetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tositumomab, ustekinumab, mogamulizumab, and vedolizumab.

[0125] Additionally, the present disclosure provides a kit comprising (i) a first pharmaceutical composition comprising the FOXP3-T cells of the present disclosure and a pharmaceutically acceptable carrier, and (ii) another therapeutic agent described herein and a pharmaceutically acceptable carrier.

[0126] 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 cellular activity), 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.

[0127] 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.

[0128] 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.

[0129] In one embodiment, a pharmaceutical composition comprising the FOXP3-T cells of the present disclosure comprising a chimeric antigen receptor (CAR) of the present disclosure can be used in cell therapy. In cell therapy, the FOXP3-T cells of the present disclosure comprising a chimeric antigen receptor (CAR) of the present disclosure can be infused into a subject in need thereof as a pharmaceutical composition or as a formulation of a therapeutically effective cell population expressing the CAR of the present disclosure. The infused effector T cells in the 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 whom the cells were obtained (autologous cell therapy), or the cells may be derived from another subject of the same species (allogeneic cell therapy).

[0130] In one embodiment, the FOXP3-T cells of the present disclosure comprising a CAR of the present disclosure, or a population thereof, can be formulated for administration to a subject using techniques known to those skilled in the art. In one embodiment, a formulation comprising a therapeutically effective effector T cell or a population thereof comprising a CAR of the present disclosure 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, tonicity agents, bulking agents, and lubricants.

[0131] The preparations comprising the therapeutically effective FOXP3-T cell of the present disclosure, comprising the CAR of the present disclosure, or a group thereof, can be administered to subjects by 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) etc.

[0132] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and may be those described in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, special edition of Experimental Medicine "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and relevant portions (possibly in their entirety) of these are incorporated herein by reference.

[0133] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other examples include Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (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). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc. No. 6,299,133, which are incorporated herein by reference in their relevant parts.

[0134] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0135] 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. [Example]

[0136] The following examples are provided, beginning with a description of the methods and materials used throughout, which may be obtained from sources other than those specifically mentioned, or may be prepared in-house.

[0137] Example 1: Example of FOXP3-T cells This example describes the production and characterization of exemplary FOXP3-T cells. The methods and materials are shown below. These methods and materials are also used appropriately in Example 2 and subsequent examples. Methods and Materials Generation of FOXP3-CAR construct and viral vector production The anti-CD19CAR backbone plasmid (pLVSIN-CD19BBz-tEGFR) (CART) contains the CD8 leader sequence-anti-CD19 single-chain variable fragment (scFv)-CD8 hinge-CD8 transmembrane domain (TM)-4-1BB intracellular domain (ICD)-CD3z-P2A-trancated EGFR (tEGFR) construct. The vectors were constructed by introducing the wild-type FOXP3 tv1 sequence (NM_014009.4) (WT CART-FOXP3) with the P2A sequence immediately upstream (WT CART-FOXP3) or various FOXP3 mutations (see Table 3) that impair the ability of FOXP3 to bind to NFAT or RUNX1. The vectors were constructed as pMSGV-CD19CAR-mutant FOXP3tv1-tEGFR (Mut#1-18). The retroviral vector was transfected into Phoenix-Ampho cells together with Lipofectamine 2000 (Invitrogen), and the retrovirus was isolated from the supernatant.

[0138] In silico structural analysis The amino acid sequences of FOXP3, RUNX1, and NFAT1 were obtained from UniProt. The FOXP3 wild-type sequences were analyzed using the Alphafold server. We predicted the dimer structure for the FOXP3 type and various FOXP3 mutations, and also predicted the structure when RUNX1 or NFAT1 binds to the FOXP3 dimer.

[0139] Gene transfer Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from heparinized whole blood using the Ficoll method (Ficoll-Paque PLUS, GE Healthcare) and then stimulated with anti-CD3 / 28 beads (Dynabeads T-Activator CD3 / CD28, Veritas). On days 3–4, 2.5 ml of retroviral supernatant was adsorbed onto a 12-well plate coated with RetroNectin (10 μg / well, Takara) and transfected into T cells for CAR gene transfer. In some experiments, CAR-positive cells were purified using tEGFR. Cultures were cultured in the presence of 100 U / ml IL-2 and either frozen for days 8–12 or directly subjected to evaluation. For shCTLA-4 and ShTGF-β1 lentiviral vectors obtained using the following method, lentiviral solution was added on day 1 for transduction. For sgCTLA-4 and sgTGF-β1, anti-CD3 / 28 beads were removed on day 5, and 1.0 × 10 7 An RNP complex containing 10 μg of Alt-R sgRNA and 40 μg of Alt-R Cas9 nuclease was prepared per cell, and electroporation was performed using BTX Gemini.

[0140] (Construction of shCTLA-4 / TGF-β1 vector and CTLA-4 / TGF-β1 knockdown) The siRNA sequences for human CTLA-4tv2 (NM_001037631.3) and human TGF-β1 (NM_000660) were determined using siDirect (siDirect v.2.0 sidirect2.rnai.jp), and an shRNA sequence with a loop structure between them was inserted between the restriction enzymes BamHI and EcoRI to create an oligo DNA (Tables 1 and 2).

[0141] [Table 1]

[0142] [Table 2]

[0143] These were inserted into the pLVSIN-mU6-MCS lentiviral vector to create lentiviral vectors, which were then transfected into 293T cells together with the packaging vectors psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) to obtain shCTLA4 and shTGF-β1 lentiviruses. CD3-positive T cells isolated from PBMCs were stimulated with anti-CD3 / 28 beads and then transfected with s hCTLA-4 lentivirus solution was added to each well, and on day 4, flow cytometry (FCM) was used to identify shCTLA-4_#4 (target sequence 1000-10 ... The shTGF-β1 lentivirus solution was added to Tl-su cells, a human adult T-cell leukemia cell line, on day 0 and cultured in X-vivo media. On day 3, 1 mol / L HCl was added to the supernatant for stimulation, and TGF-β1 was measured by ELISA using the Human TGF-β1 Quantikine ELISA Kit (R & D #DB100B). shTGF-β1_#1 (target sequence 5'-ACCAGAAATACAGCAACAATTCCTG-3' (SEQ ID NO: 24)) was used, as it contributed most to the decrease in TGF-β1.

