Effector t-cell and pharmaceutical composition containing same

Abstract

The present disclosure provides cells that exhibit competitiveness with respect to cancer cells without being starved and exhausted even in a glucose depleted environment. More specifically, the present disclosure provides cells that are effective in metabolic (for example, low glucose, high lactic acid, and high fatty acid environments) and immunological tumor environments (chemokine profiles that induce immunosuppressive immune cells, expression of checkpoint molecules, and inhibitory cell infiltration that negatively regulates antitumor immune responses, the inhibitory cells being typified by inhibitory cytokines and Treg cells) in which infiltration by normal T-cells or conventional genetically modified cells are inhibited, and in which, in addition, even if infiltration occurs, said cells cannot exhibit long-term effector functions due to starvation and exhaustion. The present disclosure also provides T-cells having an effector function, wherein the T-cells are modified so as to express Foxp3 (including mutants and partial deletions), and / or have an enhanced expression of Foxp3.

Description

Effector T cells and pharmaceutical compositions containing them

[0001] This disclosure relates to effector T cells (T ｅｆｆ ), in particular effector T cells (T) whose metabolic capacity has been modified. ｅｆｆ ), and relating to pharmaceutical compositions and related technologies containing such cells.

[0002] Cancer cells reprogram their metabolic pathways to favor their own cell proliferation, actively utilizing glycolysis, a pathway that is less efficient at producing adenosine triphosphate (ATP) even in the presence of oxygen, thereby increasing glucose uptake and lactate production (Warburg effect).

[0003] In the tumor microenvironment, many factors suppress the metabolic activity and function of anti-tumor T cells. In addition to immunosuppressive factors, metabolic competition between tumors and T cells is involved in the formation of the immunosuppressive environment. When T cells are stimulated by T cell receptors (TCRs), Ca ２＋ The intracellular concentration of increases, activating calcineurin. Activation of calcineurin causes the dephosphorylated transcription factor NFAT to translocate into the nucleus, where it interacts with other transcription factors, promoting and activating the transcription of genes such as interleukin-2 (IL-2).

[0004] On the other hand, cancer cells consume large amounts of glucose, depleting it, so T, which needs glucose as an energy source, ｅｆｆ Tumor-specific T cells of a certain type, when they invade a tumor, receive signals from the TCR, but the cells' Ca ２＋ As the concentration decreases, the translocation of NFAT into the nucleus is reduced. As a result, T cells become dysfunctional, and cell proliferation and cytokine production are suppressed.

[0005] Furthermore, when cancer cells consume large amounts of nutrients such as amino acids and fatty acids in addition to glucose, the T cells that compete for these nutrients become dysfunctional. In this way, the metabolic mechanisms that support the active proliferation of cancer cells in the tumor microenvironment inhibit the antitumor effector action 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 environments.

[0007] This disclosure relates to T cells (T) that have effector function. ｅｆｆ ), in particular effector T cells (T) whose metabolic capacity has been modified. ｅｆｆ The present invention provides cells that function without starvation or exhaustion even in a tumor environment, and pharmaceutical compositions containing such cells.

[0008] Accordingly, this disclosure provides: (Item X1) A cell population of T cells (FOXP3-T cells) that exogenously express a variant of Foxp3, have immune effector function, and contain a chimeric antigen receptor (CAR), wherein the variant has reduced DNA binding ability. (Item X1A) The cell population according to the above item, wherein the mutation in the variant is located between positions 330 and 400 in SEQ ID NO: 1. (Item X1B) The cell population according to any one of the above items, wherein the mutation in the variant includes a mutation in one or more amino acids K332, R337, M370, A372, R386, or R397. (Item X1C) The cell population according to any one of the above items, wherein the mutation in the variant includes a mutation in the amino acids K332, R337, M370, A372, R386, or R397. (Item X2) The cell population according to any one of the above items, wherein the mutant is K332D, R337Q, M370I, A372P, R386H, or R397W. (Item X3) The cell population according to any one of the above items, wherein the FOXP3-T cells are modified so that the function and / or expression of factors that negatively regulate the immune effector function induced by FOXP3 (hereinafter referred to as "negative regulators") is reduced or eliminated. (Item X4) The cell population according to any one of the above items, wherein the Foxp3 is modified so that the Foxp3-T cells are given immune effector function, or so that the immune effector function of the T cells is maintained or enhanced. (Item X5) The cell population described in any one of the above items, wherein the Foxp3 is modified such that the expression and / or function of factors that negatively regulate immune effector function (negative regulators) is reduced or eliminated, and effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced. (Item X6) The cell population described in any one of the above items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item X7) The cell population described in any one of the above items, wherein the Foxp3-T cells are cells with modified chemokine receptor expression, and / or migration to, infiltration into, and survival in the local environment.(Item X8) The cell population according to any one of the above items, wherein the FOXP3-T cells have enhanced expression of chemokine receptors related to migration to, infiltration into, and survival in the local environment. (Item X9) The cell population according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially absent in the FOXP3-T cells. (Item X10) The cell population according to 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 X11) The cell population according to Item 1, wherein the FOXP3-T cells are human effector T cells. (Item X12) The cell population according to any one of the above items, wherein the cell population includes CD4-positive cells and / or CD8-positive cells. (Item X13) The cell population according to any one of the above items, wherein the cell population includes CD4-positive cells. (Item X14) The cell population according to any one of the above items, wherein the cell population includes CD4-positive cells and CD8-positive cells. (Item X15) The cell population according to any one of the above items, wherein the cell population is enriched with CD4-positive cells. (Item X16) A pharmaceutical product comprising the cell population according to any one of the above items. (Item X16A) The pharmaceutical product according to Item X16 for treating or preventing cancer. (Item X17) A regenerative medicine product comprising the cell population according to any one of the above items. (Item X17A) A regenerative medicine product according to Item X16 for treating or preventing cancer. (Item Y1) A method for treating or preventing a disease, disorder, or symptom in a subject that requires it, or in a subject for which treatment or prevention is effective, comprising the step of administering to the subject an effective amount of a cell population of T cells (FOXP3-T cells) that exogenously express a variant of Foxp3, have immune effector function, and contain a chimeric antigen receptor (CAR), wherein the variant has reduced DNA binding ability.(Item Y1A) The method according to the above item, wherein the mutation in the variant is located between positions 330 and 400 in SEQ ID NO: 1. (Item Y1B) The method according to any one of the above items, wherein the mutation in the variant includes a mutation in one or more amino acids K332, R337, M370, A372, R386, or R397. (Item Y1C) The method according to any one of the above items, wherein the mutation in the variant includes a mutation in the amino acids K332, R337, M370, A372, R386, or R397. (Item Y2) The method according to any one of the above items, wherein the variant is K332D, R337Q, M370I, A372P, R386H, or R397W. (Item Y3) The method according to any one of the above items, wherein the FOXP3-T cells are modified so that the function and / or expression of factors that negatively regulate immune effector function induced by FOXP3 is reduced or eliminated. (Item Y4) The method according to any one of the above items, wherein the Foxp3 is modified so that immune effector function is conferred to the Foxp3-T cells, or the immune effector function of the T cells is maintained or enhanced. (Item Y5) The method according to any one of the above items, wherein the Foxp3 is modified so that the expression and / or function of factors that negatively regulate immune effector function is reduced or eliminated, and so that effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced. (Item Y6) The method according to any one of the above items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item Y7) The method according to any one of the above items, wherein the Foxp3-T cells are cells in which chemokine receptor expression and / or migration to, infiltration into and survival in the local environment have been modified. (Item Y8) The method according to any one of the above items, wherein the expression of chemokine receptors related to migration to, infiltration into and survival in the local environment is enhanced in the Foxp3-T cells. (Item Y9) The method according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially eliminated in the Foxp3-T cells.(Item Y10) The method according to any one of the above items, 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. (Item Y11) The method according to any one of the above items, wherein the FOXP3-T cells are human effector T cells. (Item Y12) The method according to any one of the above items, wherein the cell population comprises CD4-positive cells and / or CD8-positive cells. (Item Y13) The method according to any one of the above items, wherein the cell population comprises CD4-positive cells. (Item Y14) The method according to any one of the above items, wherein the cell population comprises CD4-positive cells and CD8-positive cells. (Item Y15) The method according to any one of the above items, wherein the cell population is enriched with CD4-positive cells. (Item Y16) The method according to any one of the above items, wherein the disease, disorder or symptom is cancer. (Item Z1) A cell population of T cells (FOXP3-T cells) for use as a pharmaceutical, which exogenously expresses a variant of Foxp3, has immune effector function, and contains a chimeric antigen receptor (CAR), wherein the variant has reduced DNA binding ability. (Item Z1A) The cell population according to the above item, wherein the mutation in the variant is located between positions 330 and 400 in SEQ ID NO: 1. (Item Z1B) The cell population according to any one of the above items, wherein the mutation in the variant includes a mutation in one or more amino acids of K332, R337, M370, A372, R386, or R397. (Item Z1C) The cell population according to any one of the above items, wherein the mutation in the mutant includes a mutation in the amino acid K332, R337, M370, A372, R386, or R397. (Item Z2) The cell population according to any one of the above items, wherein the mutant is K332D, R337Q, M370I, A372P, R386H, or R397W.(Item Z3) The cell population according to any one of the above items, wherein the FOXP3-T cells are modified so that the function and / or expression of factors that negatively regulate immune effector function induced by FOXP3 is reduced or eliminated. (Item Z4) The cell population according to any one of the above items, wherein the Foxp3 is modified so that immune effector function is conferred to the Foxp3-T cells, or the immune effector function of the T cells is maintained or enhanced. (Item Z5) The cell population according to any one of the above items, wherein the Foxp3 is modified so that the expression and / or function of factors that negatively regulate immune effector function is reduced or eliminated, and so that effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced. (Item Z6) The cell population according to any one of the above items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item Z7) The cell population according to any one of the above items, wherein the Foxp3-T cells are cells in which chemokine receptor expression and / or migration to, infiltration into, and survival in the local environment have been modified. (Item Z8) The cell population according to any one of the above items, wherein the expression of chemokine receptors related to migration to, infiltration into, and survival in the local environment is enhanced in the Foxp3-T cells. (Item Z9) The cell population according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially absent in the Foxp3-T cells. (Item Z10) A cell population according to any one of the above items, 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. (Item Z11) A cell population according to any one of the above items, wherein the FOXP3-T cells are human effector T cells. (Item Z12) A cell population according to any one of the above items, wherein the cell population comprises CD4-positive cells and / or CD8-positive cells.(Item Z13) The cell population described in any one of the above items, wherein the cell population includes CD4-positive cells. (Item Z14) The cell population described in any one of the above items, wherein the cell population includes CD4-positive cells and CD8-positive cells. (Item Z15) The cell population described in any one of the above items, wherein the cell population is enriched with CD4-positive cells. (Item Z16) The cell population described in any one of the above items, for use in the treatment or prevention of cancer. (Item W1) Use of a cell population of T cells (FOXP3-T cells) exogenously expressing a variant of Foxp3, having immune effector function, and containing a chimeric antigen receptor (CAR), for the manufacture of a medicine, wherein the variant has reduced DNA binding ability. (Item W1A) Use described in the above item, wherein the mutation in the variant is located between positions 330 and 400 in SEQ ID NO: 1. (Item W1B) The use according to any one of the above items, wherein the mutation in the variant includes a mutation in one or more amino acids of K332, R337, M370, A372, R386, or R397. (Item W1C) The use according to any one of the above items, wherein the mutation in the variant includes a mutation in the amino acids of K332, R337, M370, A372, R386, or R397. (Item W2) The use according to Item W1, wherein the variant is K332D, R337Q, M370I, A372P, R386H, or R397W. (Item W3) The use according to any one of the above items, wherein the FOXP3-T cells are modified to have reduced or absent function and / or expression of factors that negatively regulate immunoeffector function induced by FOXP3. (Item W4) The use described in any one of the above items, wherein Foxp3 is modified to impart immune effector function to the Foxp3-T cells, or to maintain or enhance the immune effector function of the T cells.(Item W5) The use described in any one of the above items, wherein the Foxp3 is modified such that the expression and / or function of factors that negatively regulate immune effector function is reduced or eliminated, and effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced. (Item W6) The use described in any one of the above items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item W7) The use described in any one of the above items, wherein the Foxp3-T cells are cells with modified chemokine receptor expression, and / or migration to, infiltration into, and survival in the local environment. (Item W8) The use described in any one of the above items, wherein the expression of chemokine receptors related to migration to, infiltration into, and survival in the local environment is enhanced in the FOXP3-T cells. (Item W9) The use according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially eliminated in the FOXP3-T cells. (Item W10) The use according to any one of the above items, 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. (Item W11) The use according to any one of the above items, wherein the FOXP3-T cells are human effector T cells. (Item W12) The use according to any one of the above items, wherein the cell population comprises CD4-positive cells and / or CD8-positive cells. (Item W13) The use according to any one of the above items, wherein the cell population comprises CD4-positive cells. (Item W14) The use according to any one of the above items, wherein the cell population includes CD4-positive cells and CD8-positive cells. (Item W15) The use according to any one of the above items, wherein the cell population is enriched with CD4-positive cells. (Item W16) The use according to any one of the above items, wherein the pharmaceutical is for the treatment or prevention of cancer.(Item W17) The use described in any one of the above items, wherein the pharmaceutical is a pharmaceutical product or a regenerative medicine product. (Item 1) T cells (FOXP3-T cells) that (exogenously) express Foxp3, its variants or a part thereof, and / or have enhanced (endogenous) Foxp3 expression and possess immune effector function. (Item 2) The FOXP3-T cells described in any one of the above items, wherein the FOXP3-T cells are modified so that the function and / or expression of factors that negatively regulate immune effector function induced by FOXP3 (hereinafter referred to as "negative regulators") is reduced or eliminated. (Item 3) The negative regulatory factor is a Treg cell-like immunosuppressive factor, such as an inhibitory cytokine such as TGFβ or IL-10, or an inhibitory molecule such as CTLA-4, as described in any one of the above items, FOXP3-T cell. (Item 4) The Foxp3 is modified to confer immune effector function to the Foxp3-T cell, or to maintain or enhance the immune effector function of the T cell, as described in any one of the above items, FOXP3-T cell. (Item 5) (Renouncing suppressive function + effector) The Foxp3 is modified to reduce or eliminate the expression and / or function of a factor that negatively regulates immune effector function (negative regulatory factor), and to confer effector function to the T cell, or to maintain or enhance the effector function of the T cell, as described in any one of the above items, FOXP3-T cell. (Item 6) The FOXP3-T cells described in any one of the above items, wherein the Foxp3-T cells are cells with modified metabolic capacity. (Item 7) The FOXP3-T cells described in any one of the above items, wherein the Foxp3-T cells are cells with modified chemokine receptor expression and / or migration to, infiltration into, and survival in the local environment. (Item 8) The FOXP3-T cells described in any one of the above items, wherein the local environment is the environment in which the target cells reside, and if the target cells are tumor cells, it is the tumor environment or tumor microenvironment.(Item 9) A FOXP3-T cell (FOXP3-T cell) having effector function and being modified to express Foxp3, its variants, or a portion thereof, and / or having enhanced expression of Foxp3, wherein the FOXP3-T cell is modified so that the inhibitory function of the negative regulator is reduced or eliminated, as described in any one of the above items. (Item 10) A FOXP3-T cell according to any one of the above items, wherein as a result of being modified so that the inhibitory function of the negative regulator is reduced or eliminated, the immune effector function is maintained or enhanced. (Item 11) A FOXP3-T cell according to any one of the above items, wherein the expression of chemokine receptors related to migration to, infiltration into and survival in the local environment is enhanced in the FOXP3-T cell. (Item 12) The chemokine receptor is a FOXP3-T cell as described in any one of the above items, including CCR4 and CCR8. (Item 13) The FOXP3-T cell as described in any one of the above items, satisfying at least one predetermined condition. (Item 14) The predetermined condition is that FOXP3-T cells are effective against all tumors, but are particularly effective against tumor environments in which Treg cell infiltration is enhanced and / or T cell infiltration, survival, and function are suppressed. The above condition occurs when the patient 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 that matches the metabolic mode induced by FOXP3 (low glucose, high fatty acid, high lactate conditions, etc.). To diagnose the above, the presence of abundant Treg cell infiltration histopathologically, and / or the presence of gene mutations that can induce the above tumor environment (EGFR mutations, ROHA mutations, MHC class II, and increased expression of self molecules), are effective biomarkers, and the FOXP3-T cells described in any one of the above items can be identified by tumor biopsy, gene panel testing by peripheral blood liquid biopsy, or next-generation sequencing.(Item 15) FOXP3-T cells according to any one of the above items, wherein the T cells include an externally inserted antigen receptor such as a chimeric antigen receptor (CAR). (Item 16) FOXP3-T cells according to any one of the above items, wherein the CAR is expressed on the T cells. (Item 17) FOXP3-T cells according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially absent. (Item 18) FOXP3-T cells according to 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) A human effector T cell, which is a FOXP3-T cell as described in any one of the above items. (Item 20) A method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) a step of diagnosing the local environment of a subject, wherein the diagnosis is achieved by determining the status of Treg in the subject or gene mutations of environmental factors of target cells; and C) a step of providing T cells having immune effector function and / or factors that modify the tumor environment based on the local environment. (Item 21) A method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) a step of diagnosing the local environment of a subject (for example, determining whether Treg cell infiltration is enhanced and / or whether T cell infiltration, survival, and function are suppressed), wherein the diagnosis is achieved by determining the status of Treg in the subject or gene mutations of environmental factors of target cells (for example, histopathologically, that there is abundant Treg cell infiltration and / or that the target cells have gene mutations that can induce the tumor environment (EGFR mutation, ROHA mutation, etc.)); and C) a step of providing T cells having immune effector function and / or factors that modify the tumor environment (for example, internal infiltration, chemokines, etc.) based on the local environment.(Item 22) A method for improving the immune status of a subject, comprising: A) a step of diagnosing the immune status of a subject; B) a step of diagnosing gene mutations in the subject; and C) a step of providing T cells having immune effector function according to the immune status based on the immune status and the gene mutations, and providing factors that modify the local environment (e.g., internal infiltration, BATF, chemokines, etc.) based on the gene mutations. (Item 23) A pharmaceutical product comprising T cells having immune effector function or a population of T cells, wherein the T cells are modified to express Foxp3, its variants, or a portion thereof, and / or the expression of Foxp3 is enhanced. (Item 24) A method for treating or preventing a disease associated with an abnormal immune status, comprising administering an effective amount of T cells or a population of said T cells having immune effector function, wherein the T cells are modified to express Foxp3, a variant thereof, or a portion thereof, and / or the expression of Foxp3 is enhanced, to a subject in need. (Item 25) Effector T cells (T. ｅｆｆA pharmaceutical composition comprising, wherein the T cells are modified to express Foxp3 and / or the expression of Foxp3 is enhanced. (Item 26) The composition according to any one of the above items, wherein the T cells are cells with modified metabolic capacity. (Item 27) The composition according to any one of the above items, wherein the T cells comprise a chimeric antigen receptor (CAR). (Item 28) The composition according to any one of the above items, wherein the CAR is expressed on the T cells. (Item 29) The composition according to any one of the above items, wherein the expression of at least one immunosuppressive gene is reduced or substantially eliminated. (Item 30) The composition according to any one of the above items, wherein the immunosuppressive gene comprises 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 31) The composition according to any one of the above items, wherein the T cell is a human effector T cell. (Item A1) A cell population comprising T cells (FOXP3-T cells) that (exogenously) express Foxp3, its variants or a part thereof, and / or have enhanced (endogenous) Foxp3 expression and possess immune effector function. (Item A2) The cell population described in any one of the above items, wherein the FOXP3-T cells are modified so that the function and / or expression of factors that negatively regulate the immune effector function induced by FOXP3 (hereinafter referred to as "negative regulators") is reduced or eliminated. (Item A3) The cell population described in any one of the above items, wherein the negative regulators include Treg cell-like immunosuppressive factors, such as inhibitory cytokines such as TGFβ and IL-10, and inhibitory molecules such as CTLA-4. (Item A4) The cell population described in any one of the above items, wherein the Foxp3 is modified so that the Foxp3-T cells are given immune effector function, or so that the immune effector function of the T cells is maintained or enhanced.(Item A5) (Relinquishment function abandonment + effector) The cell population described in any one of the above items, wherein the Foxp3 is modified such that the expression and / or function of factors that negatively regulate immune effector function (negative regulators) is reduced or eliminated, and effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced. (Item A6) The cell population described in any one of the above items, wherein the Foxp3-T cells are cells whose metabolic capacity has been modified. (Item A7) The cell population described in any one of the above items, wherein the Foxp3-T cells are cells whose chemokine receptor expression and / or migration to, infiltration into, and survival in the local environment have been modified. (Item A8) The cell population described in any one of the above items, wherein the local environment is the environment in which the target cells exist, and if 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 above items, wherein the T cells (FOXP3-T cells) have effector function and are modified to express Foxp3, its variants, or a portion thereof, and / or have enhanced expression of Foxp3, the FOXP3-T cells being modified so that the inhibitory function of the negative regulatory factor is reduced or eliminated. (Item A10) A cell population according to any one of the above items, as a result of modification so that the inhibitory function of the negative regulatory factor is reduced or eliminated, the immune effector function is maintained or enhanced. (Item A11) A cell population according to any one of the above items, wherein the expression of chemokine receptors related to migration to, infiltration into and survival in the local environment is enhanced in the FOXP3-T cells. (Item A12) A cell population according to any one of the above items, wherein the chemokine receptors include CCR4 and CCR8, etc. (Item A13) A cell population described in any one of the above items, satisfying at least one of the specified conditions.(Item A14) The above-mentioned conditions are a cell population described in any one of the above items, in which FOXP3-T cells are effective against all tumors, but are particularly effective against tumor environments in which Treg cell infiltration is enhanced and / or T cell infiltration, survival, and function are suppressed, or the above conditions are a cell population described in any one of the above items that occurs when the patient tumor environment is rich in ligands (CCL17, CCL22, CCL1, etc.) for chemokine receptors (CCR4 and CCR8) induced by FOXP3, and / or has a nutritional environment that matches the metabolic mode induced by FOXP3 (low glucose, high fatty acid, high lactate conditions, etc.). Alternatively, to diagnose the above, the presence of abundant Treg cell infiltration histopathologically and / or the presence of gene mutations that can induce the above tumor environment (EGFR mutations, ROHA mutations, MHC class II and increased expression of self molecules, etc.) is effective as a biomarker and can be identified by tumor biopsy, or by gene panel testing or next-generation sequencing using peripheral blood liquid biopsy, as described in any one of the above items. (Item A15) The cell population described in any one of the above items in which the T cells include an externally inserted antigen receptor such as a chimeric antigen receptor (CAR). (Item A16) The cell population described in any one of the above items in which the CAR is expressed on the T cells. (Item A17) The cell population described in any one of the above items in which the expression of at least one immunosuppressive gene is reduced or substantially absent. (Item A18) A cell population according to 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) A cell population according to any one of the above items, wherein the T cells are human effector T cells.(Item A20) A method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) a step of diagnosing the local environment of a subject, wherein the diagnosis is achieved by determining the status of Treg cells or gene mutations of environmental factors of target cells in the subject; and C) a step of providing, based on the local environment, factors that modify the T cells or cell population and / or tumor environment as described in any one of the above items. (Item A21) A method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) a step of diagnosing the local environment of a subject (for example, determining whether Treg cell infiltration is enhanced and / or whether T cell infiltration, survival, and function are suppressed), wherein the diagnosis is achieved by determining the status of Treg in the subject or gene mutations of environmental factors of target cells (for example, histopathologically, that there is abundant Treg cell infiltration and / or that the target cells have gene mutations that can induce the tumor environment (EGFR mutation, ROHA mutation, etc.)); and C) a step of providing T cells or cell populations and / or factors that modify the tumor environment (for example, internal infiltration, chemokines, etc.) as described in any one of the above items, based on the local environment. (Item A22) A method for improving the immune status of a subject, comprising: A) a step of diagnosing the immune status of a subject; B) a step of diagnosing gene mutations in the subject; and C) a step of providing T cells or a cell population according to any one of the above items, based on the immune status and the gene mutations, and providing a factor that modifies the local environment (e.g., internal infiltration, BATF / chemokine, etc.) based on the gene mutations. (Item A23) A pharmaceutical product comprising T cells or a cell population of T cells having an immune effector function, wherein the T cells are modified to express Foxp3, its variants, or a part thereof, and / or the expression of Foxp3 is enhanced.(Item A24) A method for treating or preventing a disease associated with an abnormal immune state, comprising administering to a subject in need thereof an effective amount of T cells having an immune effector function or a cell population of said T cells, wherein said T cells are modified to express Foxp3, a variant thereof, or a part of any of them, and / or the expression of Foxp3 is enhanced. (Item A25) A pharmaceutical composition comprising a cell population of effector T cells (T. ｅｆｆ ), wherein said T cells are modified to express Foxp3 and / or the expression of Foxp3 is enhanced. (Item A26) The composition according to any one of the above items, wherein said T cells are cells with modified metabolic ability. (Item A27) The composition according to any one of the above items, wherein said T cells contain a chimeric antigen receptor (CAR). (Item A28) The composition according to any one of the above items, wherein said CAR is expressed in said T cells. (Item A29) The composition according to any one of the above items, wherein the expression of at least one immunosuppressive gene is decreased or substantially disappeared. (Item A30) The composition according to any one of the above items, wherein said 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 above items, wherein said T cells are human effector T cells. (Item B1) A method for treating or preventing a disease, disorder or condition of a subject, comprising administering to the subject in need thereof an effective amount of the cell, cell population, composition or medicament according to any one of the above items. (Item C1) The cell, cell population, composition or medicament according to any one of the above items for treating or preventing a disease, disorder or condition. (Item D1) Use of the cell, cell population, composition or medicament according to any one of the above items for manufacturing a medicament for treating or preventing a disease, disorder or condition.

[0009] In this disclosure, one or more of the above features may be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary.

[0010] Furthermore, any other features and notable effects of this disclosure will become clear to those skilled in the art by referring to the following sections on embodiments of the invention and the drawings.

[0011] This disclosure describes effector T cells (T) with modified metabolic capacity. ｅｆｆ This allows us to provide such cells, and by using these cells, we can provide chimeric antigen receptor (CAR) T cells that can function without starvation or exhaustion even in a tumor environment.

[0012] The cells disclosed herein can be described as enhanced T cells, applying the mechanisms by which regulatory T cells infiltrate, survive, and function within the tumor environment to unregulated T cells. By using the transcription factor Foxp3, it is possible to simultaneously induce elements necessary for T cells to infiltrate tumors and continue to act over the long term, such as changes in chemokine expression profiles and metabolic patterns. Furthermore, by knocking out / knocking down inhibitory molecules that are byproducts of regulatory T cells, it is possible to impart only antitumor effects to T cells and induce high antitumor activity not found in conventionally modified T cells.

[0013] Figure 1 illustrates the concept of this disclosure. Typically, it shows how FOXP3 is used to induce factors useful for the effector function of T cells in non-Treg cells, thereby enhancing the antitumor effect. The effectiveness of CAR-T cell therapy in solid tumors is limited. This is because solid tumors form a unique immune microenvironment, creating a hypoxic / low-glucose / high-lactic acid environment. CAR-T cells, which rely on glycolysis for metabolism, become exhausted and undergo apoptosis, leading to dysfunction. On the other hand, regulatory T cells (Treg), unlike CAR-T cells, efficiently infiltrate and activate within the tumor microenvironment. This is because they can overcome metabolic checkpoints by using fatty acids and lactic acid present in the tumor microenvironment as nutrients, rather than solely relying on glucose. Furthermore, enhanced invasive function through increased expression of chemokine receptors such as CCR4 and CCR8 allows them to maintain invasion and activation within the harsh tumor microenvironment. This disclosure describes how expressing FOXP3, the master regulator of Treg cells, in T cells enhances effector function and increases antitumor effect by conferring diverse functions of FOXP3, such as metabolic reprogramming and chemokine receptor expression, to CAR-T cells. Figure 2 shows T cells expressing FOXP3 (FOXP3-T cells). By expressing FOXP3, the master regulator of regulatory T cells (Treg cells), in T cells, T cells are obtained that possess factors that negatively regulate effector function (negative regulators) and factors that positively regulate effector function (positive regulators), which are present in parent T cells (T cells before modification) and / or induced by FOXP3. Representative examples of the former are the expression of Treg cell-like inhibitory cytokines and inhibitory molecules, while representative examples of the latter are metabolic reprogramming, tumor invasion and survival ability, and cytotoxic activity / cytokine production ability. Figure 3 shows T cells in which negative regulatory factors of FOXP3-T cells are suppressed or abandoned, and positive regulatory factors are maintained or enhanced. As shown, in order to selectively obtain effector functions that are important for the antitumor immune response, negative regulatory factors of effector functions are suppressed or abandoned using gene editing (CRISPR / Cas9, etc.) or RNA interference (shRNA, etc.) (A).Alternatively, a modified FOXP3 derivative (mutant FOXP3 or partially deleted FOXP3) that selectively maintains or enhances positive regulatory factors without inducing negative regulatory factors is introduced (B). This establishes T cells with enhanced antitumor activity (including genetically modified T cells such as CAR-T cells). 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, whose expression is controlled by the transcription factor FOXP3 (here, the genes for the metabolic transporters CD36, 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 makes it impossible to introduce multiple factors that enhance effector function controlled by the numerous FOXP3-regulated factors in a cumulative manner. Figure 5 shows the expression of immunosuppressive molecules induced by introduction of wild-type FOXP3 and the induction of reduced expression using RNA interference. When comparing the traits of cCAR and wild-type FOXP3-introduced CAR-T (WT), we confirmed an increase in the expression of Treg-derived immunosuppressive molecules such as CTLA4 and CD25. Furthermore, we confirmed that the expression of immunosuppressive molecules such as CTLA4 and TGFβ1 can be simultaneously suppressed using an ShRNA system. We demonstrate the increased expression of factors (immunosuppressive molecules) that negatively regulate effector function as a side effect of introduction of wild-type FOXP3, and the suppression or abandonment of these factors using RNA interference. When comparing the characteristics of conventional CAR-T cells (normal CAR-T cells without FOXP3-related manipulation, cCAR-T cells) and wild-type FOXP3-introduced CART cells (WT), we confirmed an increase in the expression of Treg cell-like immunosuppressive molecules that can negatively regulate FOXP3-induced effector functions, such as CTLA4 and CD25. We also confirmed that the expression of immunosuppressive molecules such as CTLA4 and TGFβ1 could be simultaneously suppressed by RNA interference using ShRNA. Figure 6 shows a diagram illustrating the characteristics of wild-type FOXP3-introduced CAR-T cells. The characteristics of conventional CAR-T cells (CAR-T cells without FOXP3 introduction, labeled as cCAR) and wild-type FOXP3-introduced CAR-T cells (CART-FOXP3, labeled as WT) were compared.FCM analysis revealed that in the WT (Wet Tumor) stage, FOXP3 expression was increased (a), the memory T cell fraction, which ensures long-term antitumor activity, was increased (b), the proportion of exhaustion markers PD-1+ and TIM-3+ cells was increased (c), TOX expression was decreased (d), and chemokine receptor (CCR4, CCR8) expression, which induces T cell migration within tumors, was increased (e). Furthermore, in the WT stage, impaired glycolysis was observed, including decreased expression of the glucose transporter GLUT1 and decreased glucose uptake capacity (2-NBDG). On the other hand, increased CPT1A expression and increased fatty acid uptake capacity (BODIPY-FL) suggested enhanced fatty acid oxidation function (f). Analysis using an extracellular flux analyzer showed increased oxidative phosphorylation (OCR) activity under a low-sugar environment (Glu 0.5 mM RPMI) (g). Furthermore, when fatty acid oxidation was suppressed by adding etomoxir, the maximum value of OCR decreased only in WT, suggesting that the enhancement of oxidative phosphorylation activity in a low-sugar environment in WT may be dependent on fatty acid oxidation (h). GSEA using scRNAseq also showed increased expression of genes involved in oxidative phosphorylation and fatty acid oxidation in WT compared to cCAR (i). Figure 7 shows an example of a CAR construct incorporating FOXP3 (CAR-FOXP3 construct). Based on a second-generation anti-CD19CAR with 4-1BB and CD3z as signaling molecules, wild-type FOXP3 was incorporated via 2A sequencing to create an all-in-one construct expressing CD19CAR, wild-type FOXP3, and a gene transfer marker (tEGFR) (A). To suppress negative regulators of effector function, constructs were developed combining RNA interference, the CRISPR / Cas9 system (B), and dominant negative TGFβR (C). Furthermore, CAR-FOXP3 constructs were developed incorporating mutant FOXP3 (D) and partially deleted FOXP3 (E) that suppress negative regulators of effector function and selectively maintain or enhance positive regulators. CD19scFv can be replaced with other scFvs (any scFv, including anti-mesothelin, anti-ROR1, and anti-EGFRRvIII). Figure 8 shows the increase in chemokine receptor expression after FOXP3 introduction.As shown, FCM was used to compare the expression of CCR4, CCR5, CCR8, and CXCR3 in CD19 CART cells (CART) and FOXP3-introduced CD19 CART cells (CART-FOXP3). Compared to 19 CART (CART), increased expression of the above chemokine receptors (CCR4, CCR5, CCR8, CXCR3) was observed in 19 CART-FOXP3 cells. Figure 9 shows the cytokine production capacity of FOXP3-introduced cells. The cytokine production capacity of CART-FOXP3 cells was evaluated by intracellular staining. CART-FOXP3 cells showed cytokine production capacity equivalent to or greater than that of conventional CD19 CART cells. Figure 10 shows the antitumor effect of FOXP3-expressing CAR-T in an NSG mouse model. (a) Human leukemia cell line Nalm-6 was infused into NSG mice, and the antitumor effects of Unrunsduced T cells (UTD), cCAR, and WT were confirmed. WT showed a reduction in tumor volume compared to UTD, but tumor recurrence was observed earlier compared to cCAR. (b) 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, and the antitumor effect of WT did not show a significant difference compared to UTD. Figure 11 shows the identification of highly efficient sh-CTLA4 and sh-TGFb. As shown, in this disclosure, multiple shRNAs were constructed (see Tables 2 and 3), and high-efficiency shRNAs were identified for each. Figure 12 shows the identification of high-efficiency sg-CTLA4 and sg-TGFb. Multiple shRNAs were constructed (see Tables 3 and 4), and complete removal of CTLA-4 and TGFb was successfully achieved using CRISPR-Cas9. Figure 13 shows the enhancement of CART cell amplification by suppression of CTLA-4 and TGFb. CTLA-4 and TGFb were knocked down in CD19CART-Foxp3 cells using shRNA. Both molecule knockout CD19CART-Foxp3 cells showed cell amplification superior to that of normal CART cells. Figure 14 shows TGFb signal blocking by the dominant-negative TGFb receptor (dnTGFbR). When dnTGFRbR was added to CD19CART-Foxp3 cells, TGFb signaling was blocked, and an increase in IFN-g production capacity was observed.Figure 15 shows an example of the structure of FOXP3 and the site of mutation introduction. (a) The three-dimensional structure of FOXP3 predicted using Alphafold3 is shown. FOXP3 forms a leucine zipper structure by dimers and is suggested to bind to RUNX1 and NFAT1 via the Forkhead region. It is thought that through the formation of these complexes, FOXP3 induces transcriptional repression of inflammatory cytokines and increased expression of immunosuppressive molecules. (b), (c) Structure of FOXP3 and site of mutation introduction. By introducing a FOXP3 mutation from the leucine zipper region to the Forkhead region into CAR, it was thought that complex formation with RUNX1 and NFAT1 could be inhibited, allowing CAR-T cells to acquire the tumor-local survival function of regulatory T cells while reducing the decrease in immunosuppressive function derived from FOXP3 and improving the antitumor effect. Furthermore, we investigated the possibility of decreased immunosuppressive function by introducing FOXP3 mutations, which are observed in IPEX syndrome, a fatal autoimmune disease associated with FOXP3 mutations. Figure 16 shows an example of the antitumor effect demonstrated by mutant FOXP3-expressing CAR-T cells (FOR-containing FOXP3-T cells) using an NSG mouse model. (a) Human pancreatic cancer cell line Aspc-1 (tumor burden 90-725 mm3) was infused into NSG mice, and the antitumor effects of Untruncated T cells (UTD), cCAR, WT, F325D, F331D, F331D, K332D, H334D, R337Q, F340D, Y342F, W348Q, M370I, A372P, R386H, R397W, E399R / E401A, and D409A mutations were examined. The R397W mutation showed a significant tumor reduction effect compared to all other groups. The K332D, R337Q, M370I, A372P, and R386H mutations showed antitumor effects equivalent to those of cCAR. (b) Human pancreatic cancer cell line Aspc-1 (tumor burden 20-180 mm3) was infused into NSG mice, and the antitumor effects of the UTD, cCAR, WT, K332D, R337Q, Y342F, R356E, F367L, M370I, A372P, R386H, V396E, R397W, V398E, and V408E mutations were evaluated. Similar antitumor effects were observed for the cCAR, R356E, F367L, M370I, A372P, R386H, and R397W mutations.(c) Intractable tumors (tumor load 180-400 mm3) in which the oncogene MYC, which is involved in enhanced glycolysis activation and increased cell proliferation, was forcibly expressed in the human pancreatic cancer cell line Aspc-1 were infused into NSG mice, and the antitumor effects of cCAR, K332D, M370I, and R397W mutations were confirmed. It has been reported that the MYC expression model induces a low-sugar, high-lactic acid state in the tumor microenvironment by enhancing glycolysis function. In this model, the R397W mutation showed a higher tumor reduction effect than all other groups. Figure 17 shows an example of fatty acid metabolic activity in a high-fatty acid environment. (a) Established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 for 72 hours in a CAR-T:Tumor = 10:1, low-sugar, high-fatty acid environment (Glucose 0 mM, Palmitic acid 2 mM). (b) Changes in CAR-T cell counts in the culture environment were evaluated in three donors. WT, R356E, F367L, A372P, and R397W mutations tended to have significantly higher viable cell counts compared to 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 compared to cCAR. (d) Palmitic acid was added to Glucose 0 mMRPMI (0.2 mM, 0.5 mM, 1.0 mM), and the human leukemia cell line Nalm6 was reacted with CAR-T:Tumor = 10:1. The cells were then co-cultured for 72 hours, and CPT1A expression was confirmed. WT, F367L, and R397W mutations tended to increase CPT1A expression in a fatty acid concentration-dependent manner, but this trend was not observed in cCAR. (e) Nalm6 cells were stained with Celltrace violet 2.5 μM before stimulation, and expression was evaluated after 72 hours. F367L, M370I, A372P, and R397W mutations had higher proliferative capacity compared to cCAR. Figure 18 shows an example demonstrating the evaluation of metabolic activity in a high-fatty acid environment.Established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 for 48 hours in a CAR-T:Tumor ratio of 10:1 and a low-sugar, high-fatty acid environment (Glucose 0 mM, Palmitic acid 2 mM). After co-culture, the CAR-T cells were transferred to Glu 0 mM RPMI, 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). Donor 2 showed a tendency for increased OCR after BAM15 stimulation in WT, K356E, and R397W mutations, while Donor 3 showed a tendency for increased OCR after BAM15 stimulation in WT, F367L, M370I, A372P, and R386H mutations. Figure 19 shows another example demonstrating oxidative phosphorylation activity in a high-lactic acid environment. (a) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc1 for 96 hours in a CAR-T:Tumor = 10:1 low-sugar, high-lactic acid environment (Glucose 0 mM, Lactate 40 mM). (b) Changes in the number of CAR-T cells in the culture environment were evaluated in one donor. WT, F367L, M370I A372P, and R397W mutations tended to have higher viable cell counts compared to cCAR. (c) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc-1 for 96 hours in a CAR-T:Tumor ratio of 10:1 and a low-sugar, high-fatty acid environment (Glucose 0 mM, Lactate 40 mM). After that, the CAR-T cells were transferred to Glu 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, and R386H mutations showed a tendency for increased OCR compared to cCAR cells upon FCCP stimulation. Figure 20 shows examples demonstrating the proliferative capacity and phenotypic changes of CAR-T cells upon repeated stimulation. (a) Established CAR-T cells were co-cultured with the pancreatic cancer cell line Aspc-1 in a CAR-T:Tumor ratio of 1:1, and stimulation with Aspc-1 was repeated every 96 hours. (b) Changes in the number of CAR-T cells due to repeated stimulation (doubling) were evaluated. Cell proliferation tended to be higher in F367L and R397W mutations compared to cCAR.