[0144] Creation of sgCTLA-4 / TGF-β1 and CTLA-4 / TGF-β1 knockout sgRNA sequences for human CTLA-4tv2 (NM_001037631.3) and human TGF-β1 (NM_000660) were used as CRISPR-Cas9 guides. The sequence was determined based on the RNA design checker (https: / / sg.idtdna.com / site / order / designtool / index / CRISPR_SEQUENCE) (Tables 3 and 4).

[0145] [Table 3]

[0146] [Table 4]

[0147] An RNP complex containing 8 μg of Alt-R sgRNA and 32 μg of Alt-R Cas9 nuclease was prepared and transfected into Tl-su cells (1.0 × 10 6 ) were electroporated using BTX Gemini. CTLA-4 expression was evaluated for sgCTLA4-transfected cells on Day 3 using FCM. sgTGF-β1-transfected cells were cultured using X-vivo media, and TGF-β1 was quantified by ELISA using the same method as for ShTGF-β1 Knockdown. Based on the results of the above, we adopted sgCTLA4_#1 (target sequence 5'-GTGCGGCAACCTACATCATGGGG-3' (SEQ ID NO: 25)) and sgTGF-β1_#3 (target sequence 5'-CGGGAGAGCAACACGGGTTC-3' (SEQ ID NO: 26)), which contributed most to the reduction of CTLA4 and TGF-β1 expression.

[0148] Phenotype analysis (FCM) The antibodies used in the FCM analysis are shown in Table 5.

[0149] [Table 5]

[0150] Cells were washed with 4% FBS-PBS. After dead cell staining, surface staining was performed, followed by fixation, perforation, and cytoplasmic staining using the FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cells were analyzed using an LSRFortessa X20 to acquire data. FACSDiva (v8.0.1, BD Analysis was performed using FlowJo (TreeStar) software.

[0151] Phenotype analysis (FCM) The antibodies used for FCM analysis are listed in Table 1. Cells were washed with 4% FBS-PBS. Dead cell staining was followed by surface staining, followed by fixation, perforation, and cytoplasmic staining using a FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cells were analyzed using an LSRFortessa X20 or BD FACS. Data were acquired using ymphony A3 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0152] Cytokine analysis (FCM) CART cells 1 x 10 6 Cells were prepared from 2 × 10 CD19-positive leukemia cell line (Nalm6). 6 Cells were cultured in PMA 50ng / ml (SIGMA P8139) / Ionomycin 1μM (SIGMA 10634) or medium, stimulated for 6 hours in the presence of 5μg / ml Monensin (BD 554724 BD Bioscience), and then cultured in eBioscience Foxp3 / Transcription Factor (TF)-1 medium. Intracellular cytokines were stained using Staining Buffer Set (Thermo Fisher Scientific), and data were acquired using an LSRFortessaX20 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0153] Cytotoxic activity analysis In a 96-well plate, 1 x 10 target cells (GFP-positive CD19-expressing Aspc-1 cells) were added per well. 4Cells were seeded and co-cultured with CART cells at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1 in high-glucose medium (RPMI + 10% human AB serum) or at E:T ratios of 10:1, 3:1, 1:1, and 0.3:1 in low-glucose medium (glucose-free RPMI [Wako] + 0.5 mM glucose [Wako] + 15 mM lactate [Sigma] + 10% human AB serum). After 24 hours, cells were lysed with Cell Culture Lysis Reagent (Promega). Luciferase activity was measured using a GloMax Navigator microplate luminometer (Promega), and cytotoxicity was calculated.

[0154] Cell culture in a high fatty acid environment CART cells 1 x 10 6 Cells were prepared from a CD19-positive leukemia cell line (Nalm6) at 1 × 10 5 Cells were cultured in a low-sugar, high-fat medium (glucose-free RPMI [Wako] + 0.5 mM glucose [Wako] + 0.2, 0.5, 1, or 2 mM palmitic acid [Fuji]) + 10% human AB serum in the presence of IL-2 100 U / ml for 72 hours. After culture, the cells were subjected to metabolic analysis using an XF96 Cell Flux Analyzer (Bioscience), phenotypic analysis (FCM), and CFSE T cell proliferation assay to evaluate their function.

[0155] Cell culture in a high lactic acid environment CART cells 1 x 10 6 The cells were a pancreatic cancer cell line (Aspc-1) at 1 × 10 5 Cells were cultured in a low-sugar, high-fat medium (glucose-free RPMI [Wako] + 0.5 mM glucose [Wako] + 40 mM lactate [Sigma]) + 10% human AB serum in the presence of IL-2 100 U / ml for 96 hours. After culture, the cells were subjected to metabolic analysis using an XF96 Cell Flux Analyzer (Bioscience), phenotypic analysis (FCM), and CFSE T cell proliferation assay to evaluate their function.

[0156] Cell culture with repeated stimulation CART cells 1 x 10 6 The cells were a pancreatic cancer cell line (Aspc-1) at 1 × 10 6 The cells were cultured in RPMI + 10% human AB serum in the presence of IL-2 100 U / ml. Under the same conditions, 1 × 10 pancreatic cancer cell line (Aspc-1) was added every 96 hours. 6 The stimulation with cells was repeated a total of nine times. These cells were subjected to phenotypic analysis (FCM), CFSE T cell proliferation assay, and functional evaluation.