(c) Cellrace violet 2.5 μM staining was performed before Aspc-1 stimulation, and expression was evaluated on Day 12 (after 3 stimulations). WT, F367L, and A372P mutations showed higher proliferative capacity compared to cCAR. (d) Ki-67 expression after 1 Aspc-1 stimulation was confirmed by FCM in two donors. WT, K356E, F367L, M370I, A372P, R386H, and R397W mutations tended to show higher Ki-67 expression compared to cCAR. (e)-(f) Tim-3, TOX expression levels and the percentage of PD-1+, Tim-3+ cells after 3 Aspc-1 stimulation were confirmed by FCM in one donor. In the WT, K356E, F367L, A372P, R386H, and R397W mutations, Tim-3 expression tended to be lower compared to cCAR. In the WT, R356E, F367L, and R397W mutations, TOX expression tended to be lower compared to cCAR. In addition, the proportion of PD-1+ and Tim-3+ cells tended to be lower in the WT, K356E, F367L, A372P, R386H, and R397W mutations. Figure 21 shows the changes in phenotype in the tumor environment. After engraftment of the pancreatic cancer cell line Aspc1 in NSG mice, CAR-T cells were administered, and tumors were excised 10 days later. The phenotype of CAR-T cells contained within the tumors was evaluated. Regarding the proportion of PD-1+ and Tim-3+ cells, a decrease in the proportion of both positive cells was observed in the Y342F, R386H, and R397W mutations (a). Furthermore, TOX expression tended to decrease in the Y342F, R386H, and R397W mutations (b), while CCR8 expression tended to increase in the M370I and A372P mutations. Figure 22 shows the changes in gene expression profiles by RNA sequencing. RNA extraction and RNA sequencing were performed 12 days after CAR-T cell establishment. (a) Heatmap revealed three groups: one showing gene expression patterns similar to Th2 cells, such as IL13 (A372P, R337Q, R397W), one showing gene expression patterns similar to WT (F367L, Y342F, R386H, K332D, K356E), and one showing gene expression patterns similar to cCAR (M370I). (b) The gene expression patterns of WT and each mutant FOXP3-CART were compared with cCAR, and a volcanoplot was created.The R397W mutation showed a characteristic gene expression pattern, including upexpression of IL13, as well as high expression of TNFRSF8 (CD30) and BATF3. (c) The gene expression patterns of each mutant FOXP3-CART were compared with those of the WT, and volcanoplots were created. The Y342F, F367L, and R386H mutations showed decreased expression of genes involved in the suppression function of regulatory T cells, such as MYB and CTLA4, compared with the WT. The Y342F, R386H, and R397W mutations showed decreased expression of genes involved in CAR-T cell dysfunction, such as ID3 and SOX4. The R337Q, A372P, and R397W showed gene expression profiles similar to those of Th2 cells, such as IL13 and IL4, and the R397W mutation in particular showed characteristic increases in the expression of BATF3 and TNFRSF8. 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 signal (d), and IFN-γ response (e), and changes in gene expression patterns compared to cCAR were evaluated. Decreased expression of PI3-AKT-mTOR signaling, glycolysis, and the MYC target gene was observed in the WT, K332D, Y342F, F367L, and R386H mutations. Decreased gene expression related to IL-2-STAT5 signal and IFN-γ response was also observed in the WT, K332D, Y342F, F367L, and R386H mutations. Figure 24 shows the analysis of various mutants. This is a gene ontology analysis. Gene ontology analysis (MF) was performed, and changes in molecular function due to RNA sequencing were compared with cCAR. WT, K356E, and F367L mutations showed increased expression of genes involved in cell invasion, such as chemokine receptor activity. On the other hand, R337Q, A372P, and R397W mutations showed increased expression of genes involved in ATP hydrolysis activity and protein kinase activity. Figure 25 shows the changes in the binding pattern of FOXP3 dimer and RUNX1 induced by Alphafold3. Alphafold3 was used to predict the complex structure of FOXP3 dimer and RUNX1 for each FOXP3 mutation.The F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A mutations show altered binding with RUNX1, and in particular, the F367L mutation results in loss of binding between RUNX1 and the FOXP3 Forkhead region. Human CD19-expressing human pancreatic cancer cell line AsPC-1 was subcutaneously infused into NSG mice, and after engraftment, 1 x 10 cells were grown. ６ Untransformed T cells (UTDs), CAR-positive cells, 1 x 10⁶ ６ Unsorted R397W mutant FOXP3-introduced CAR-T cells (bulk), 1 x 10⁶ ６ CAR-T cells (CD4) purified to be CD4-positive using magnetic beads of cells, or 1 x 10⁶ of the same. ６The antitumor effect was evaluated by administering purified R397W mutant TOXP3-introduced CAR-T cells (CD8) that were CD8-positive cells via tail vein. The results are shown in Figure 26. Figure 26(A) shows the change in tumor volume, and (B) shows the survival curve. (C) shows the tumor volume 22 days after treatment. (D) shows the complete remission rate for each group at 56 days after treatment. In (B), the log-rank test was performed, where * means <p=0.05; ** means <p=0.01. In (C), the One-way ANOVA test was performed, where ns means not significant; * means <p=0.05. Compared to UTD, effective tumor reduction and survival effects were observed in all Bulk, CD4, and CD8 groups. On the other hand, compared to the CD8 monotherapy group, the CD4 and Bulk groups tended to show higher antitumor effects and remission rates, and the survival rate at 56 days after T cell administration, the end date of the experiment, was 100% in both the Bulk and CD4 groups. In conventional CAR-T cells (cCAR), wild-type FOXP3 (WT), and CAR-T cells with each FOXP3 mutant, the expression of CTLA-4, TGF-β, and ICOS, which are known to be induced by FOXP3 and negatively regulate the effector function of T cells, and the expression of IL-2, which is known to be suppressed by FOXP3 and is necessary for or directly involved in the effector function of T cells, was evaluated by flow cytometry (FCM) and ELISA. In CAR-T (WT) cells introduced with wild-type FOXP3, compared to conventional CAR-T cells (cCAR), increased expression of CTLA-4, TGF-β, and ICOS (factors that negatively regulate T cell effector function), and decreased expression of IL-2 (a factor necessary for or directly involved in T cell effector function) were observed. On the other hand, in mutant FOXP3-introduced CAR-T cells, the increased expression of CTLA-4, TGF-β, and ICOS observed in wild-type FOXP3-introduced CAR-T cells was canceled, and IL-2 expression was also restored.In conventional CAR-T cells (cCAR), wild-type FOXP3 (WT), and each FOXP3 mutant-introduced CAR-T cell, the anti-apoptotic effect (a) after CD95 stimulation that induces apoptosis, the oxidative phosphorylation intensity (b), and the expression of fatty acid transporter (CPT-1A) (c), which are known to be characteristic of regulatory T cells induced by FOXP3 and having FOXP3 as a master regulator, and are also advantageous factors when T cells exert effector functions, were evaluated by the increased Annexin-v expression, flux analyzer, and FCM after CD95 stimulation, respectively. After CD95 stimulation, apoptosis was suppressed in wild-type FOXP3-introduced CAR-T cells compared to conventional CAR-T cells (cCAR). In mutant-type FOXP3 (F397L, R397W), the apoptosis-suppressing effect (a) observed in wt was also observed. Also, in wild-type FOXP3-introduced CAR-T cells, enhanced oxidative phosphorylation dependence (b) and enhanced expression of fatty acid transporter (c) were observed compared to conventional CAR-T cells (cCAR). In mutant-type FOXP3 (F397L, R397W), these changes observed in wt were maintained.

[0014] Hereinafter, the present disclosure will be described while showing the best mode. Throughout this specification, it should be understood that the singular expressions also include the plural concepts thereof unless otherwise specified. Therefore, singular articles (for example, in English, "a", "an", "the", etc.) should be understood to also include the plural concepts thereof unless otherwise specified. Also, the terms used in this specification should be understood to be used in the meanings commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains. In case of contradiction, this specification (including the definitions) shall prevail.

[0015] The definitions of the terms specifically used in this specification and / or the basic technical content will be appropriately described below.

[0016] In this specification, "approximately" means ±10% of the following number. For example, "approximately 20" includes "18 to 22". A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. "Approximately" in relation to a range applies to both endpoints of that range. Therefore, for example, "approximately 20 to 30" includes "18 to 33".

[0017] In this specification, "immune effector function" means the function of directly or indirectly damaging or suppressing target cells (e.g., tumor cells, virus-infected cells, antigen-positive cells, etc.). In T cells, the presence of 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, etc., and gene expression profiles by RNA sequencing. Examples of immune effector function include, for example, preferential infiltration of the site of inflammation and sustained function in the tumor microenvironment, which is unfavorable for immune system effector cells (such as killer T cells) due to conditions such as hypoxia and malnutrition. Outside of T cells, "immune effector function" may include biological activity originating from the Fc region of antibodies, and such immune effector functions include, for example, C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and activation of B cells.

[0018] In this specification, "conferring" immune effector functions means adding or enhancing a specific function to a particular target. Specific examples include introducing molecules that induce an immune response in cells or tissues. Methods that induce the expression of immune-related genes using chemical substances or biological reagents are also included. Furthermore, gene editing techniques used to impart specific functions to immune cells also fall under this category. Other possible examples include protein modifications and peptide introductions aimed at regulating the immune system. It should be noted that "conferring" is not limited to artificial methods but also applies to utilizing naturally occurring processes.

[0019] In this specification, "maintenance" of "immune effector function" etc. means stably maintaining the function in a specific subject. As a specific example, adjustment for constantly exerting the functions possessed by immune cells even under the influence of external environments or internal factors is included. Also included is a method of appropriately supplying nutrients, cytokines, or other physiologically active substances so as not to impair the homeostasis of the immune system. Furthermore, control of mechanisms for stabilizing gene expression and preventing protein degradation also falls under this. In addition, therapeutic or preventive interventions for not reducing the functions of adaptive immunity or innate immunity may also be applicable. Note that "maintenance" is applied not only to cases by external intervention but also to methods of supporting the intrinsic mechanisms of the subject.

[0020] In this specification, "enhancement" of "immune effector function" etc. means exerting the function in a specific subject at a higher level than normal. As a specific example, a method of increasing the secretion amount of cytokines by promoting the activation of immune cells is included. Also included are chemical or genetic engineering techniques for strengthening the functions of antigen-presenting cells. Furthermore, methods of amplifying signal transduction between immune cells and strongly inducing an immune response also fall under this. In addition, boosting the ability to eliminate pathogens using drugs or treatment methods that stimulate the immune system may be included in some cases. Note that "enhancement" is not limited to cases by external intervention, and enhancement of naturally occurring immune responses is also applicable.

[0021] In this specification, "modification" of genes, etc., refers to all operations performed on a specific gene or related element with the aim of changing its function or properties. This "modification" includes not only changes to the base sequence of the gene itself, but also operations that affect the regulation of gene expression and the function of gene products (e.g., proteins). Specific examples of "conferring" function include operations that add a new tumor immune response by introducing artificial receptors (CARs) that specifically recognize tumor cells into T cells. Regarding "maintaining" function, this includes operations that introduce stable promoters to stably sustain gene expression even under cell division or stress conditions. Furthermore, regarding "enhancing" function, this includes operations that improve the catalytic efficiency of enzymes by modifying specific amino acid sequences. CRISPR / Cas9 is a method for achieving these modifications. This technology makes it possible to add function by inserting novel sequences into specific gene sequences, or to edit epigenetic regulatory regions to stabilize expression. Furthermore, gene vector modification is widely used, involving the introduction of new genes to confer novel functions, as well as the design of vectors that enable long-term expression to maintain gene expression. Modification of promoters is also common. By designing specific promoters or enhancers, it is possible to add new expression patterns or maintain stable expression. Additionally, chemical induction systems can be used to introduce mechanisms that allow gene expression only in the presence of specific chemical substances, thereby controlling and enhancing expression. These modification techniques are broadly applied not only to manipulating gene function but also to improving the properties of gene products and adding novel biological functions.