[0157] metabolic analysis The metabolic activity of the cells was measured using an XF96 Cellular Flux Analyzer (Bioscience) with the Seahorse XF T Cell Metabolic Profiling Kit (Agilent Technologies) and the Seahorse XF Substrate Oxidation Stress Test Kits (Agilent Technologies) according to the manufacturer's protocol. On the day before analysis, 96-well flat-bottom plates for analysis were coated with poly-L-ornithine (0.1 mg / ml) and the sensor cartridge was hydrated in a CO2-free incubator. On the day of analysis, the cells for analysis were washed with analysis medium (Seahorse XF RPMI Medium, D(+)Glucose 0.5 mM, L-Glutamine 2 mM), and 2 x 10 CART cells were plated onto the poly-L-ornithine-coated analysis plate. 5After seeding, cells were incubated at 37°C in a CO2-free atmosphere for 60 minutes. The Seahorse XF T Cell Metabolic Profiling Kit contained 13.5μM oligomycin A, 25μM Bam15, and 5.5μM rctenone / antimycin A. The Seahorse XF Substrate Oxidation Stress Test Kit contained 40μM etomoxir, 15μM oligomycin, 20μM FCCP, and 5μM rctenone / antimycin A. Cells were then analyzed using a flux analyzer to assess metabolic function based on OCR, ECAR, and PER.

[0158] Bodipy-FL analysis For evaluation of fatty acid uptake and content, 1 × 10 CART cells were 6 Cells were seeded onto 96-well plates at 100 μL each, and Bodipy-FL (Thermo Fisher Scientific) was added to 25 μM of 200 μL of glucose-free RPMI 1640 (Wako). The cells were then incubated at 37°C for 30 minutes. After washing twice with PBS, the cells were analyzed by FCM. Data were acquired using an LSR Fortessa X20 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0159] 2-NBDG analysis For evaluation of fatty acid uptake and content, 1 × 10 CART cells were 6 Cells were seeded onto 96-well plates at 100 μM 2-NBDG (Thermo Fisher Scientific) in 200 μl of glucose-free RPMI 1640 (Wako) and incubated at 37°C for 30 minutes. After washing twice with PBS, cells were analyzed by FCM. Data were acquired using an LSR Fortessa X20 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0160] CFSE T cell proliferation assay 1 × 10 CART cells 6 The cells were prepared in equal numbers, mixed with PBS and CFSE at a concentration of 5 μM, and stained for 30 minutes in the dark. 6 Mix with cells and add various media ((1) RPMI (Wako) + 10% hu (2) Glucose-free RPMI [wako] + Glucose 0.5 mM [Wako] + 15 mM lactic acid [Sigma] + 10% human AB serum, (3) glucose-free RPMI [wako] + Glucose 0.5mM Cells were cultured for 72 hours in the presence of [Wako] + 0.12 mM palmitic acid [Fuji] + 10% human AB serum. After washing twice with PBS, cells were analyzed by FCM. Data were acquired using an LSR Fortessa X20 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0161] Quantitative PCR analysis The primers used for PCR are shown in Table 2. CART cells were cultured at 3 × 10 6 RNA was prepared according to the number of cells, and purified using RNeasy Kits (Quigen). 100 ng of purified RNA was mixed with 2 μl of PrimeScript RT Master Mix (Takara) and subjected to reverse transcription. The resulting cDNA was mixed with 10 μl of SYBR Green Realtime PCR Master Mix (Applied Biosynthemes) and 0.4 μl of primer adjusted to 10 μM, diluted with Ambion Nuclease-Free Water (Life Technologies) to a total volume of 20 μl, and analyzed using QuantStudio 6 (Applied Biosynthemes). mRNA was quantified using a PCR-based PCR system.

[0162] scRNAseq cCART and WT CART-FOXP3 1 × 10 5 Cells were subjected to emulsion formation, cDNA purification, and library purification using the 10x Chromium 3' Library and Gel Bead Kit (10x Genomics) according to the manufacturer's protocol. Subsequently, sequencing was performed using an Illumina Novaseq 6000, and FASTQ files were obtained using Cell Ranger (Ver. 4.0.0, 10x Genomics). The generated data were preprocessed and purified using the R (Ver. 4.3.2) packages Seurat (Ver. 5.0.1) and ScTransform (Ver. 2). Gene expression profiles were clustered using principal component analysis (PCA), and the results were displayed using UMAP. The resulting gene expression profiles were compared using Single Cell Gene Set Enrichment Analysis (scGSEA) using the Escape package (Ver. 1.12.0).

[0163] RNAseq CAR-T cells 4×10 6 RNA was extracted from the cells using the RNeasy Mini Kit (QIAGEN). Libraries were prepared using the TruSeq® Stranded Total RNA Library Prep Kit (Illumina), and RNA sequencing was performed using NovaSeq X Plus. RNA sequencing data was paired-end sequenced and mapped to hg38 using bowtie2. Gene expression levels were measured using feature counts, and comparisons between two groups were performed using edgeR. GSEA analysis was performed using the fgsea package (Ver. 1.30.0).

[0164] Xenograft mouse model 2×10 human pancreatic cancer cell line Aspc-1 expressing CD19 antigen was administered to 6-8 week-old NOD / scid / IL2rγ- / - (NSG) mice. 6 After subcutaneous administration of cells, 2 × 10 CAR-T cells were administered 4 weeks later. 6 The cells were intravenously administered. In addition, MSCV Myc T58A puro (Addgene) was transfected into Aspc-1 cells to generate cMYC-high-expressing Aspc-1 cells. 6 After subcutaneous administration of cells, 2 × 10 CAR-T cells were administered 4 weeks later. 6 Cells were administered intravenously. The tumor diameter was calculated as the long diameter x short diameter. 2 Measurements were taken once a week as a ÷2.

[0165] Isolation and analysis of tumor-infiltrating lymphocytes NSG mice engrafted with the human pancreatic cancer cell line Aspc-1 were treated with CAR-T cells, and tumors were excised one week after administration. The tumor tissue was then reacted with TTDR (BD Bioscience) for 30 minutes with stirring. After adding a reaction stop solution, the tissue fluid was filtered to isolate tumor-infiltrating lymphocytes. After staining for dead cells, cells were stained for surface staining, followed by fixation, perforation, and cytoplasmic staining using the FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cells were acquired using an LSRFortessa X20 or BD FACSymphony A3 and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0166] The primers used are as follows:

[0167] [Table 6]

[0168] [Table 7]

[0169] (result) A schematic diagram summarizing the results is shown in Figure 1. As shown in Figure 1, the concept of this disclosure is to use FOXP3 to induce factors useful for T cell effector function in non-Treg cells, thereby enhancing antitumor efficacy. The efficacy of CAR-T cell therapy in solid tumors is limited. This is because the unique immune microenvironment formed in solid tumors creates a hypoxic, low-glucose, and high-lactate environment, which causes CAR-T cells, which rely on glycolysis for metabolism, to become exhausted and apoptotic, resulting in dysfunction. In contrast, regulatory T cells (Tregs), unlike CAR-T cells, efficiently infiltrate and activate in the tumor microenvironment. This is because they are not dependent solely on glucose but can overcome metabolic checkpoints using fatty acids and lactate present in the tumor microenvironment as nutrient sources. Furthermore, enhanced infiltration function due to increased expression of chemokine receptors such as CCR4 and CCR8 allows them to maintain their infiltration and activation in the hostile tumor microenvironment. In this disclosure, by expressing FOXP3, the master regulator of Tregs, in T cells, CAR-T cells are endowed with the diverse functions of FOXP3, such as metabolic reprogramming and chemokine receptor expression, thereby enhancing effector function and enhancing anti-tumor effects.

[0170] Figure 2 shows a FOXP3-expressing CAR-T (CAR-containing FOXP3-T cells of the present disclosure). In this example, FOXP3, a master regulator of Tregs, and truncated EGFR were linked to the CAR gene via the P2A and T2A sequences to establish FOXP3-expressing CAR-T cells. It was anticipated that forced expression of FOXP3 would confer various Treg-specific functions, such as metabolic reprogramming and chemokine receptor expression, to CAR-T cells. However, it was anticipated that FOXP3 introduction would result in the expression of immunosuppressive molecules, such as CTLA4 expression and TGFβ1 production, which could impair antitumor efficacy. Therefore, we investigated whether it would be possible to efficiently reduce immunosuppressive molecules and enhance antitumor efficacy by using the CRISPR / Cas9 system or RNA interference to reduce CTLA4 and TGFβ-1 expression or by introducing mutations into FOXP3.

[0171] As shown in Figure 2, FOXP3-expressing T cells (FOXP3-T cells) are obtained by expressing FOXP3, a master regulator of regulatory T cells (Treg cells). This results in T cells possessing factors that negatively regulate effector functions (negative regulators) and factors that positively regulate effector functions (positive regulators) that are inherent in parental T cells (unmodified T cells) and / or induced by FOXP3. The former are typified by the expression of Treg-like inhibitory cytokines and inhibitory molecules, while the latter are typified by metabolic reprogramming, tumor invasion and survival, cytotoxic activity, and cytokine production.

[0172] Figure 3 shows T cells in which negative regulatory factors of FOXP3-T cells were suppressed or abandoned, and positive regulatory factors were maintained or enhanced. As shown, to selectively obtain effector functions important for anti-tumor immune responses, gene editing (CRISPR / Cas9, etc.) and RNA interference (shRNA) techniques are being used. (A) By using a gene encoding a FOXP3 gene (e.g., FOXP3-mutant FOXP3) to suppress or eliminate negative regulatory factors of effector function, or (B) by introducing a FOXP3 derivative (e.g., mutant FOXP3 or partially deleted FOXP3) that has been modified to selectively maintain or enhance positive regulatory factors without inducing negative regulatory factors, T cells (including genetically modified T cells such as CAR-T cells) with enhanced anti-tumor activity can be established.

[0173] (Detailed results) Figure 4 shows the results of directly introducing factors that enhance effector function into T cells. As shown in Figure 4, factors that positively regulate effector function, the expression of which is controlled by the transcription factor FOXP3 (here, the metabolic transporter genes CD36 and MCT1, and the chemokine receptor CCR4) were introduced into T cells individually or in combination, resulting in increased expression of each factor. However, when two or three factors were introduced simultaneously, the expression efficiency of each factor gradually decreased, suggesting that this method is not feasible for the cumulative gene introduction of numerous factors that enhance effector function controlled by FOXP3.

[0174] Figure 5 shows the increased expression of factors (immunosuppressive molecules) that negatively regulate effector function as a side effect of wild-type FOXP3 transfection, and the suppression or abrogation of these factors using RNA interference. When comparing the characteristics of conventional CAR-T cells (normal CAR-T cells, cCAR-T cells, without FOXP3-related manipulation) with wild-type FOXP3-transfected CAR-T cells (WT), we confirmed increased expression of Treg cell-like immunosuppressive molecules such as CTLA4 and CD25, which can negatively regulate FOXP3-induced effector function. We also confirmed that RNA interference using shRNA can simultaneously suppress the expression of immunosuppressive molecules such as CTLA4 and TGFβ1.

[0175] Figure 6 shows the phenotype of wild-type FOXP3-transduced CAR-T cells. We compared the phenotypes of conventional CAR-T cells (CAR-T cells without FOXP3 transduction, abbreviated as cCAR) with those of wild-type FOXP3-transduced CAR-T (CAR-FOXP3, abbreviated as WT). FCM analysis revealed increased FOXP3 expression (a), an increased fraction of memory T cells (responsible for long-term antitumor activity) (b), decreased proportions of exhaustion markers PD-1+ and TIM-3+ cells (c) and TOX expression (d), and increased expression of chemokine receptors (CCR4 and CCR8) (e), which induce intratumoral T cell migration. Furthermore, WT cells exhibited decreased glycolytic function, including decreased expression of the glucose transporter GLUT1 and decreased glucose uptake (2-NBDG). On the other hand, increased CPT1A expression and increased fatty acid uptake (BODIPY-FL) suggested enhanced fatty acid oxidation (f). Analysis using an extracellular flux analyzer revealed increased oxidative phosphorylation (OCR) activity in a low-glucose environment (Glu 0.5mM RPMI) (g). Furthermore, suppression of fatty acid oxidation by adding etomoxir reduced the maximum OCR only in WT, suggesting that the increased oxidative phosphorylation activity in WT under a low-glucose environment may be dependent on fatty acid oxidation (h). GSEA using scRNAseq also revealed increased expression of genes involved in oxidative phosphorylation and fatty acid oxidation in WT compared to cCAR (i).