[0022] In this specification, "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 a crucial role in regulating cell movement, migration, and function. "Chemokine receptor expression" is achieved through gene transcription, translation, and subsequent protein transport to the cell membrane. Specifically, the expression of CCR5 and CXCR4 in immune cells contributes to cell migration to inflammatory sites and immune responses to pathogens. Furthermore, the expression of chemokine receptors in tumor cells may be involved in tumor progression and metastasis. Methods for regulating chemokine receptor expression include the following: Firstly, there is a method of manipulating the chemokine receptor gene using CRISPR / Cas9 to enhance or control its expression. Secondly, it is possible to induce receptor expression under specific conditions by modifying the promoter region. Thirdly, techniques that control post-translational modifications of chemokine receptors to improve their transport efficiency to the cell membrane are sometimes used. "Chemokine receptor expression" significantly influences cell function, making it an important concept in the fields of immune response research, therapeutic drug development, and tumor treatment.

[0023] In this specification, "migration to 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, a site of tissue injury, etc.). This migration occurs in response to chemical, physical, or biological stimuli and is an essential process for cells to move to the appropriate location and perform their functions. Factors that induce cell migration mainly include secreted molecules such as chemokines and cytokines. For example, chemokines secreted at inflammatory sites (e.g., CXCL12) promote migration to the local environment by attracting immune cells that express specific chemokine receptors (e.g., CXCR4). Physical factors such as substrate stiffness and cell-cell adhesion can also affect cell migration. Techniques for regulating "migration to a local environment" include the following: Firstly, enhancing or controlling the expression of chemokine receptors to improve migratory ability. Secondly, controlling the concentration of chemokines or cytokines to selectively induce specific cells. Thirdly, techniques that modify cell surface molecules to optimize migration efficiency are sometimes used. "Migration to the local environment" is a concept that plays an important role in a wide range of fields, including inflammatory diseases, tumor immunotherapy, and regenerative medicine, and is indispensable for understanding cell function and developing therapeutic methods. Chemokine receptors are transmembrane proteins that bind to specific signaling molecules called chemokines, and play an important role in regulating cell movement, migration, and function. There are various types of chemokine receptors, each exerting its function by binding to a specific chemokine. Specific examples of C-C motif chemokine receptors (CCR family) include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10. Furthermore, the C-X-C motif chemokine receptors (CXCR family) include CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7. In addition, there is CX3CR1 as a C-X3-C motif chemokine receptor and XCR1 as an X-C motif chemokine receptor.Other specific examples of chemokine receptors include Duffy antigen receptor (DARC), C-C motif chemokine receptor-like 1 (CCRL1), C-C motif chemokine receptor-like 2 (CCRL2), abnormal chemokine receptor 1 (ACKR1), and abnormal chemokine receptor 2 (ACKR2). These chemokine receptors play a crucial role in the mechanisms by which cells migrate 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 encompassing these functions and roles.

[0024] In this specification, "ligand for a chemokine receptor" refers to a molecule that binds to a specific chemokine receptor and induces, modulates, or inhibits its activity. Ligands for chemokine receptors are mainly proteins called chemokines, which play an important role in regulating cell movement, migration, and immune responses. Specific examples of ligands that bind to C-C motif chemokine receptors (CCR family) include: CCL17 (TARC) and CCL22 (MDC) as ligands for CCR4; CCL1 (I-309) for CCR8; and CCL2 (MCP-1) as a ligand for CCR2, and CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) as ligands for CCR5. CCR6 binds to CCL20 (MIP-3α), and CCR7 binds to CCL19 and CCL21, respectively. Furthermore, ligands that bind to 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. CX3CR1 binds to the C-X3-C motif chemokine CX3CL1 (fractalkine), and XCR1 binds to the X-C motif chemokine XCL1 (lymphothiatin). These ligands induce specific cells in the local environment through binding to chemokine receptors, contributing to inflammatory responses, immunomodulation, tumor microenvironment formation, and tissue repair. Furthermore, the intensity and duration of the signal change depending on the ligand concentration and affinity to the receptor, thus determining the biological role of each. The relationship between chemokine receptors and their ligands is extremely important in physiological and pathological processes.

[0025] In this specification, "gene mutations that can induce a tumor environment" refers to gene mutations that affect the properties of cells and their surrounding environment, contributing to the formation and maintenance of the tumor microenvironment (TME). These gene mutations affect intracellular and extracellular signaling, immune response evasion, angiogenesis, metabolic regulation, and intercellular interactions, and may promote tumor progression and metastasis. For example, mutations in KRAS, PIK3CA, and BRAF cause abnormal activation of signaling pathways, promoting tumor formation by inducing cell proliferation and the secretion of inflammatory cytokines. Furthermore, as mutations related to immune evasion, TP53 deficiency or mutation impairs tumor suppressor function and creates an environment that evades immune surveillance. In addition, mutations that cause overexpression of CD274 (PD-L1) and abnormalities in the JAK / STAT pathway are factors that construct an immunosuppressive cellular environment. Mutations in VEGFA and HIF1A are involved in promoting angiogenesis, which plays an important role in the tumor microenvironment. These mutations promote the formation of neovascularization, which supplies oxygen and nutrients to tumors, thus supporting tumor growth. Other gene mutations associated with changes in tumor metabolism include IDH1 / IDH2 mutations. These create an immunosuppressive environment in tumors through the accumulation of abnormal metabolites. Similarly, abnormalities in SDH (succinate dehydrogenase) and FH (fumarate hydratase) induce metabolic conditions favorable to tumor formation. Furthermore, MMP (matrix metalloproteinase) related genes are examples of gene mutations that promote extracellular matrix (ECM) remodeling. These accelerate the degradation of the extracellular matrix, facilitating tumor cell invasion and metastasis. In addition, abnormalities in the NF-κB pathway secrete inflammatory cytokines (e.g., IL-6 and CXCL8), inducing oncogenic inflammation. Epigenetic abnormalities are also important in tumor environment formation; abnormalities in DNMT3A cause abnormal DNA methylation, suppressing the expression of tumor suppressor genes. Furthermore, EZH2 mutations alter chromatin modifications, promoting tumor progression and immune evasion. These gene mutations affect a wide range of processes in the tumor microenvironment, creating conditions favorable for tumor growth and progression.Understanding these mutations is crucial for developing new approaches to tumor treatment and tumor control strategies.

[0026] In this specification, "immunosuppressive genes" refer to genes that encode molecules that play a role in suppressing or regulating the immune response. These genes play an important role in suppressing excessive reactions of the immune system and maintaining immune homeostasis. They are also involved in physiological and pathological processes such as the tumor microenvironment, autoimmune diseases, and the suppression of graft rejection. Specifically, immunosuppressive genes include the following: CTLA-4 is a molecule that directly suppresses T cell activation and plays an important role in regulating the immune response. TGF-β is a cytokine that promotes the formation of an immunosuppressive environment and has the effect of suppressing the proliferation and activity of immune cells. CD25 (IL-2Rα) is also involved in regulating the proliferation and activity of T cells. Furthermore, OX40 and GITR contribute to the maintenance of regulatory T cells (Tregs) that have immunosuppressive functions. ICOS is involved in maintaining the function of regulatory T cells and balancing the immune response. Furthermore, CD80 (B7-1) and CD86 (B7-2) are co-stimulatory molecules that induce immunosuppression through interaction with CTLA-4. The TGF-β receptor (TGFβR) suppresses the activity of immune cells through TGF-β signaling. IDO (indoleamine 2,3-dioxygenase) is a molecule that promotes immunosuppression through tryptophan metabolism. In addition, IL-10 and IL-35 function as anti-inflammatory cytokines, forming an immunosuppressive environment. Also, CD39 and CD73 degrade ATP to produce adenosine, resulting in immunosuppressive effects. Such genes play a particularly important role in tumor immune evasion and the formation of an immunosuppressive environment. Understanding the function of immunosuppressive genes and developing techniques to regulate their effects is extremely important in cancer immunotherapy, the treatment of autoimmune diseases, and even transplant medicine. The development of therapies targeting these genes will form the basis for new medical technologies based on immune regulation.

[0027] In this specification, "enhanced Treg cell infiltration and / or suppressed T cell infiltration, survival, and function" refers to a state in which regulatory T cells (Treg) selectively infiltrate specific local environments, such as the tumor microenvironment or inflammatory sites, resulting in enhanced immunosuppression, while simultaneously hindering the infiltration of effector T cells (T cells) or inhibiting their survival and function.

[0028] Treg cells are cells that negatively regulate the immune response, and their invasion is enhanced in the tumor microenvironment by the induction of immunosuppressive chemokines (e.g., CCL22, CCL28). This invasion strengthens immunosuppression in the local environment, promoting immune evasion by tumor cells and tumor progression. On the other hand, effector T cells (e.g., CD8-positive T cells and Th1 cells) play an important role in eliminating tumors and infected cells, but the suppression of their invasion, survival, and function significantly reduces the immune response in the local environment. The suppression of T cell invasion, survival, and function is often caused by the following factors. First, immunosuppressive factors (e.g., TGF-β, IL-10) secreted from tumor cells and Treg cells inhibit the migration and activity of T cells in the local environment. Second, metabolic conditions in the tumor microenvironment, such as low glucose and high lactate, limit the energy supply to T cells and impair their function. Furthermore, overexpression of immune checkpoint molecules such as PD-L1 and CTLA-4 suppresses T cell proliferation and cytotoxic activity. Specifically, Treg cells selectively infiltrate inflammatory sites and the tumor microenvironment, physically inhibiting the infiltration of effector T cells. Even when T cells reach the tumor microenvironment, inhibitory signals from tumor cells and Treg cells induce apoptosis or significantly reduce their cytotoxic activity and cytokine secretion. These factors play a crucial role not only in tumor immune evasion and progression but are also associated with the development of chronic inflammatory diseases and autoimmune diseases. This invention provides a novel strategy for cancer immunotherapy and the treatment of immune-related diseases by targeting these immunosuppressive mechanisms.

[0029] In this specification, "factors that modify the tumor environment" refers to factors that influence 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 from 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. First, internal invasion factors can be cited as factors that modify the tumor environment. These include factors that promote the invasion of Treg cells and macrophages into the tumor microenvironment. Specifically, chemokines CCL22 and CCL28 induce Treg cell invasion and enhance the immunosuppressive environment in tumors. In addition, CSF-1 (colony-stimulating factor 1) promotes macrophage invasion and facilitates tumor progression. Next, chemokines are factors that control 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 play a role in promoting tumor cell migration and metastasis. Furthermore, immunosuppressive cytokines are also important factors in modifying the tumor environment. TGF-β and IL-10 suppress the activity of effector T cells while simultaneously promoting the increase of immunosuppressive cells such as Treg cells and M2 macrophages. This creates an environment in which tumors can easily evade immune attacks. Metabolic factors also contribute to the modification of the tumor microenvironment. Lactate produced by tumor cells activating aerobic glycolysis creates a local acidic environment, reducing the function of effector T cells. In addition, under hypoxic conditions, the expression of HIF-1α (hypoxia-inducible factor 1α) increases, promoting angiogenesis and tumor cell adaptation. Furthermore, VEGF (vascular endothelial growth factor) can be cited as an angiogenic factor. VEGF helps supply nutrients and oxygen to tumor cells by forming new blood vessels, but the heterogeneous vascular structure hinders the invasion of immune cells. In addition, matrix metalloproteinases (MMPs), which are remodeling factors of the extracellular matrix (ECM), facilitate the invasion and metastasis of tumor cells by degrading the ECM.Finally, exosomes and extracellular vesicles secreted by tumor cells and immune cells contain chemokines, cytokines, and microRNAs, which contribute to the modification of the tumor environment. These factors interact with each other to form the tumor microenvironment, creating conditions favorable for tumor growth and progression. Furthermore, the development of therapies targeting these factors offers new possibilities for tumor treatment.

[0030] In this specification, "subject's immune status" refers to the overall state indicating how the subject's immune system is functioning at a given time. This immune status reflects the dynamic balance of the immune system, including the composition and degree of activation of immune cells, cytokine secretion patterns, and suppression or enhancement of the immune response. A subject's immune status includes not only normal immune function in healthy individuals, but also abnormal immune function that may occur due to disease progression or the effects of treatment. For example, in tumor patients, the immune status is characterized by the proportion of immunosuppressive cells (e.g., regulatory T cells (Treg), M2 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 element of the immune status. Specifically, the following elements can be used as indicators of the immune status. Firstly, the composition of immune cells includes the proportions 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-positive T cells, CD8-positive T cells, and Treg cells). Secondly, the activation state of immune cells is important, and this includes the expression of cell surface markers (e.g., PD-1, CTLA-4) and activation markers (e.g., CD69, HLA-DR), as well as effector functions (e.g., cytokine secretion, cytotoxic activity). Thirdly, 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 evaluating the immune state. Furthermore, the presence and activity of antibodies against specific pathogens and antigens, as well as the function of memory T cells and memory B cells, are also evaluated from the perspective of immunological memory. In tumor patients and those with chronic inflammatory conditions, the proportion and function of immunosuppressive cells (e.g., Treg cells, myeloid-derived suppressor cells (MDSCs)) are crucial factors in the immune status. These factors are measured using methods such as blood and tissue sample analysis, flow cytometry, ELISA, real-time PCR, and immunofluorescence staining. Appropriate assessment of the immune status plays a vital role in understanding the disease, formulating treatment strategies, and confirming the effectiveness of new therapies.

[0031] In this specification, "diagnosing gene mutations" means detecting, identifying, and analyzing the presence or absence of gene mutations, such as changes in the base sequence, deletions, insertions, or duplications, in a specific gene of a subject. This diagnosis is performed for the purpose of risk assessment, monitoring of progression, selection of treatment methods, and prognosis estimation for diseases involving gene mutations. Gene mutation diagnosis is performed by various methods. First, there is base sequence analysis. In this method, the base sequence of the target gene is directly analyzed using next-generation sequencing (NGS) or Sanger sequencing, making it possible to identify mutations with high accuracy. It is also common to amplify a specific gene region using the PCR method (polymerase chain reaction) and rapidly detect the presence or absence of mutations. By using real-time PCR (qPCR) or allele-specific PCR, specific gene mutations can be diagnosed with high sensitivity. Furthermore, hybridization methods use DNA microarrays or Southern blotting to detect mutations using probes complementary to a specific gene sequence. This method is effective when comprehensively evaluating large-scale genomic mutations. Furthermore, a new diagnostic method utilizing the CRISPR-Cas system has emerged, enabling rapid and accurate detection of specific gene mutations. This method leverages CRISPR's target recognition capabilities, offering both high accuracy and efficiency. In addition, methods that indirectly diagnose gene mutations are employed, such as measuring the expression and function of abnormal proteins caused by the mutations. This includes analyses using Western blotting and mass spectrometry. Diagnosing gene mutations plays a crucial role in the early detection of diseases such as cancer, genetic disorders, and infectious diseases, as well as in determining treatment strategies. For example, diagnosing mutations in tumor-related genes such as TP53, KRAS, and PIK3CA in cancer allows for the evaluation of tumor characteristics and sensitivity to therapeutic drugs. Additionally, diagnosing mutations in BRCA1 and BRCA2 can predict the risk of developing breast and ovarian cancer.

[0032] In this specification, "immune state" refers to the current activity and condition of an individual's immune system. The present invention selectively provides T cells with immune effector functions according to this immune state. These T cells play a role in regulating the immune response and achieving specific therapeutic objectives. "Genetic mutation" refers to structural or functional changes in the DNA sequence. The present invention provides factors that modify the local environment based on these genetic mutations. These factors include internal infiltration, BATF (Basic Leucine Zipper ATF-like Transcription Factor), and chemokines, and play a role in regulating local environments such as tumor environments and inflammatory sites.

[0033] In this specification, "nutritional environment consistent with the metabolic mode induced by FOXP3" 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 a crucial role in the survival and maintenance of function of regulatory T cells (Tregs) controlled by FOXP3, with specific examples including low glucose, high fatty acid, and high lactate conditions. FOXP3 is a transcription factor essential for Treg differentiation and function, and it controls multiple cellular processes, including energy metabolism. Tregs have metabolic characteristics different from normal immune cells, with a low dependence on glycolysis and instead a strong dependence on fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). Therefore, they can secure energy supply even in a low glucose environment, while efficiently utilizing fatty acids as an energy source under high fatty acid conditions. Furthermore, high lactate conditions are known to be environmental factors that promote Treg differentiation and inhibitory function. In tumor microenvironments and inflammatory sites, lactate is often abundant as a byproduct of cellular metabolism. Under these conditions, Treg cells can utilize lactate in their metabolism, maintaining their suppressive function without competing with other immune cells. Specifically, FOXP3 regulates the expression of metabolism-related genes (e.g., CPT1A, ACADL) and promotes fatty acid oxidation. Furthermore, it suppresses the activation of glycolysis through the inhibition of the mTOR pathway, shifting metabolism towards fatty acid oxidation. Through these metabolic adaptations, Treg cells can ensure a sustained energy supply and exert their immunosuppressive function even under malnutrition conditions. A nutritional environment that matches the metabolic pattern induced by FOXP3 is of significant importance in the pathogenesis of tumors and inflammatory diseases. In particular, in tumor microenvironments, the characteristic metabolic state of low glucose and high lactate enhances Treg cell dominance and contributes to the formation of an immunosuppressive environment. These findings provide important guidelines for the development of new therapies targeting the metabolic characteristics of Treg cells.