[0176] Figure 7 shows an example of a CAR construct incorporating FOXP3 (CAR-FOXP3 construct). Based on a second-generation anti-CD19CAR that uses 4-1BB and CD3z as signaling molecules, wild-type FOXP3 was incorporated via the 2A sequence to create an all-in-one construct expressing CD19CAR, wild-type FOXP3, and a gene transfer marker (tEGFR) (A). To suppress negative regulatory factors for effector function, constructs were constructed that combine RNA interference, the CRISPR / Cas9 system (B), and dominant negative TGFβR (C). CAR-FOXP3 constructs were also constructed that incorporate mutant FOXP3 (D) or partially deleted FOXP3 (E), which suppress negative regulatory factors for effector function and selectively maintain or enhance positive regulatory factors. We constructed a CD19 scFv vector. The CD19 scFv can be replaced with other scFvs (anti-mesothelin, anti-ROR1, anti-EGFR RvIII, etc.). As shown in Figure 7, we used the metabolically enhanced CAR construct 41BB and a second-generation anti-CD19 CAR with CD3z as the signaling molecule. We then integrated Foxp3 via the 2A sequence to create an all-in-one vector co-expressing CD19 CAR, Foxp3, and a gene transfer / selection marker (tEGFR). Furthermore, we integrated shCTLA-4 and shTGFb under the U6 promoter, and knocked out the inhibitory factor using CRISPR / Cas9. Additionally, we constructed a dnTGFb construct to block TGF-b signaling. The CD19 scFv can also be replaced with other scFvs (anti-mesothelin, etc.).

[0177] Figure 8 shows the increase in chemokine receptor expression due to FOXP3 transfection. As shown, FCM was used to compare the expression of CCR4, CCR5, CCR8, and CXCR3 in CD19CART cells (CART) and FOXP3-transfected CD19CART cells (CART-FOXP3). Compared to 19CART (CART), 19CART-FOXP3 (CART-FOXP3) showed increased expression of the above chemokine receptors (CCR4, CCR5, CCR8, and CXCR3).

[0178] Figure 9 shows the cytokine production ability of FOXP3-transfected cells. The cytokine production ability of CART-FOXP3 cells was evaluated by intracellular staining. CART-FOXP3 cells exhibited cytokine production equal to or greater than that of conventional CD19CART cells.

[0179] Figure 10 shows the antitumor activity of FOXP3-transduced CD19CART cells (19CART-Foxp3). As shown, FOXP3-transduced CD19CART cells (19CART-Foxp3) exhibited cytotoxic activity comparable to that of conventional CART cells (CART). Furthermore, suppression of the expression of PD-1, an immune checkpoint molecule, was observed. Furthermore, tumor suppression was observed in a xenograft model.

[0180] The identification of highly efficient sh-CTLA4 and sh-TGFb is shown in Figure 11. As shown, in this example, multiple shRNAs were constructed (see Tables 2 and 3), and highly efficient shRNAs were identified for each.

[0181] The identification of highly efficient sg-CTLA4 and sg-TGFb is shown in Figure 12. We constructed multiple shRNAs (see Tables 3 and 4) and successfully eliminated CTLA-4 and TGFb completely using CRISPR-Cas9.

[0182] Figure 13 shows the enhanced proliferation of CART cells by suppressing CTLA-4 and TGF-β. CTLA-4 and TGF-β were knocked down in CD19CART-Foxp3 cells using shRNA. CD19CART-Foxp3 cells lacking both molecules exhibited greater proliferation than normal CART cells.

[0183] Figure 14 shows the blockade of TGFb signaling by dominant-negative TGFb receptor (dnTGFRbR). Introducing dnTGFRbR into CD19CART-Foxp3 cells blocked TGFb signaling and increased IFN-γ production.

[0184] Example 2: Examples of FOPX3 mutations In this example, the structure of FOXP3 and the mutation introduction site were investigated as shown in FIG.

[0185] (a) The three-dimensional structure of FOXP3 predicted using Alphafold3. It is suggested that FOXP3 forms a dimeric leucine zipper structure and binds to RUNX1 and NFAT1 via the forkhead domain. Through the formation of these complexes, FOXP3 is thought to induce transcriptional repression of inflammatory cytokines and increased expression of immunosuppressive molecules.

[0186] (b) and (c) Structure of FOXP3 and mutation site. By introducing FOXP3 mutations from the leucine zipper region to the forkhead region into CARs, we believe that complex formation with RUNX1 and NFAT1 can be inhibited, allowing CAR-T cells to acquire the survival function of regulatory T cells in tumor localization while reducing the decreased immunosuppressive function induced by FOXP3, thereby improving antitumor efficacy. We also investigated the possibility of reducing immunosuppressive function by introducing FOXP3 mutations observed in IPEX syndrome, a fatal autoimmune disease associated with FOXP3 mutations.

[0187] Example 3: Antitumor effect of mutant FOXP3-expressing CAR-T (FOXP3-T cells containing CAR) using an NSG mouse model In this example, as shown in Figure 16, the antitumor effect of mutant FOXP3-expressing CAR-T (FOXP3-T cells containing CAR) was demonstrated using an NSG mouse model.