[0034] In this specification, "invasion into the local environment" refers to the phenomenon of specific cells physically entering a tissue or microenvironment. This "invasion" refers to the process by which cells pass through or degrade the surrounding intercellular matrix and tissue structure to reach a specific local environment (e.g., inflammation site, tumor tissue, injury site, etc.). Invasion is a phenomenon in which cell motility, adhesion, and matrix-degrading ability are important factors. For example, in the invasion of tumor cells, enzymes that degrade the extracellular matrix (e.g., matrix metalloproteinases (MMPs)) play an important role. Also, when immune cells invade an inflammation site, chemokines and cytokines induce migration, and matrix-degrading enzymes enable invasion into local tissue. The following are examples of methods for regulating "invasion into the local environment": Firstly, there are methods to improve or suppress invasiveness by regulating the expression of extracellular matrix-degrading enzymes. Secondly, there are operations that control the expression and activity of chemokine receptors to increase the efficiency of specific cells invading the target local environment. Thirdly, methods are employed to enhance cell motility through genetic modification or to regulate the expression levels of adhesion molecules. Thus, "invasion into the local environment" is a concept that plays a crucial role in many physiological and pathological processes, including tumor progression and metastasis, induction of immune responses, and promotion of tissue repair. Understanding and regulating its mechanisms is essential for developing therapeutic strategies.

[0035] In this specification, “survival in a local environment” refers to the ability of specific cells to survive under stressful and unfavorable conditions in a specific local environment (e.g., inflammatory sites, tumor microenvironment, hypoxic tissues, injury sites, etc.). This “survival” is an essential element for cells to maintain their function and contribute to physiological or pathological processes while adapting to the environment. Factors contributing to survival in a local environment include securing nutrient supply, regulating the oxidative stress response, increasing the expression of anti-apoptotic molecules, and the action of immunosuppressive factors. For example, tumor cells can survive under unfavorable conditions such as hypoxia and nutrient deficiency by increasing the expression of hypoxia-inducing factor (HIF-1α). Immune cells, on the other hand, evade apoptotic pathways and contribute to inflammation and repair under the influence of local cytokines. The following are examples of methods that promote or regulate “survival in a local environment.” Firstly, there are manipulations that avoid cell death by enhancing the expression of anti-apoptotic molecules (e.g., Bcl-2 family proteins). Secondly, methods to control the oxidative stress response include regulating the expression of antioxidants and related transcription factors (e.g., Nrf2). Thirdly, there are techniques to support survival in the local environment by modifying molecules involved in the extracellular matrix and nutrient supply. Thus, "survival in the local environment" is a central element of physiological and pathological processes such as tumor progression, immune response, and tissue repair, and elucidating and controlling its mechanisms is of great significance in the development of new therapeutic methods.

[0036] In this specification, "(immune) effector cell" or "(immune) effector T cell" means an immune cell or T cell that exerts cytotoxic effects on target cells. Whether a cell is an "effector" can be determined using 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 cell-mediated 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] In this specification, “chimeric antigen receptor (CAR)” means a modified receptor capable of conferring 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 CARs of this disclosure comprise at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0038] In this specification, "Foxp3," "FOXP3," and "foxp3" (also referred to as Scruffin) are all interchangeable and refer to gene names, such as NP_001107849 (protein) or NM_001114377 (mRNA), used without species distinction, and refer to a transcription factor that regulates the immune response and is a protein mainly expressed in regulatory T cells (Tregs). In this specification, control sequences such as mutants are denoted by NM_014009.4 (mRNA) and NP_054728.2 (protein) (SEQ ID NO: 27). Foxp3 has a forkhead-type 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 abnormal expression of Foxp3 is associated with autoimmune diseases, allergic diseases, or tumor progression. In this specification, Foxp3 includes the protein encoded by the human FOXP3 gene, and proteins synthesized based on sequences identical or substantially identical to its nucleotide sequence, and also includes its physiologically active derivatives or variants. When it is necessary to specify a species, it shall be indicated as mouse Foxp3 or human FOXP3, for example.

[0039] In this specification, a “variant” of a gene (protein or nucleic acid molecule) means a protein or nucleic acid molecule derived from that gene (protein or nucleic acid molecule), and includes substitutions, deletions, insertions, or additions of one or more amino acids or nucleotides. However, these changes maintain some or all of the biological functions of the FOXP3 protein.

[0040] Let's explain the case of FOXP3. FOXP3 is a transcription factor that plays an important role in the expression and function of regulatory T cells (Tregs). The “mutants of FOXP3” referred to herein include, but are not limited to, the following specific modifications: 1) Amino acid substitutions: e.g., naturally occurring mutations (e.g., Phe367Ser) or artificial mutations (e.g., Arg48Ala). 2) Amino acid deletions: e.g., deletion of 1 to 10 amino acids in the Exon 7 region. 3) Amino acid insertions: e.g., insertion of 1 to 5 amino acids within the Forkhead domain. 4) Modifications: e.g., addition of phosphorylation sites or glycosylation modifications, and any combination thereof. Examples of such modifications include the following: Examples of mutants include: Phe367Ser mutant: an example in which Phe367 is replaced with Ser, resulting in enhanced inhibitory ability of regulatory T cells; Exon 2 deletion mutant: an example in which the Exon 2 region is deleted, resulting in decreased Treg expression but partial maintenance of the regulatory immune response; Arg48Ala mutant: a mutant in which Arg48 in the Forkhead domain is replaced with Ala, resulting in reduced DNA binding ability. It should be understood that the scope of this specification regarding "FOXP3 mutants" is clearly defined through specific examples of amino acid sequence changes and descriptions based on their biological functions. Furthermore, it should be understood that by presenting specific examples of the biological functions of the mutants, it can be easily implemented by those skilled in the art.

[0041] In this specification, "part" of a gene (protein or nucleic acid molecule) refers to a partial element or attribute that constitutes the whole of the function or structure in question, and includes partial components to the extent necessary to achieve a specific function or characteristic. "Part" 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 total length of FOXP3). Specifically, sequences including 20 amino acids, 50 amino acids, 100 amino acids, 200 amino acids, 300 amino acids, or the full length are possible in increments of 10 amino acids. The subjects listed as "part" are described to clarify elements relevant to the scope of the invention from a structural or functional standpoint. In this specification, "part" refers to, for example, the following: functional part: a portion of the FOXP3 protein responsible for specific functional characteristics such as DNA binding ability, transcriptional repression ability, or nuclear translocation ability; structural part: a specific region of the FOXP3 protein, such as the Forkhead domain, Exon 2 region, or transcription start site; sequence part: a specific amino acid sequence or motif (e.g., the sequence around Phe367).

[0042] In exemplary embodiments, examples of a portion of a gene (protein or nucleic acid molecule) include: a portion of 10 amino acids: a 10-amino acid sequence within the Forkhead domain of the FOXP3 protein; a portion of 20 amino acids: a 20-amino acid sequence contained in the Exon 2 region; a portion of 50 amino acids: a region contributing to the transcriptional repressive ability of FOXP3; a portion of 100 amino acids: a range including the entire Forkhead domain of the FOXP3 protein; a portion of 200 amino acids: a sequence including the entire Exon 7 region; and the full length (396 amino acids in humans): the entire FOXP3 protein. The definition and description of "a portion" in this specification are intended to prevent ambiguity in the scope of the invention and are written in a way that is easily understood and implementable by those skilled in the art, based on specific examples and literature. Furthermore, the meaning of "a portion" is clearly indicated structurally, functionally, and sequentially, so that the subject matter of the invention can be identified.

[0043] In this specification, "metabolic capacity" refers to the ability to consume energy to maintain and / or activate T cell function. Each T cell activates subtype-specific metabolic mechanisms, and impairment of these mechanisms leads to a decrease in T cell survival rate and function. For example, peripheral blood CD4+, CD8+ effector T cells (T ｅｆｆ In T cells, glycolysis is enhanced by mTOR signaling, whereas in regulatory T cells (Treg), oxidative phosphorylation in mitochondria following fatty acid oxidation is enhanced by AMPK signaling. The tumor microenvironment is characterized by low glucose and hypoxia, and T cells infiltrating the tumor site are also in this environment. Therefore, the metabolic capacity of T cells can also be defined as their ability to adapt to the environment using various molecular mechanisms. Metabolic capacity can be evaluated by methods such as flux analyzer analysis, glucose uptake analysis (2-NBDG uptake assay), flow cytometry, and RNA sequencing for the expression of metabolism-related factors.

[0044] In this specification, “(immune) effector function negative regulator” or “deregulator” means a molecule, gene, or protein that is interchangeable and plays a role in suppressing, inhibiting, or attenuating a specific immune effector function (e.g., immune response, signaling, cell proliferation, or metabolic activity) in a cell or tissue. These factors may take the form of transcription factors, signaling molecules, enzymes, or non-coding RNAs.

[0045] In this specification, "local environment" refers to the environment in which target cells reside, encompassing the surrounding environment and representing a complex environment including physical, chemical, and biological factors. When target cells are tumor cells, it may be called the tumor environment or tumor microenvironment. The characteristics of the local environment vary depending on the type of target cells; for example, when target cells are tumor cells, the local environment is called the tumor environment or tumor microenvironment. The tumor microenvironment includes stromal cells, immune cells, vascular endothelial cells, and the extracellular matrix surrounding the tumor cells. Oxygen concentration, pH, nutritional status, and secretory factors (such as cytokines and chemokines) are also important elements characterizing the tumor microenvironment. The tumor microenvironment significantly influences tumor progression, metastasis, and therapeutic response. For example, the presence of immunosuppressive cell populations (such as regulatory T cells and tumor-associated macrophages) in the tumor microenvironment suppresses the immune response to the tumor. Conversely, even in normal tissues, the local environment is related to maintaining tissue homeostasis, regeneration, and pathological changes. Therefore, in this specification, “local environment” is interpreted to include the surrounding conditions and elements in which target cells exist and which influence their biological behavior.

[0046] In this specification, "inhibitory function" refers to the action of attenuating, inhibiting, or halting a specific physiological or biochemical process, or a function inherent in cells or tissues, and factors having such a function are called "inhibitory factors." This "inhibitory function" is achieved by factors, structures, or pathways that function at the molecular, cellular, or systemic level. For example, immunosuppressive function means the action of reducing the activation, proliferation, or production of effector molecules of immune cells. Signal transduction suppression function may refer to the function of suppressing the transmission of signal molecules or the activity of receptors.

[0047] In this specification, 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 and regulatory factors that control their expression. Furthermore, factors with "inhibitory function" may also act as non-coding RNA or specific epigenetic modifiers. In the present invention, the manipulation of factors with "inhibitory function" may reactivate or enhance the function of specific cells or tissues.

[0048] In this specification, “abandonment of inhibitory function” means a reduction or cessation of the effect of inhibiting a particular physiological or biochemical process (hereinafter referred to as “inhibitory function”), and is used herein with the same meaning as “reduction or cessation” of “inhibitory function.” This “abandonment of inhibitory function” or “reduction or cessation of inhibitory function” occurs at the molecular, cellular, or tissue level and means a state in which the function of the inhibitory factor in question is not detected at all.

[0049] The following are some of the mechanisms that can cause the "abandonment of suppressive function" or "reduction or loss of suppressive function" of an inhibitory factor: Gene knockout: The gene encoding the target inhibitory factor is destroyed, completely stopping the production of the factor. RNA interference (RNAi): The expression of the factor is inhibited by degrading or repressing the translation of the mRNA of the inhibitory factor. Antibody neutralization: The activity of the inhibitory factor is inhibited by an antibody that specifically binds to the inhibitory factor. Small molecule inhibitors: The function of the inhibitory factor is lost by a molecule that acts on the active site of the inhibitory factor. "Abandonment of suppressive function" differs from a state in which the suppressive function is simply reduced, and requires the complete loss of the suppressive function in question. For example, in the case of an immunosuppressive factor, it means that the factor is completely inactivated and the suppression of the immune response is released. In the case of a signal transduction inhibitor, it means that the suppressive effect on the signaling pathway is completely removed and the signal is 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. By abandoning the inhibitory function of these factors, for example, activation of the immune response or improvement of certain disease conditions can be expected.

[0051] Furthermore, the definitions of "abandonment of inhibitory function" or "reduction or loss of inhibitory function" in this specification are not intended to limit the scope of the claims, but rather to illustrate specific embodiments of the invention.

[0052] In this specification, "negative regulators" refer to factors that negatively regulate immune effector function induced by Foxp3. These factors play a role in suppressing or regulating the immune response, particularly in the immunosuppressive mechanisms 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. These include molecules whose expression is directly or indirectly regulated by Foxp3, such as secretory 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 can also be useful target molecules from the perspective of transplant immunology and the treatment of autoimmune diseases. In this specification, "negative regulators" refer to the entire group of molecules that negatively regulate immune effector function, encompassing their physiological or pathological roles. Negative regulators include Treg cell-like immunosuppressive factors, such as inhibitory cytokines like TGFβ and IL-10, and inhibitory molecules like CTLA-4.

[0053] Furthermore, "abandonment of negative regulatory expression (factors that negatively regulate immunoeffector function induced by FOXP3)" or "decreased or absent expression of negative regulatory expression" refers to a state in which the transcription or translation, function and / or expression of the factor in question is reduced or absent, resulting in a substantially reduced or substantially undetectable level of the factor in cells or tissues. Also, "reduction of negative regulatory expression" or "decreased or absent expression of negative regulatory expression" means that the transcription, translation, or functional expression level of the factor in question is significantly lower than the normal or baseline state.

[0054] In this specification, specific examples of "factors that negatively regulate (immune) effector function" 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. The molecules targeted in this invention include, but are not limited to, the following inhibitory molecules / receptors, cytokines, and other molecules. 1. Inhibitory molecules / receptors The following are examples of inhibitory molecules or receptors, but are not limited to these: 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, TGF-β, IL-10, and IL-35. 3. Other molecules Examples of other molecules include, but are not limited to, CD39 and CD73. Other negative regulators in the tumor microenvironment 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 the tumor environment and induces apoptosis of T cells. These molecules may play important roles in regulating immune responses, forming immunosuppressive environments, or in disease, and are applicable to the present invention.

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

[0056] In this specification, "(immune) effector function" refers to a function that, when suppressed by a negative regulatory factor, is expected to be activated, promoted, or restored by a decrease or elimination of the expression of that factor. These definitions in this specification are illustrative only and do not limit the interpretation of the claims.

[0057] In this specification, “modified metabolic capacity” means a state or structure in which metabolic capacity has been altered by any means, meaning that at least some or all of its function has been lost or enhanced. Modified cells in this disclosure may also have modified target capacity and are therefore understood to constitute part of this disclosure. This means an altered state or structure of a molecule or cell of the present invention. The metabolic capacity of T cells can be modified in many ways, such as chemically, structurally, and functionally. T cells can also have their metabolic capacity modified by the introduction of nucleic acids. Modified metabolic T cells can exhibit high metabolic capacity, for example, in a low-glucose environment (e.g., in the tumor microenvironment), and therefore modified metabolic T cells may exhibit superior physiological activity (e.g., in the presence of a therapeutic antibody, under low-glucose conditions, etc., in the tumor microenvironment), such as cell proliferation, activation (e.g., increased cytokine production, e.g., increased production of IL-2 or IFNγ), cytotoxicity, and / or in vivo antitumor activity.

[0058] In this specification, "immunosuppressive gene" refers to a gene that expresses a molecule that functions suppressively in the immune system, and can directly or indirectly suppress or downregulate immunity, and can be used synonymously with negative regulatory factors. 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] In this specification, "substantially" means that the value may vary within any amount that contributes to the measurement error that may occur in the embodiments, unless otherwise specified. Accordingly, "substantially absent" means, for example, that the manifestation has disappeared to the extent that the effects of this disclosure are achieved.

[0060] In this specification, "modification" with respect to genes (nucleic acids, proteins, etc.) means inserting a base sequence into DNA within a cell, deleting a base sequence from DNA within a cell, or a combination thereof.

[0061] In this specification, "modification to express" Foxp3, etc. means modifying a cell that was not substantially expressing such a cell marker (usually below the detection limit) so that its expression is observable (or present to a functional level). In this specification, "enhancement" of Foxp3, etc. means increasing the expression level of such a cell marker and modifying its expression so that its function is enhanced. Such methods can be achieved by introducing the FoxP3 gene into an external invader, or by using the CRISPR-dCas Activation system, etc.

[0062] In this specification, "T cell" refers to lymphocytes that have been produced in the bone marrow, migrated to the thymus, and matured. T cells may be CD45-positive and CD3-positive cells from the normal fraction of peripheral blood and bone marrow-derived mononuclear cells.

[0063] (Preferred Embodiments) Preferred embodiments of the Disclosure are described below. The embodiments provided below are provided for a better understanding of the Disclosure, and the scope of the Disclosure should not be limited to the descriptions below. It will be clear that those skilled in the art can make appropriate modifications within the scope of the Disclosure by taking into consideration the descriptions herein. Furthermore, the embodiments of the Disclosure below can be used individually or in combination.

[0064] (FOXP3-T cells) In one aspect, the present disclosure provides T cells (FOXP3-T cells) that are modified to express Foxp3, its variants or any part thereof, and / or have enhanced Foxp3 expression and possess immune effector function, as well as pharmaceutical compositions containing the same and other related technologies.