[0188] (a) Human pancreatic cancer cell line Aspc-1 (tumor volume 90-725 mm3) was infused into NSG mice, and the antitumor effects of untrunced T cells (UTD), cCAR, WT, F325D, F331D, F331D, K332D, H334D, R337Q, F340D, Y342F, W348Q, M370I, A372P, R386H, R397W, E399R / E401A, and D409A mutations were confirmed. The R397W mutation demonstrated significant tumor regression compared with all other groups. The K332D, R337Q, M370I, A372P, and R386H mutations demonstrated antitumor effects equivalent to those of cCAR.

[0189] (b) Human pancreatic cancer cell line Aspc-1 (tumor volume 20-180 mm3) was infused into NSG mice, and the antitumor effects of UTD, cCAR, WT, K332D, R337Q, Y342F, R356E, F367L, M370I, A372P, R386H, V396E, R397W, V398E, and V408E mutations were evaluated. cCAR, R356E, F367L, M370I, A372P, R386H, and R397W mutations showed comparable antitumor effects.

[0190] (c) MYC, an oncogene involved in increased glycolytic activity and cell proliferation, was overexpressed in the human pancreatic cancer cell line Aspc-1, and refractory tumors (tumor volume 180-400 mm3) were infused into NSG mice to confirm the antitumor effects of cCAR, K332D, M370I, and R397W mutations. MYC-expressing models have been reported to induce a low-glucose, high-lactate tumor microenvironment through increased glycolytic activity. In this model, the R397W mutation demonstrated greater tumor regression than all other groups.

[0191] Example 4: Fatty acid metabolic activity in a high fatty acid environment This example demonstrates fatty acid metabolic activity in a high fatty acid environment. As shown in Figure 17, fatty acid metabolic activity in a high fatty acid environment is carried out as follows: did.

[0192] (a) Established CAR-T cells were treated with the human leukemia cell line Nalm6 in a CAR-T:Tumor ratio of 10:1 in a low-sugar, high-fat environment (Glucose 0 mM, Palmitic The cells were co-cultured with 2 mM of acetic acid for 72 hours.

[0193] (b) Changes in CAR-T cell numbers in the culture environment were evaluated in three donors. WT, K356E, F367L, A372P, and R397W mutations significantly increased the number of CAR-T cells compared to cCAR. The number of surviving cells tended to be high.

[0194] (c) CPT1A expression was assessed by FCM in three donors at 72 hours after co-culture. CPT1A expression tended to be higher in K356E, F367L, M370I, A372P, R386H, and R397W compared with cCAR.

[0195] (d) Palmitic acid was added to 0mM glucose RPMI (0.2mM, 0.5mM, 1.0mM) and the human leukemia cell line Nalm6 was incubated with CAR-T:Tumor = 10:1 for 72 hours to confirm CPT1A expression. WT, F367L, and R397W mutant cells showed a tendency to increase CPT1A expression in a fatty acid concentration-dependent manner, but this tendency was not observed in cCAR.

[0196] (e) Celltrace violet 2.5 μM staining was performed before Nalm6 stimulation, and expression was assessed 72 hours later. The F367L, M370I, A372P, and R397W mutations had higher division ability than cCAR.

[0197] (f) The established CAR-T cells were treated with the human leukemia cell line Nalm6 in a CAR-T:Tumor ratio of 10:1 in a low-sugar, high-fat environment (Glucose 0 mM, Palmitic After 48 hours of co-culture with 2 mM Glutamate (2 mM), CAR-T cells were transferred to 0 mM RPMI, and their metabolic function was analyzed using an extracellular flux analyzer (Tcell Metabolic Profiling Kit) in two donors. In Donor 2, WT, K356E, and R397W mutations, and in Donor 3, WT, F367L, M370I, A372P, and R386H mutations, tended to increase OCR after BAM15 stimulation.

[0198] Example 5: Evaluation of metabolic activity in a high fatty acid environment This example demonstrates the evaluation of metabolic activity in a high fatty acid environment.

[0199] Figure 18 shows an example demonstrating metabolic activity in a high lactic acid environment.

[0200] The established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 at a CAR-T:Tumor ratio of 10:1 in a low-glucose, high-fat environment (0 mM glucose, 2 mM palmitic acid) for 48 hours. After this, the CAR-T cells were transferred to RPMI with 0 mM Glu, and their oxidative phosphorylation activity (oxygen consumption rate [OCR]) and glycolysis activity (proton efflux rate [PER]) were analyzed in two donors using an extracellular flux analyzer (Tcell Metabolic Profiling Kit). In Donor 2, WT, K356E, and R397W mutations, and in Donor 3, WT, F367L, M370I, A372P, and R386H mutations, OCR tended to increase after BAM15 stimulation.

[0201] Example 6: Oxidative phosphorylation activity in a high lactic acid environment This example demonstrates the oxidative phosphorylation activity of cells of the present disclosure in a high lactate environment. Figure 19 shows another example of oxidative phosphorylation activity in a high lactate environment. (a) The established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc1 at a CAR-T:Tumor ratio of 10:1 in a low-glucose, high-lactic acid environment (glucose 0 mM, lactate 40 mM) for 96 hours. (b) Changes in CAR-T cell numbers in the culture environment were evaluated in one donor. WT, F367L, M370I A372P, and R397W mutations tended to have higher viable cell numbers than cCAR. (c) The established CAR-T cells were treated with the human pancreatic cancer cell line Aspc-1 in a CAR-T:Tumor ratio of 10:1 in a low-sugar, high-fat environment (glucose 0 mM, lactate 4 mM). After 96 hours of co-culture in 0 mM Glu, CAR-T cells were transferred to 0 mM RPMI, and their oxidative phosphorylation activity (oxygen consumption rate [OCR]) and glycolysis activity (extracellular oxidation rate [ECAR]) were analyzed in one donor using an extracellular flux analyzer (Mito Stress test). WT, K356E, F367L, M370I, A372P, R386H, and R397W mutants showed aerobic respiration-dominated metabolic activity compared to cCAR upon FCCP stimulation.

[0202] Example 7: Proliferative potential and phenotypic changes of CAR-T cells upon repeated stimulation In this example, we demonstrated the proliferation ability and phenotypic changes of CAR-T cells upon repeated stimulation.