[0065] In one aspect of this disclosure, it was surprising to learn that T cells (FOXP3-T cells) modified to express Foxp3, its variants, or parts thereof, and / or with enhanced Foxp3 expression and immune effector function, are not conventionally conceivable. By introducing Foxp3, the master regulator of regulatory T cells (Treg), it is possible to induce Treg-like chemokine expression patterns and metabolic modes while maintaining cytotoxic activity as an (immune) effector, rather than as a suppressor cell. Furthermore, this disclosure is noteworthy in that it establishes effector T cells with higher antitumor activity by suppressing the expression of T cell suppressors as a byproduct.

[0066] In one embodiment, the present disclosure provides a T cell (FOXP3-T cell) that (exogenously) expresses Foxp3, a variant thereof, or any part thereof, or has enhanced (endogenous) Foxp3 expression and possesses immune effector function.

[0067] The FOXP3-T cells of this disclosure are modified to have reduced or absent function and / or expression of factors that negatively regulate FOXP3-induced immune effector function (also known as "negative regulators").

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

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

[0070] The Foxp3 used in this disclosure is modified such that the expression and / or function of factors that negatively regulate immune effector function (negative regulators) is reduced or eliminated, and effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced.

[0071] In one embodiment, the Foxp3-T cells of this disclosure are cells with modified metabolic capacity.

[0072] In one embodiment, the Foxp3-T cells of this disclosure are cells whose chemokine receptor expression and / or migration to, infiltration into, and survival in the local environment have been modified.

[0073] In this specification, the local environment refers to the environment in which target cells exist, and if the target cells are tumor cells, it may be called the tumor environment or tumor microenvironment.

[0074] In one particular embodiment, the Disclosure provides a T cell (FOXP3-T cell) having immunoeffector function and being modified to express Foxp3, its variants, or any part thereof, and / or having enhanced Foxp3 expression, wherein the FOXP3-T cell is modified to have reduced or absent negative regulatory function.

[0075] In one embodiment, the inhibitory function of the negative regulatory factor used in this disclosure is reduced or eliminated as a result, while the immune effector function is maintained or enhanced.

[0076] In one embodiment, the expression of chemokine receptors in FOXP3-T cells is enhanced, specifically those associated with migration to, infiltration into, and survival in the local environment. In this particular embodiment, the chemokine receptors include, but are not limited to, CCR4 and CCR8.

[0077] In another embodiment, the disclosure provides FOXP3-T cells that satisfy at least one predetermined condition, where FOXP3-T cells are effective against any tumor, but are particularly effective against tumor environments in which Treg cell infiltration is enhanced and / or T cell infiltration, survival, and function are suppressed. The above condition occurs when the patient tumor environment is rich in ligands (such as CCL17, CCL22, CCL1) for chemokine receptors (CCR4 and CCR8) induced by FOXP3, and / or has a nutritional environment that matches the metabolic mode induced by FOXP3 (such as low glucose, high fatty acid, and high lactate conditions). To diagnose the above, the presence of abundant Treg cell infiltration histopathologically, and / or the presence of gene mutations that can induce the above tumor environment (EGFR mutations, ROHA mutations, MHC class II mutations, and increased expression of 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 this disclosure include an externally inserted antigen receptor, such as a chimeric antigen receptor (CAR).

[0079] In one embodiment, the CAR of the Disclosure is expressed in the FOXP3-T cells of the Disclosure.

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

[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 this disclosure are human effector T cells.

[0083] This disclosure also relates to chimeric antigen receptor (CAR)-expressing T cells (FOXP3-T cells) and cell populations that possess superior viability and invasiveness while maintaining or enhancing immune effector function, achieved by exogenously expressing specific Foxp3 mutants. Normally, the expression of wild-type Foxp3 induces T cells into suppressive regulatory T cells (Tregs). However, the disclosers have found that by using a specific mutant of Foxp3 with reduced DNA binding ability, Treg formation can be avoided while conferring the metabolic regulation and survival benefits inherent in Foxp3 to effector T cells. Traditionally, the introduction of Foxp3 into T cells has meant "conversion to suppressive T cells" and has been considered a forbidden technique in the production of CAR-T cells for cancer treatment. However, because the mutant in this disclosure selectively reduces only DNA binding ability, it does not activate the transcription of the Treg-specific suppressor gene set, thus maintaining the attacking power of CAR-T cells. Foxp3 has the ability to stabilize T cell lipid metabolism and mitochondrial activity through protein-protein interactions that do not involve DNA binding. CAR-T cells expressing the mutant described herein show strong resistance to "exhaustion" that wild-type effector T cells suffer in the tumor microenvironment, achieving both "high cytotoxicity" and "long-term survival and memory formation," which were difficult to achieve with conventional technologies. By combining the suppression of negative regulatory factors and the enhancement of chemokine receptors, T cells reliably infiltrate even refractory solid tumors and continue their attack for a long period, producing remarkable therapeutic effects that could not have been predicted by those skilled in the art. This disclosure opens up a new phase in cell therapy by using Foxp3 with specific structural modifications.

[0084] In one aspect, the present disclosure provides a cell population of T cells (FOXP3-T cells) that exogenously express a variant of Foxp3, have immune effector function, and contain a chimeric antigen receptor (CAR), wherein the variant preferably has reduced DNA binding ability.

[0085] In another context, the present disclosure provides a population of cells of the present disclosure for use as a pharmaceutical.

[0086] In another aspect, the Disclosure provides the use of the cell populations of the Disclosure for the manufacture of a medicine and a method for the treatment or prevention using the same.

[0087] In one aspect, the present disclosure provides a method for treating or preventing a disease, disorder, or symptom in a subject in need or in which treatment or prevention would be effective, comprising administering to the subject an effective amount of a population of T cells (FOXP3-T cells) exogenously expressing a variant of Foxp3, having immune effector function, and containing a chimeric antigen receptor (CAR), preferably the variant having reduced DNA binding ability.

[0088] In one embodiment, the mutation in the variant is located between positions 330 and 400 in SEQ ID NO: 1 (wild-type human Foxp3). In particular, the mutation includes mutations in one or more amino acids: K332, R337, M370, A372, R386, or R397. In a specific embodiment, the variant has one or more of the following: K332D, R337Q, M370I, A372P, R386H, or R397W. These mutations are located in the forkhead (FKH) domain of Foxp3 and inhibit binding to specific DNA sequences, while maintaining the ability to interact with other proteins (such as transcription factors). The mutations at positions 330-400 (FKH domain) of SEQ ID NO: 1 provided herein have been shown to be important for physical contact with DNA through crystal structure analysis and biochemical analysis. For example, substitutions of residues such as K332 and R397 can be confirmed by EMSA (gel shift assay) to significantly reduce DNA binding ability compared to the wild type. Furthermore, in T cells into which CARs (e.g., anti-CD19 CAR, anti-MESO CAR, etc.) have been introduced, the expression of these mutants has been shown to be equivalent to or better than the empty vector control group in terms of cytokine production ability (IFN-γ, TNF-α) and cytotoxic activity against target cells.

[0089] In one embodiment, the FOXP3-T cells are modified to either reduce the function and expression of negative regulatory factors, or to maintain or enhance effector function. This blocks the immunosuppressive circuit mediated by Foxp3, maximizing cytotoxicity. In another embodiment, the cells have modified metabolic capacity (such as mitochondrial function) or chemokine receptor expression (local migration and survival ability). This enables long-term survival and invasion within the tumor microenvironment (TME).

[0090] In one embodiment, the expression of immunosuppressive genes such as CTLA-4, TGFβ, CD25, and PD-1 is reduced or eliminated (e.g., knocked out). In one embodiment, the cells are human effector T cells and include CD4-positive and / or CD8-positive cells. In particular, introducing this mutant into CD4-positive cells yields a cell population that possesses both potent helper function and cytotoxicity.

[0091] In a preferred embodiment, the target disease, disorder, symptom, or indication for the pharmacopoeia is "cancer."

[0092] (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) a step of diagnosing the local environment of a subject (subject) (for example, determining whether Treg cell infiltration is enhanced and / or whether T cell infiltration, survival, and function are suppressed), wherein the diagnosis is achieved by determining gene mutations of the Treg status or target cell environmental factors (for example, histopathologically rich Treg cell infiltration and / or the target cells having gene mutations that can induce the tumor environment (such as EGFR mutations or ROHA mutations)); and C) a step of providing T cells having immune effector function and / or factors that modify the tumor environment (for example, internal infiltration, chemokines, etc.) based on the local environment. Embodiments of this method may employ any features or combinations thereof described in (FOXP3-T Cells).

[0093] In another aspect, this disclosure provides a method for improving the immune status of a subject, comprising: A) diagnosing the immune status of a subject; B) diagnosing gene mutations in the subject; and C) providing T cells having immune effector functions according to the immune status based on the immune status and the gene mutations, and providing factors that modify the local environment (e.g., internal infiltration, BATF / chemokines, etc.) based on the gene mutations. Embodiments of this method may employ any features or combinations thereof described in (FOXP3-T cells).

[0094] In another aspect, the present disclosure provides a pharmaceutical product comprising T cells having immune effector function, wherein the T cells are modified to express Foxp3, its variants, or any part thereof, and / or have enhanced Foxp3 expression. Embodiments of the pharmaceutical product may employ any feature or combination thereof described in (FOXP3-T cells).

[0095] In another aspect, the present disclosure relates to a method for treating or preventing a disease associated with an abnormal immune status, comprising administering an effective amount of T cells having immune effector function, wherein the T cells are modified to express Foxp3, a variant thereof, or a portion thereof, and / or have enhanced Foxp3 expression, to a subject in need. Embodiments of this method may employ any feature or combination thereof described in (FOXP3-T cells).

[0096] In another context, this disclosure relates to effector T cells (T ｅｆｆ The present invention provides a pharmaceutical composition comprising (FOXP3-T cells), wherein the T cells are modified to express Foxp3 and / or the expression of Foxp3 is enhanced. Embodiments of this composition may employ any features or combinations thereof described in (FOXP3-T cells).

[0097] In one embodiment, the T cells in this disclosure are cells with modified metabolic capacity.

[0098] In one embodiment, the T cells in this disclosure include a chimeric antigen receptor (CAR).

[0099] In one embodiment, the CAR in this disclosure is expressed in the T cells.

[0100] In one embodiment, the expression of at least one immunosuppressive gene in this disclosure is reduced or substantially eliminated.

[0101] In one embodiment, the immunosuppressive gene in this 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.

[0102] In one embodiment, the T cells in this disclosure are human effector T cells.

[0103] In one embodiment, it is advantageous that the cells of this disclosure have modified metabolic capacity.

[0104] <Modification of Metabolic Capacity> One advantageous embodiment will be described. Cancer cells reprogram their metabolic systems to be advantageous for their own cell proliferation, actively utilizing glycolysis, which is inefficient in producing ATP even in the presence of oxygen, thereby increasing glucose uptake and lactate production (Warburg effect). As a result, cancer cells consume large amounts of glucose and deplete it, so T, which needs glucose as an energy source, ｅｆｆ Tumor-specific T cells of a certain type, when they invade a tumor, receive signals from the TCR, but the cells' Ca ２＋ The concentration decreases, leading to starvation and exhaustion.

[0105] Accordingly, in one embodiment of the present disclosure, FOXP3-T cells are provided that have been genetically engineered to have modified metabolic capacity in order to promote aerobic glycolysis and enhance the competitiveness and / or compatibility of immune cells in a glucose-depleted tumor microenvironment (TME).

[0106] In one embodiment, modification of metabolic capacity can be achieved by modifying the organism to express Foxp3.

[0107] Regulatory T cells (Tregs) are involved in immunosuppression and are important cells in tumor immunity. Tregs express the master gene Foxp3, which activates the mitochondrial electron transport chain, enhances mitochondrial function, and shifts Treg metabolism toward OXPHOS and FAO. Furthermore, Foxp3 suppresses c-Myc expression and inhibits glycolysis. Conversely, when Treg glycolysis is enhanced, Foxp3 expression decreases in an activation-dependent manner of the mTOR pathway, and the number of Tregs in the tumor also decreases. Thus, there is a close relationship between metabolism and the master transcription factor Foxp3. In addition, the induction of OXPHOS by Foxp3 increases the intracellular NAD+ / NADH ratio and converts lactate to pyruvate, thus escaping the inhibitory effects of lactate under high lactate conditions at the tumor site. In this way, Treg adapts to local environments such as the tumor microenvironment by suppressing glycolysis and increasing OXPHOS.

[0108] In one embodiment of this disclosure, such metabolic reprogramming function by Foxp3 can be utilized to utilize the metabolic mechanism of Treg cells in the FOXP3-T cells of this disclosure.

[0109] While typical effector T cells depend on glucose metabolism, as disclosed herein, T ｅｆｆ By modifying the cells to express Foxp3 and / or to enhance Foxp3 expression, metabolic function is altered, enabling them to take up lactate and fatty acids in addition to glucose, and to use them as metabolic and nutrient sources. Furthermore, it is possible to induce a chemokine receptor expression profile that can respond to chemokines that are abundant in the tumor environment. Therefore, the FOXP3-T cells (T) of this disclosure ｅｆｆ By modifying the drug to express Foxp3 and / or to enhance Foxp3 expression, it is possible to obtain the advantage of being able to infiltrate T cells and maintain antitumor activity even in the metabolic environment of tumors.

[0110] In typical embodiments, the FOXP3-T cells of this disclosure are T cells that play a central role in the immune response, playing an important role in pathogen elimination, tumor cell suppression, or immunomodulation. These cells can attack or modulate specific targets through cytokine production or direct cytotoxicity.

[0111] In a typical embodiment, the FOXP3-T cells of this disclosure are a type of T cell with effector function and typically have the following characteristics: FOXP3 expression: These cells are genetically modified to express the FOXP3 (Forkhead box P3) transcription factor, or their FOXP3 expression is enhanced. FOXP3 is commonly known as a marker for regulatory T cells (Treg) and plays a role in immunosuppressive function. Purpose of modification: By having T cells with immune effector function express FOXP3 (FOXP3-T cells of this disclosure), conventional T cells are modified. ｅｆｆ The ability to fine-tune the immune response while suppressing the pro-inflammatory effects and excessive immune responses of cells is conferred. Characteristics and design of FOXP3-T cells Modification of FOXP3 expression: These cells are created by exogenously introducing the FOXP3 gene or by enhancing the endogenous expression of FOXP3. This modification is achieved using gene editing technology (e.g., CRISPR / Cas9) or viral vectors (e.g., retroviruses, lentiviruses). Expression of mutants or parts: Not only the full-length protein of FOXP3, but also parts of it (e.g., functional domains) or mutants may be expressed, making it possible to emphasize only specific functions. Immunological characteristics of FOXP3-T cells Immunosuppressive function: FOXP3 expression makes these cells different from conventional T cells ｅｆｆ Unlike cells, they may have the function of suppressing excessive inflammatory responses. Differentiation flexibility: By expressing FOXP3, FOXP3-T cells have some characteristics similar to conventional Treg cells, but also T ｅｆｆIt can retain cell-specific effector functions. Application Fields FOXP3-T cells can be applied to various medical fields such as: Autoimmune diseases: They may be able to suppress excessive immune responses and treat autoimmune diseases (e.g., rheumatoid arthritis, type 1 diabetes, multiple sclerosis). Transplant medicine: Used to suppress rejection reactions in organ transplantation and hematopoietic stem cell transplantation. Cancer immunotherapy: Conventional T ｅｆｆ By maintaining the antitumor activity of cells while preventing excessive inflammatory responses, it improves the safety and efficacy of immunotherapy. In chronic inflammatory diseases such as Crohn's disease and ulcerative colitis, it is expected to suppress pathogenic immune responses.

[0112] In a typical embodiment of this disclosure, FOXP3-T cells may be produced and evaluated in the following steps: Gene transfer: The FOXP3 gene is introduced into T cells. Selection and amplification: Cells expressing FOXP3 are selected and amplified as necessary. Functional evaluation: Immunosuppressive activity and retention of effector function are evaluated as indicators of cytokine production and cytotoxicity. Precautions While FOXP3-T cells have immunomodulatory functions, if not properly controlled, excessive immunosuppression may occur, potentially increasing the risk of infection and tumors. Therefore, it is necessary to carefully evaluate their safety and efficacy.

[0113] In one embodiment of the present disclosure, FOXP3-T cells (T ｅｆｆ ) can be defined as having reduced or substantially absent expression of at least one immunosuppressive gene. As described above, effector T cells (T ｅｆｆ By modifying the T cells to express Foxp3 and / or to enhance Foxp3 expression, effector T cells (T) can be modified. ｅｆｆ In this case, the metabolic mechanism of Treg cells can also be utilized. On the other hand, the function of T cells may also become like that of Treg cells, and changes in effector function may occur. Therefore, in one embodiment of this disclosure, in order to maintain effector function while preventing the exertion of immunosuppressive function like that of Treg cells, effector T cells (T ｅｆｆ) can also be modified and / or derivatives of Foxp3 introduced into them. In other embodiments, FOXP3-T cells (T) of the Disclosure ｅｆｆ In this context, effector function can also be maintained by reducing or substantially eliminating the expression of immunosuppressive genes.

[0114] In one embodiment, the immunosuppressive gene is not particularly limited as long as it is a gene that expresses a molecule that functions suppressively 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.

[0115] In one embodiment of this disclosure, the FOXP3-T cells of this disclosure can be human effector T cells.