[0203] Figure 20 shows the proliferation ability and phenotypic changes of CAR-T cells after repeated stimulation. (a) The established CAR-T cells were co-cultured with the pancreatic cancer cell line Aspc-1 at a CAR-T:Tumor ratio of 1:1, and stimulation with Aspc-1 was repeated every 96 hours. (b) Changes in CAR-T cell numbers (doubling) due to repeated stimulation were evaluated. Compared to cCAR, F367L and R397W mutations tended to result in higher cell proliferation. (c) Cells were stained with 2.5 μM Celltrace violet before Aspc-1 stimulation, and expression was assessed on Day 12 (after three stimulations). WT, F367L, and A372P mutant cells had higher division ability than cCAR. (d) Ki-67 expression after a single stimulation with Aspc-1 was confirmed by FCM in two donors. Ki-67 expression tended to be higher in WT, K356E, F367L, M370I, A372P, R386H, and R397W mutants compared to cCAR. (e)-(f) The expression levels of Tim-3 and TOX and the percentage of PD-1+ and Tim-3+ cells after three rounds of stimulation with Aspc-1 were confirmed by FCM in one donor. Tim-3 expression tended to be lower in WT, K356E, F367L, A372P, R386H, and R397W mutants compared to cCAR. TOX expression tended to be lower in WT, R356E, F367L, and R397W mutants compared to cCAR. In addition, the percentage of PD-1+ and Tim-3+ cells tended to be lower in WT, K356E, F367L, A372P, R386H, and R397W mutants.

[0204] Example 8: Changes in traits in the tumor environment This example demonstrates the alteration of traits in the tumor environment.

[0205] Figure 21 shows the changes in traits in the tumor environment.

[0206] After engrafting the pancreatic cancer cell line Aspc1 into NSG mice, CAR-T cells were administered and the tumors were removed 10 days later, and the characteristics of the CAR-T cells contained within the tumors were evaluated.

[0207] The percentage of PD-1+ and Tim-3+ cells was decreased in the Y342F, R386H, and R397W mutations (a). Furthermore, TOX expression tended to decrease in the Y342F, R386H, and R397W mutations (b), and CCR8 expression tended to increase in the M370I and A372P mutations (c).

[0208] Example 9: Changes in gene expression profiles by RNA sequencing In this example, we demonstrate changes in gene expression profiles by RNA sequencing.

[0209] As shown in Figure 22, changes in gene expression profiles by RNA sequencing are shown.

[0210] RNA was extracted 12 days after the establishment of CAR-T cells, and RNA sequencing was performed. (a) Heatmap revealed three groups: a group showing gene expression patterns similar to Th2 cells, including IL13 (A372P, R337Q, R397W), a group showing gene expression patterns similar to WT (F367L, Y342F, R386H, K332D, K356E), and a group showing gene expression patterns similar to cCAR (M370I). (b) Gene expression patterns of WT and each mutant FOXP3-CART were compared with cCART to create volcanoplots. The R397W mutation showed characteristic gene expression patterns, including elevated expression of TNFRSF8 (CD30) and BATF3, in addition to increased expression of IL13. (c) Volcanoplots were created to compare the gene expression patterns of each mutant FOXP3-CART with WT. Compared to WT, Y342F, F367L, and R386H mutations showed decreased expression of genes involved in the suppressive function of regulatory T cells, such as MYB and CTLA4. Y342F, R386H, and R397W mutations showed decreased expression of genes involved in CAR-T cell dysfunction, such as ID3 and SOX4. R337Q, A372P, and R397W showed a gene expression profile similar to Th2 cells, including IL13 and IL4, and the R397W mutation in particular showed characteristic increased expression of genes such as BATF3 and TNFRSF8.

[0211] Example 10: Gene ontology analysis In this example, gene ontology analysis is shown.

[0212] Gene ontology analysis was performed as shown in FIG. Gene ontology analysis (MF) was performed to compare changes in molecular function with cCAR by RNA sequencing. WT, K356E, and F367L mutations showed increased expression of genes related to cell invasiveness, such as chemokine receptor activity. On the other hand, R337Q, A372P, and R397W mutations showed increased expression of genes related to ATP hydrolysis and protein kinase activity.

[0213] Example 11: Changes in binding pattern of FOXP3 dimer and RUNX1 by Alphafold3 In this example, the analysis of various mutants is shown. The methods and materials are as follows.

[0214] (method materials) Using Alphafold3, we predicted the complex structure of FOXP3 dimer and RUNX1 for each FOXP3 mutation and demonstrated that Alphafold3 altered the binding pattern of FOXP3 dimer and RUNX1. (result) The results are shown in Figure 24. In the amino acid sequence shown in SEQ ID NO: 27, the following mutations were observed: F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A, altering the binding to RUNX1. In particular, the F367L mutation abolished the binding of RUNX1 to the FOXP3 forkhead region.

[0215] As shown in Figure 25, the complex structure of the FOXP3 dimer and RUNX1 is shown. The red circle (R) represents FOXP3, and the blue circle (B) represents RUNX1. The change in the binding pattern of the FOXP3 dimer and RUNX1 by Alphafold3 is shown.

[0216] Using Alphafold3, we predicted the complex structure of FOXP3 dimers and RUNX1 for each FOXP3 mutation. Mutations F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A altered the binding of FOXP3 to RUNX1. In particular, the F367L mutation altered the binding of RUNX1 to FOXP3 F. Binding of the orkhead region is lost.

[0217] Example 12: Enhancement of Foxp3 expression Endogenous Foxp3 expression was induced and / or enhanced using the CRISPR-dCas Activation system. Cells with enhanced Foxp3 expression were subjected to phenotypic analysis (FCM), cytokine analysis (FCM), cytotoxic activity analysis, metabolic analysis, and Bodipy analysis as in Example 1 to evaluate enhanced Foxp3 expression, enhanced immune effector function, and altered metabolic capacity as described in Example 1.