[0116] <Chimeric Antigen Receptor (CAR)> In other aspects of this disclosure, T cells are provided in which the FOXP3-T cells of this disclosure are modified to include a chimeric antigen receptor (CAR). The CAR included in the FOXP3-T cells of this disclosure may be included as a protein or as a nucleic acid molecule expressing the CAR, insofar as it is able to perform its function as a CAR. In one embodiment, the CAR of this disclosure may be expressed in effector T cells.

[0117] The CARs disclosed herein include at least one extracellular domain capable of binding to an antigen, at least one transmembrane domain, and at least one intracellular domain.

[0118] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody linked to a T cell signaling domain via a transmembrane domain. A key characteristic of CARs is their ability to redirect the specificity and responsiveness of T cells toward a selected target by leveraging the antigen-binding properties of monoclonal antibodies, independently of MHC (microcellular carcinoma). This MHC-independent antigen recognition can give CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus evading tumor immune escape.

[0119] The intracellular T cell signaling domain of a CAR may 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 the CAR that includes the intracellular domain of the T cell receptor, such as the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand, required for the efficient response of lymphocytes to an antigen.

[0120] (Extracellular Domain) In one embodiment, the CAR used in FOXP3-T cells of the present disclosure includes an antigen-binding domain or a portion thereof. The antigen-binding domain or a portion thereof can be appropriately selected depending on the type and number of ligands on the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR of the present disclosure include tissue-specific markers, tumor-specific markers, viral, bacterial and parasitic infections, autoimmune diseases, and those associated with cancer cells.

[0121] The extracellular binding domain of CAR may consist of a single-chain variable fragment (scFv) obtained by fusing the variable weight and light regions of a mouse or humanized monoclonal antibody. Alternatively, a Fab-derived scFv (obtained from a Fab library, for example, rather than from an antibody) may be used. The scFv can be fused to the transmembrane domain and then to the intracellular signaling domain.

[0122] In one embodiment, the antigen-binding domain portion of the CAR of this disclosure includes: (1) alloantigens including MHC class I and MHC class II; (2) extracellular autoantigens including TSHR (thyroid stimulating hormone receptor), DSG3 (desmoglein 3), and Cytokeratin 8; (3) exogenous antigens including Gliadin and Ara h2; and (4) CD4, CD8, CD19, BCMA, CD68, MSLN (mesothelin), and MadCam1 (mucosal vascular addressin cell adhesion molecule). 1) While it is possible to target antigens containing targeting molecules such as the above, the antigen-binding domain portion of the CAR of this disclosure is not limited to these antigens that can be targeted.

[0123] In one embodiment, depending on the desired antigen to be targeted, the CAR of this 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 may be used as the antigen-binding domain in the CAR. Not limited to the following examples of cancer antigens: CD19, CD20, CD30, CD33, CD38, CD133, BCMA, TEM8, EpCAM, ROR1, folic acid receptor, CD70, MAGE-1, MAGE-2, MAGE-3, MAGE A-10, MAGE-C2, MAGE-A12, CEA, Tyrosinase, Midoquin-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, IL13Ralph, IL13Ralph2, AIM-2, AIM-3, NY-ESO-1, C9orfl l2, SART1, SART2, SART3, BRAP, RTN4, GLEA2, TNKS2, KIAA0376, ING4, HSPH1, C13orf24, RBPSUH, C6orfl53, NKTR, NSEP1, U2AF1L, CYNL2, TPR GOLGA, BMI1, COX-2, EGFRvIII, EZH2, LICAM, Livin, LivinP, MRP-3, Nestin, OLIG2, AR T1, ART4, B cycling, Grill, Cav-1, Cathepsin B, CD74, E-Cadherin, EphA2 / Eck, Fra-1 / Fosl Examples include GAGE-1, ganglioside / GD2, GnT-V, pl, 6-N, Ki67, Ku70 / 80, PROXI, PSCA, SOXIO, SOX11, Survivin, phCG, WT1, mesoserine, melan A, NY-BR-1, NY-CO-58, MN (gp250), telomerase, SSX-2, PRAME, PLK1, VEGF-A, VEGFR2, and Tie-2. In some embodiments, the effector cells disclosed herein are engineered to express one or more CARs to recognize one or more antigens.

[0124] (Transmembrane domains) The CAR used in the FOXP3-T cells of this disclosure may include one or more transmembrane domains fused to the extracellular domain.

[0125] In one embodiment, a linker domain derived from an extracellular domain may be linked to a transmembrane domain. The transmembrane domain may be natural or synthetic, and a natural transmembrane domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions particularly used in this disclosure may be derived from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19, etc.

[0126] In one embodiment, the CAR used in the inducible regulatory T cells disclosed herein may also have a spacer domain positioned 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 the binding of the CAR to the antigen and enhances signal transduction into the cell.

[0127] (Intracellular Domain) The cytoplasmic signaling domain (or intracellular signaling domain) of CAR is involved in the activation of at least one normal effector function of immune cells expressing CAR. The intracellular signaling domain refers to a portion of a protein that transmits effector function signals and instructs cells expressing CAR to perform specialized functions. The intracellular signaling domain may include any complete, mutant, or cleaved portion of the intracellular signaling domain of a given protein sufficient to transmit signals that induce or block immune cell effector function.

[0128] In one embodiment, an example of an intracellular signaling domain used in a CAR is a cytoplasmic signaling sequence of a T cell receptor (TCR) and a co-receptor that induces signaling after antigen receptor binding.

[0129] (Alloantigens, allergens, and haptens related to rejection) The CARs used in effector T cells disclosed herein include those related to alloantigens, allergens, and haptens related to rejection.

[0130] <Pharmaceutical Uses of FOXP3-T Cells Containing Chimeric Antigen Receptors (CARs)> In one aspect of this disclosure, a pharmaceutical composition comprising FOXP3-T cells of the Disclosure is provided, wherein the T cells are modified to express Foxp3 and / or the expression of Foxp3 is enhanced and the immune effector function is enhanced. In another aspect of this disclosure, a pharmaceutical composition comprising FOXP3-T cells of the Disclosure is provided, wherein the T cells contain a chimeric antigen receptor (CAR), and the T cells are modified to express Foxp3 and / or the expression of Foxp3 is enhanced and the immune effector function is enhanced. In one embodiment of this disclosure, the FOXP3-T cells of the Disclosure may have one or more features of the FOXP3-T cells of the Disclosure as described above.

[0131] In another aspect, the present disclosure provides a therapeutic agent comprising FOXP3-T cells or a cell population, which diagnoses a target disease and selects an appropriate CAR included in the effector T cells or the cell population based on the diagnosis.

[0132] In another embodiment, the Disclosure provides a pharmaceutical composition comprising any of the FOXP3-T cells described herein and a pharmaceutically acceptable carrier. If 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 to a tumor antigen, a pathogen antigen, or an autoantigen. The pathogen antigen may be a bacterial antigen, a viral antigen, or a fungal antigen.

[0133] In one embodiment, the Fc-containing therapeutic agent is adalimumab, adtrastuzumab emtansine, alemtuzumab, basiliximab, bevacizumab, belimumab, brentuximab, canakinumab, cetuximab, certolizumab, daclizumab, denosumab, dinutuximab, eculizumab, efalizumab, epratuzumab, gemtuzumab, golimumab, hu14.18K322A, ibrit Therapeutic antibodies may include, but are not limited to, momab, infliximab, ipilimumab, rabetuzumab, muromonab, natalizumab, obinutuzumab, ofatumumab, omalizumab, palivizumab, panitumumab, pertuzumab, ramucirumab, ranibizumab, rituximab, tocilizumab, trastuzumab, tocitumomab, ustekinumab, mogamulizumab, and vedolizumab.

[0134] Furthermore, the Disclosure provides a kit comprising (i) a first pharmaceutical composition comprising FOXP3-T cells and a pharmaceutically acceptable carrier as described herein, and (ii) other therapeutic agents and pharmaceutically acceptable carriers as described herein.

[0135] In other aspects of the Disclosure, methods are provided for inhibiting cells expressing a target antigen in a subject (e.g., reducing the number of such cells, inhibiting cell proliferation, and / or suppressing cell activity), which include administering the cells, cell populations, and / or pharmaceutical compositions of the Disclosure to the subject. In one embodiment, at least a portion of the cells expressing the target antigen may be placed in a low-glucose environment.

[0136] In one embodiment, the subjects treated by the method of this disclosure may be human patients suffering from cancer, such as carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Exemplary target cancers include, but are not limited to, B-cell 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 cancers include B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin lymphoma.

[0137] In addition to treating targeted diseases or disorders such as cancer or infectious disorders, the use of the T cells or cell populations of this disclosure to manufacture pharmaceutical products for the medical treatment of interest is also within the scope of this disclosure.

[0138] In one embodiment, a pharmaceutical composition comprising the FOXP3-T cells of the Disclosure, comprising the chimeric antigen receptor (CAR) of the Disclosure, can be used in cell therapy. In cell therapy, the FOXP3-T cells of the Disclosure, comprising the chimeric antigen receptor (CAR) of the Disclosure, can be injected into a subject in need, either as a pharmaceutical composition or as a preparation of a therapeutically effective cell population expressing the CAR of the Disclosure. The injected effector T cells in the subject may treat a target disease or disorder, such as cancer or infectious disorder, in that subject. The subject may be the same subject from which the cells were obtained (autologous cell therapy), or the cells may be derived from a different subject of the same species (allogeneic cell therapy).

[0139] In one embodiment, the FOXP3-T cells or population thereof, including the CAR of the Disclosure, may be formulated for administration to a subject using techniques known to those skilled in the art. In one embodiment, a formulation containing therapeutically effective effector T cells or population thereof, including the CAR of the Disclosure, may contain pharmaceutically acceptable excipients (carriers or diluents). The excipients included in the formulation may serve different purposes, for example, depending on the properties of the antigen-binding domain of the CAR of the 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, isotonic agents, bulking agents, and lubricants.

[0140] Formulations comprising therapeutically effective FOXP3-T cells or populations thereof, including CARs, may be administered to a subject using methods and techniques known to those skilled in the art. Exemplary methods include, but are not limited to, intravenous injection. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular (articular), intrasynovial (synovial fluid segment), intracranial, intraspinal, and intrathecal (cerebrospinal fluid) administration.

[0141] (General Techniques) The molecular biological, biochemical, and microbiological techniques used herein are well-known and commonly used in the field, 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, and the special issue of Experimental Medicine, "Experimental Methods for Gene Transfer & Expression Analysis," Yodosha, 1997, are described herein, and relevant parts (possibly all) of these are referenced.

[0142] For DNA synthesis techniques and nucleic acid chemistry to produce artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other resources include, for example, 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, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press. This is described in Hermanson, GT(I996). Bioconjugate Techniques, Academic Press, etc., and the relevant parts of these are incorporated herein by reference.

[0143] In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else". In this specification, when "within the range" of "two values" is specified, that range includes the two values ​​themselves. References cited in this specification, such as scientific literature, patents, and patent applications, are incorporated herein by reference in the same degree as they are described specifically.

[0144] The present disclosure has been described above with reference to preferred embodiments for ease of understanding. The present disclosure will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present disclosure. Accordingly, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0145] Examples are described below. First, the methods and materials used throughout are explained. The materials can be obtained from sources other than those specifically mentioned, or they can be prepared in-house.

[0146] (Example 1: Example of FOXP3-T cells) This example describes the preparation and characterization of an example of FOXP3-T cells. The methods and materials are shown below. These methods and materials will also be used as appropriate in Examples 2 and beyond. (Methods and Materials) Preparation of FOXP3-CAR construct and viral vector Preparation of a wild-type FOXP3 tv1 sequence (NM_014009.4) (WT) with a P2A sequence positioned directly above an anti-CD19 CAR backbone plasmid (pLVSIN-CD19BBz-tEGFR) (CART) having a CD8 leader sequence-anti-CD19 single-chain variable region (scFv)-CD8 hinge-CD8 transmembrane region (TM)-4-1BB intracellular domain (ICD)-CD3z-P2A-transcribed EGFR (tEGFR) structure We constructed pMSGV-CD19CAR-mutant FOXP3tv1-tEGFR retroviral vectors (Mut #1-18) by introducing CART-FOXP3 or various FOXP3 mutations (see Table 3) that induce loss of binding ability between FOXP3 and NFAT and RUNX1. The retroviral vectors were transfected into Phoenix-Ampho cells with Lipofectamine 2000 (Invitrogen), and retroviruses were obtained from the supernatant.

[0147] In silico structural analysis, amino acid sequences of FOXP3, RUNX1, and NFAT1 were obtained from UniProt. Using Alphafold Server, Dimer structures were predicted for wild-type FOXP3 and various FOXP3 mutants using Alphafold3, and further structural predictions were made for when RUNX1 or NFAT1 is bound to the FOXP3 dimer.

[0148] Gene-transfected peripheral blood mononuclear cells from healthy individuals 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 / well of retroviral supernatant was adsorbed onto 12-well plates coated with RetroNectin (10 μg / well, Takara), and then T cells were transfected to introduce the CAR gene. In some experiments, CAR-positive cells were purified using tEGFR. Cells were cultured in the presence of IL-2 100 U / ml and then cryopreserved or directly subjected to evaluation on days 8-12. For the shCTLA-4 and ShTGF-β1 lentiviral vectors obtained by the following method, a lentiviral solution was added on day 1 and transduction was performed. For sgCTLA-4 and sgTGF-β1, after removing the anti-CD3 / 28 beads on day 5, 1.0 × 10⁶ beads were added on day 6. ７ RNP complexes were prepared using 10 μg of Alt-R sgRNA and 40 μg of Alt-R Cas9 nuclease per cell, and electroporation was performed using BTX Gemini.

[0149] (Preparation 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 oligoDNA was created by inserting the shRNA sequence, with a loop structure in between, between the restriction enzymes BamHI and EcoRI (Tables 1 and 2).

[0150]

[0151]

[0152] These were inserted into a pLVSIN-mU6-MCS lentiviral vector to create a lentiviral vector, which was then transfected into 293 T cells along with the package 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. After stimulation, shCTLA-4 lentivirus solution was added to each cell on day 1, and on day 4, flow cytometry (FCM) was used to select shCTLA-4_#4 (target sequence 5'-GGGAATTCATCTCTCTTTTAATAT-3' (SEQ ID NO: 23)), which showed the lowest expression of CTLA-4. Furthermore, Tl-su cells, a cell line for adult human T-cell leukemia, were cultured on day 0 with shTGFβ1 lentivirus solution added, and X-vivo was used as the media. On day 3, the supernatant was stimulated with 1 mol / l HCl, and TGF-β1 levels were measured by ELISA using the Human TGF-β1 Quantikine ELISA Kit (R&D#DB100B). The shTGF-β1_#1 (target sequence 5'-ACCAGAAATACAGCAACAAATTCCTG-3' (SEQ ID NO: 24)), which contributed most to the decrease in TGF-β1, was selected.

[0153] The sgRNA sequences for sgCTLA-4 / TGF-β1 were determined based on the CCRISPR-Cas9 guide RNA design checker (https: / / sg.idtdna.com / site / order / designtool / index / CRISPR_SEQUENCE) and CTLA-4 / TGF-β1 knockout human CTLA-4tv2 (NM_001037631.3) and human TGF-β1 (NM_000660) (Tables 3 and 4).

[0154]

[0155]

[0156] An RNP complex was created using 8 μg of Alt-R sgRNA and 32 μg of Alt-R Cas9 nuclease, and Tl-su cells (1.0 × 10⁶) were used. ６ Electrification was performed on the cells using BTX Gemini. For Sg CTLA4-transformed cells, CTLA-4 expression was evaluated using FCM on Day 3. For sg TGF-β1-transformed cells, X-vivo was cultured as the medium, and TGF-β1 was quantified by ELISA using the same method as ShTGF-β1 Knockdown. Based on these results, sgCTLA4_#1 (target sequence 5'-GTGCGGCAACCTACATCATGGGG-3' (SEQ ID NO: 25)) and sgTGF-β1_#3 (target sequence 5'-CGGGAGAGCAACACGGGGTTC-3' (SEQ ID NO: 26)), which contributed most to the decrease in CTLA4 and TGF-β1 expression, were selected.

[0157] Phenotype analysis (FCM) The antibodies used in the FCM analysis are shown in (Table 5).

[0158]

[0159] Cell washing was performed using 4% FBS-PBS. After dead cell staining, surface staining was performed, followed by fixation wells and cytoplasmic staining using the FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cell data was acquired using LSR Fortessa X20 and analyzed using FACS Diva (v8.0.1, BD Biosciences) and FlowJo (TreeStar) software.

[0160] Phenotype Analysis (FCM) The antibodies used for FCM analysis are shown in Table 1. Cell washing was performed using 4% FBS-PBS. After dead cell staining, surface staining was performed, followed by fixation wells and cytoplasmic staining using the FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cell data was acquired using LSR Fortessa X20 or BD FACSymphony A3, and the data was analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0161] Cytokine analysis (FCM) CART cells 1 x 10 ６ The cells were divided into two sets of 2 x 10⁶ cells from a CD19-positive leukemia cell line (Nalm6). ６ Cells were cultured in PMA 50 ng / ml (SIGMA P8139) / Ionomycin 1 μM (SIGMA 10634) or a medium, stimulated for 6 hours in the presence of 5 μg / ml Monensin (BD 554724 BD Bioscience), and intracellular cytokines were stained with eBiosscience Foxp3 / Translation Factor Staining Buffer Set (Thermo Fisher Scientific). Data were acquired using LSR Fortessa X20 and analyzed using FACS Diva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0162] Cytotoxic activity analysis: 1 x 10⁶ target cells (GFP-positive CD19-expressing Aspc-1 cells) per well in a 96-well plate.４ Cells were seeded and co-cultured with CART cells under high-sugar conditions (RPMI + 10% human AB serum) at E:T ratios of 3:1, 1:1, 0.3:1, and 0.1:1, and under low-sugar conditions (glucose-free RPMI [Wako] + Glucose 0.5 mM [Wako] + lactate 15 mM [Sigma] + 10% human AB serum) at E:T ratios of 10:1, 3:1, 1:1, and 0.3:1. After 24 hours, the cells were thawed with Cell Culture Lysis Reagent (Promega), and luciferase activity was measured using a GloMax Navigator microplate luminometer (Promega), and cytotoxic activity was calculated.