[0218] Example 13: Use of other CARs We will use various structural modifications of the CD19CAR (CD19CAR-FOXP3mut.) containing the FOXP3 derivative shown in Example 1 to examine whether T cell enhancement similar to that observed with CD19CART-FOXP3mut. cells is induced. Specifically, we will replace the CAR with single-chain antibodies reactive to other antigens, such as CD19, 20, 22, mesotherin, EGFRvIII, GD2, claudin 6, and claudin 9, or with zetakine CARs, such as IL15Ra, with CARs with other hinges, such as CD8 and IgG4, or with CARs with other intracellular activation domains, such as 4-1BB, CD28, CD27, and ICOS, and establish CAR-T cells using the resulting plasmids. Furthermore, we will replace the CAR from the lentiviral vector system with a retroviral vector system or a non-viral vector plasmid system to establish various CART-FOXP3 cells.

[0219] We will analyze the T cell phenotype, chemokine expression, and exhaustion / activation marker expression of each CART-FOXP3 cell by flow cytometry and RNA sequencing. We will also co-culture each cell with each antigen-positive cell line (e.g., luciferase-expressing AsPC1, Nalm-6, Raji, and U87d cell lines) to assess cytotoxic activity by luciferase activity, intracellular cytokine production by intracellular staining and ELISA, and CAR-T cell expansion and apoptosis by repeated co-culture. We will also perform metabolic analysis using a flux analyzer.

[0220] Regardless of the CAR structure or gene transfer method, the above CAR-T cells exhibited the same pattern of increased effector function, enhanced memory function, altered chemokine receptor profile, and metabolic changes as CD19CART-FOXP3mut.

[0221] Using a CAR different from that used in Example 1, the induction of Foxp3 expression and alteration of metabolic capacity will be evaluated.

[0222] The following CAR configurations are available: Single-chain antibodies that react with other antigens, such as CD19, 20, 22, mesotherin, EGFRvIII, GD2, claudin 6, and claudin 9, and zetakine CARs, such as IL15Ra. CARs with other hinges, such as CD8 and IgG4 CARs with other intracellular activation domains such as 4-1BB, CD28, CD27, and ICOS A combination of the above These CARs are expressed in T cells using lentivirus, retrovirus, or non-viral vectors. The resulting CAR T cells are used for evaluation in the same manner as in Example 1.

[0223] Example 14: Inhibition of expression of immunosuppressive genes It suppresses the expression of immunosuppressive genes.

[0224] The immunosuppressive genes whose expression is suppressed include the following:

[0225] [Table 8]

[0226] The cells obtained by expression suppression are compared with CART cells that have not been suppressed to confirm that they have greater cell proliferation than non-suppressed CART cells.

[0227] (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 in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0228] According to the present disclosure, even in glucose-depleted environments such as the tumor microenvironment (TME), This method can provide cells that are competitive with cancer cells without starving or exhausting them, and therefore is expected to be applied in the medical field, as it can develop strategies to improve the effectiveness of cell-based immunotherapy. [Sequence List Free Text]

[0229] SEQ ID NOs: 1 to 6: shCTLA-4 sequences SEQ ID NOs: 7 to 10: shTGFb1 sequences SEQ ID NOs: 11 to 13: CTLA-4 CRISPR-Cas9 guide RNA sequences SEQ ID NOs: 14 to 16: TGFb CRISPR-Cas9 guide RNA sequences SEQ ID NOs: 17 to 22: Primer sequences used in the examples SEQ ID NO: 23: shCTLA-4_#4 target sequence SEQ ID NO: 24: shTGF-β1_#1 target sequence SEQ ID NO: 25: sgCTLA4_#1 target sequence SEQ ID NO: 26: sgTGF-β1_#3 target sequence SEQ ID NO: 27: Amino acid sequence of human FOXP3 sequence (NP_054728.2)

Claims

1. exogenously expressing a mutant of Foxp3; and It has immune effector functions, chimeric antigen receptors (CARs), A cell population of T cells (FOXP3-T cells), wherein the mutant has reduced DNA binding ability.

2. The cell population described in claim 1, wherein the mutant is K332D, R337Q, M370I, A372P, R386H or R397W.

3. The cell population of claim 1, wherein the FOXP3-T cells have been modified to reduce or eliminate the function and / or expression of a factor that negatively regulates FOXP3-induced immune effector function (hereinafter referred to as a "negative regulatory factor").

4. The cell population of claim 1, wherein the Foxp3 has been modified to confer an immune effector function to the Foxp3-T cells, or to maintain or enhance the immune effector function of the T cells.

5. The cell population of claim 1, wherein the Foxp3 has been modified to reduce or eliminate expression and / or function of a factor that negatively regulates immune effector function (negative regulatory factor), and to confer effector function to the T cells, or to maintain or enhance the effector function of the T cells.

6. The cell population of claim 1, wherein the Foxp3- T cells are cells with altered metabolic capacity.

7. The cell population of claim 1, wherein the Foxp3-T cells are cells with altered chemokine receptor expression and / or altered migration, infiltration, and survival in a local environment.

8. The cell population of claim 1, wherein the FOXP3-T cells have enhanced expression of a chemokine receptor associated with migration, infiltration, and survival in the local environment.

9. The cell population described in claim 1, in which expression of at least one immunosuppressive gene is reduced or substantially eliminated in the FOXP3-T cells.

10. The cell population of claim 9, wherein the immunosuppressive gene comprises at least one selected from the group consisting of CTLA-4, TGFβ, CD25 (IL-2Ra), OX40, GITR, ICOS, CD80 (B7-1), CD86 (B7-2), TGFβ receptor (TGFβR), IDO, IL-10, IL-35, CD39, and CD73.

11. The cell population of claim 1, wherein the FOXP3-T cells are human effector T cells.

12. A pharmaceutical comprising the cell population described in claim 1.

13. A pharmaceutical comprising the cell population described in claim 2.

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