[0163] Cell culture in a high-fatty acid environment: CART cells 1 × 10 ６ The cells were divided into CD19-positive leukemia cell line (Nalm6) 1 x 10⁶ ５ Cells were cultured for 72 hours in a low-sugar, high-fatty acid environment (glucose-free RPMI [Wako] + Glucose 0.5 mM [Wako] + palmitic acid 0.2, 0.5, 1, 2 mM [Fuji]) + 10% human AB serum in the presence of IL-2 100 U / ml. After culture, metabolic analysis, phenotypic analysis (FCM), and CFSE T cell proliferation assay were performed on the cultured cells using an XF96 cell flux analyzer (Biosscience) to evaluate their function.

[0164] Cell culture in a high-lactic acid environment: CART cells 1 × 10 ６ The cells were divided into a pancreatic cancer cell line (Aspc-1) 1 x 10⁻¹⁰ ５ Cells were cultured for 96 hours in a low-sugar, high-fatty acid environment (glucose-free RPMI [Wako] + Glucose 0.5 mM [Wako] + Lactate 40 mM [Sigma]) + 10% human AB serum in the presence of IL-2 100 U / ml. After culture, metabolic analysis, phenotypic analysis (FCM), and CFSE T cell proliferation assay were performed on the cultured cells using an XF96 cell flux analyzer (Biosscience) to evaluate their function.

[0165] Cell culture by repeated stimulation: CART cells 1 x 10 ６The cells were divided into a pancreatic cancer cell line (Aspc-1) 1 x 10⁻¹⁰ ６ Cells were cultured in RPMI + 10% human AB serum in the presence of IL-2 100 U / ml. Under similar conditions, pancreatic cancer cell line (Aspc-1) 1 × 10⁶ cells were cultured every 96 hours. ６ Cell stimulation was repeated a total of nine times. Phenotype analysis (FCM) and CFSE T cell proliferation assay were performed on these cells to evaluate their function.

[0166] Metabolic activity of cells for metabolic analysis was measured according to the product protocol using the XF96 Cell Flux Analyzer (Biosscience), Seahorse XF T Cell Metabolic Profiling Kit (Agilent Technologies), and Seahorse XF Substrate Oxidation Stress Test Kits (Agilent Technologies). On the day before analysis, the 96-well flat-bottom plates for analysis were coated with poly-L ornithine (0.1 mg / ml), and the sensor cartridges were hydrated in a CO2-free incubator. On the day of analysis, the cells for analysis were washed using analysis medium (Seahorse XF RPMI Medium, D(+) Glucose 0.5 mM, L-Glutamine 2 mM), and then 2 × 10⁶ CART cells were placed on an analysis plate coated with poly-L-ornithine. ５ After sowing the cells one by one, they were left to stand for 60 minutes at 37°C in a CO2-free environment. The Seahorse XF T Cell Metabolic Profiling Kit was adjusted to contain 13.5 μM of Oligomycin A, 25 μM of Bam15, and 5.5 μM of Rcteneone / Antimycin A. The Seahorse XF Substrate Oxidation Stress Test Kit was adjusted to contain 40 μM of Etomoxir, 15 μM of Oligomycin, 20 μM of FCCP, and 5 μM of Rcteneone / Antimycin A. Metabolic function was then evaluated using a flux analyzer, with OCR, ECAR, and PER being used for analysis.

[0167] Bodypy-FL analysis: To evaluate fatty acid uptake and content, CART cells were analyzed at a rate of 1 × 10⁶ ６ Cells were seeded in 96-well plates, and 200 μl of gluten-free RPMI 1640 (Wako) was mixed with 25 μM of Bodypy-FL (Thermo Fisher Scientific) and incubated at 37°C for 30 minutes. After washing twice with PBS, analysis was performed using FCM. Data was acquired using LSR Fortessa X20 and analyzed using FACS Diva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0168] 2-NBDG analysis: To evaluate fatty acid uptake and content, CART cells were analyzed at a rate of 1 × 10⁶ ６ Cells were seeded in 96-well plates, and 200 μl of glucose-free RPMI1640 (Wako) was mixed with 100 μM of 2-NBDG (Thermo Fisher Scientific) and incubated at 37°C for 30 minutes. After washing twice with PBS, analysis was performed using FCM. Data was acquired using LSR Fortessa X20 and analyzed using FACS Diva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0169] CFSE T cell proliferation assay: CART cells 1 × 10 ６ Cells were prepared in batches, and PBS was mixed with CFSE to a concentration of 5 μM. The cells were stained under light-shielding conditions for 30 minutes. Subsequently, the CD19-expressing leukemia cell line Nalm6 was used for staining. (0.3 × 10⁶ cells) ６Cells were mixed with various mediums and cultured for 72 hours in the presence of (1) RPMI (Wako) + 10% human AB serum, (2) glucose-free RPMI [Wako] + Glucose 0.5 mM [Wako] + lactate 15 mM [Sigma] + 10% human AB serum, (3) glucose-free RPMI [Wako] + Glucose 0.5 mM [Wako] + palmitic acid 0.12 mM [Fuji] + 10% human AB serum. After washing twice with PBS, the cells were analyzed by FCM. Data was acquired using an LSR Fortessa X20 and analyzed using FACS Diva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0170] Quantitative PCR analysis. The primers used for PCR are shown in Table 2. CART cells were analyzed in 3 × 10⁶ steps. ６ We prepared cells in batches and performed RNA purification using RNeasy Kits (Quigen). 100 ng of purified RNA was mixed with 2 μl of PrimeScript RT Master Mix (Takara) and reverse transcription was performed. 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. This mixture was then diluted with Ambion Nuclease-Free Water (Life Technologies) to a total volume of 20 μl, and mRNA was quantified using QuantStudio6 (Applied Biosynthemes).

[0171] scRNAseq cCART and WT CART-FOXP3 1×10 ５Cells were subjected to emulation, cDNA purification, and library purification using the 10x Chromium 3' Library and Gel Bead Kit (10x Genomics) according to the product protocol. Subsequently, sequencing was performed using Illumina Novaseq 6000, and FASTQ files were obtained using Cell Ranger (Ver. 4.0.0, 10x Genomi). 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 by principal component analysis, and the results were displayed using UMAP. The obtained gene expression profiles were compared using Single Cell Gene Set Enrichment Analysis (scGSEA) with the Escape package (Ver. 1.12.0).

[0172] RNAseq CAR-T cells 4×10 ６ RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN). Library preparation was performed using the TruSeq® stranded Total RNA Library Prep. Kit (illumina), and RNAseq was performed using NovaSeq X plus. RNAseq data were paired-end sequenced and mapped to hg38 using bowtie2. Gene expression levels were measured using feature counts, and intergroup comparisons were performed using edgeR. GSEA analysis was performed using fgsea package (Ver. 1.30.0).

[0173] Xenograph mouse model: Human pancreatic cancer cell line Aspc-1 expressing CD19 antigen in 6-8 week old NOD / scid / IL2rγ- / - (NSG) mice, 2 x 10⁻¹⁰ ６ After subcutaneous administration of cells, CAR-T cells 2 x 10⁶ were observed 4 weeks later. ６Cells were administered intravenously. In addition, MSCV Myc T58A puro (Addgene) was introduced into Aspc-1 to create cMYC-high-expression Aspc-1, and 2 × 10⁻¹⁰ cells were produced. ６ After subcutaneous administration of cells, CAR-T cells 2 x 10⁶ were observed 4 weeks later. ６ Cells were administered intravenously. Tumor size was measured as long diameter × short diameter. ２ The measurement was taken once a week, divided by two.

[0174] Isolation and Analysis of Tumor-Infiltrating Lymphocytes: In NSG mice engrafted with the human pancreatic cancer cell line Aspc-1, tumors were excised one week after administration to CAR-T cells. The shredded tumor tissue was reacted with TTDR (BD bioscience) for 30 minutes with agitation, then a reaction stop solution was added, and the tissue fluid was filtered to isolate tumor-infiltrating lymphocytes. The isolated tumor-infiltrating lymphocytes were stained for dead cells, followed by surface staining, and then fixation holes and cytoplasmic staining using a FOXP3 transcription factor staining buffer set (Thermo Fisher Scientific). After washing, cells were acquired using LSR Fortessa X20 or BD FACSymphony A3, and analyzed using FACSDiva (v9.0, BD Biosciences) and FlowJo (TreeStar) software.

[0175] The primers and other materials used are as follows:

[0176]

[0177]

[0178] (Results) 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 the effector function of T cells in non-Treg cells, thereby enhancing the antitumor effect. The effect of CAR-T cell therapy in solid tumors is limited. This is because solid tumors form a unique immune microenvironment, creating a hypoxic / low glucose / high lactate environment, which leads to exhaustion and apoptosis of CAR-T cells, whose metabolism depends on glycolysis, resulting in dysfunction. On the other hand, regulatory T cells (Treg), unlike CAR-T cells, efficiently infiltrate the tumor microenvironment and become activated. This is because they can overcome metabolic checkpoints by using fatty acids and lactate present in the tumor microenvironment as nutrients, rather than relying solely on glucose, and because enhanced infiltration function through increased expression of chemokine receptors such as CCR4 and CCR8 allows them to maintain infiltration and activation in the harsh tumor microenvironment. In this disclosure, by expressing FOXP3, the master regulator of Treg, in T cells, the effector function of CAR-T cells is enhanced and the antitumor effect is increased by conferring various functions of FOXP3, such as metabolic reprogramming and chemokine receptor expression, to CAR-T cells.

[0179] Figure 2 shows FOXP3-overexpressing CAR-T cells (CAR-containing FOXP3-T cells as disclosed herein). In this embodiment, FOXP3, the master regulator of Treg, and Truncated EGFR were linked to the CAR gene via P2A and T2A sequences to establish FOXP3-overexpressing CAR-T cells. It was expected that overexpression of FOXP3 would confer a variety of Treg-specific functions, such as metabolic reprogramming and chemokine receptor expression, to CAR-T cells. On the other hand, it is conceivable that the introduction of FOXP3 may impair the antitumor effect by stimulating the expression of immunosuppressive molecules such as CTLA4 expression and TGFβ1 production. Therefore, we decided to investigate whether it is possible to efficiently reduce only immunosuppressive molecules and enhance the antitumor effect by inducing a decrease in the expression of CTLA4 and TGFβ-1 using the CRISPR / Cas9 system or RNA interference, or by introducing mutations into FOXP3.

[0180] As shown in Figure 2, T cells expressing FOXP3 (FOXP3-T cells) are produced by expressing FOXP3, the master regulator of regulatory T cells (Treg cells), in T 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 present in parental T cells (T cells before modification) and / or induced by FOXP3. Representative examples of the former include the expression of Treg cell-like inhibitory cytokines and inhibitory molecules, while representative examples of the latter include metabolic reprogramming, tumor invasion and survival ability, and cytotoxic activity / cytokine production ability.

[0181] Figure 3 shows T cells in which negative regulatory factors of FOXP3-T cells are suppressed or abandoned, and positive regulatory factors are maintained or enhanced. As shown, in order to selectively obtain effector functions that are important for the antitumor immune response, negative regulatory factors of effector functions are suppressed or abandoned using gene editing (CRISPR / Cas9, etc.) or RNA interference (shRNA, etc.) (A). Alternatively, FOXP3 derivatives modified to selectively maintain or enhance positive regulatory factors without inducing negative regulatory factors (mutant FOXP3 or partially deleted FOXP3) are introduced (B). These methods establish T cells with enhanced antitumor activity (including genetically modified T cells such as CAR-T cells).

[0182] (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, whose expression is controlled by the transcription factor FOXP3 (here, the genes for the metabolic transporters CD36, 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 makes it impossible to introduce multiple factors that enhance effector function controlled by the numerous FOXP3-regulated factors in a cumulative manner.

[0183] Figure 5 shows the increased expression of factors (immunosuppressive molecules) that negatively regulate effector function as a side effect of introducing wild-type FOXP3, and the suppression and abandonment of these factors using RNA interference. When comparing the characteristics of conventional CAR-T cells (normal CAR-T cells and cCAR-T cells without FOXP3-related manipulation) with wild-type FOXP3-introduced CART cells (WT), it was confirmed that the expression of Treg cell-like immunosuppressive molecules that can negatively regulate FOXP3-induced effector function, such as CTLA4 and CD25, increased. It was also confirmed that the expression of immunosuppressive molecules such as CTLA4 and TGFβ1 could be simultaneously suppressed by RNA interference using ShRNA.

[0184] As shown in Figure 6, the characteristics of wild-type FOXP3-introduced CAR-T cells are explained. The characteristics of conventional CAR-T cells (CAR-T cells without FOXP3 introduction, labeled as cCAR) and wild-type FOXP3-introduced CAR-T cells (CART-FOXP3, labeled as WT) were compared. FCM analysis showed that WT cells exhibited increased FOXP3 expression (a), an increase in the memory T cell fraction that ensures long-term antitumor activity (b), a decrease in the proportion of exhaustion markers PD-1+ and TIM-3+ cells (c), decreased expression of TOX (d), and increased expression of chemokine receptors (CCR4, CCR8) that induce T cell migration within tumors (e). Furthermore, WT cells showed impaired glycolysis, including decreased expression of the glucose transporter GLUT1 and decreased glucose uptake capacity (2-NBDG). On the other hand, increased CPT1A expression and increased fatty acid uptake capacity (BODIPY-FL) suggested enhanced fatty acid oxidation function (f). Analysis using an extracellular flux analyzer revealed enhanced oxidative phosphorylation (OCR) activity under a low-sugar environment (Glu 0.5 mM RPMI) (g). Furthermore, when fatty acid oxidation was suppressed by adding etomoxir, the maximum OCR value decreased only in WT, suggesting that the enhanced oxidative phosphorylation activity in a low-sugar environment in WT may be dependent on fatty acid oxidation (h). GSEA using scRNAseq also showed increased expression of genes involved in oxidative phosphorylation and fatty acid oxidation in WT compared to cCAR (i).

[0185] 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, we incorporated wild-type FOXP3 via 2A sequencing to create an all-in-one construct that expresses CD19CAR, wild-type FOXP3, and a gene transfer marker (tEGFR) (A). To suppress negative regulators of effector function, we constructed constructs combining RNA interference, the CRISPR / Cas9 system (B), and dominant negative TGFβR (C). In addition, we constructed CAR-FOXP3 constructs incorporating mutant FOXP3 (D) and partially deleted FOXP3 (E) that suppress negative regulators of effector function and selectively maintain or enhance positive regulators. CD19scFv is replaceable with other scFvs (any scFv, such as anti-mesothelin, anti-ROR1, anti-EGFRRvIII, etc.). As shown in Figure 7, an all-in-one vector was created that simultaneously expresses three elements: CD19CAR, Foxp3, and a gene transfer / selection marker (tEGFR), by incorporating Foxp3 via a 2A sequence into a second-generation anti-CD19CAR construct that uses the metabolism-enhancing CAR construct 41BB and CD3z as signaling molecules. Furthermore, methods for incorporating shCTLA-4 and shTGFb under the U6 promoter, and knocking out repressors with CRISPR / Cas9 were also employed. In addition, a dnTGFb construct that blocks the TGF-β signal was also constructed. CD19scFv is replaceable with other scFvs (such as anti-mesothelin).

[0186] Figure 8 shows the increased expression of chemokine receptors after FOXP3 introduction. As shown, FCM was used to compare the expression of CCR4, CCR5, CCR8, and CXCR3 in CD19 CART cells (CART) and CD19 CART cells introduced with FOXP3 (CART-FOXP3). Compared to 19 CART (CART), increased expression of the above chemokine receptors (CCR4, CCR5, CCR8, CXCR3) was observed in 19 CART-FOXP3 (CART-FOXP3).

[0187] Figure 9 shows the cytokine production capacity of FOXP3-introduced cells. The cytokine production capacity of CART-FOXP3 cells was evaluated by intracellular staining. CART-FOXP3 cells showed cytokine production capacity equivalent to or better than that of conventional CD19 CART cells.

[0188] Figure 10 shows the antitumor activity of FOXP ART cells. As shown, FOXP3-introduced CD19 CART cells (19CART-Foxp3) showed comparable cytotoxic activity to conventional CART cells (CART). Furthermore, suppression of PD-1 expression, an immune checkpoint molecule, was observed. In addition, a tumor suppression effect was observed in a xenograft model.

[0189] Figure 11 shows the identification of high-efficiency sh-CTLA4 and sh-TGFb. As shown, in this example, multiple shRNAs were constructed (see Tables 2 and 3), and high-efficiency shRNAs were identified for each.

[0190] Figure 12 shows the highly efficient identification of sg-CTLA4 and sg-TGFb. Multiple shRNAs were constructed (see Tables 3 and 4), and complete removal of CTLA-4 and TGFb was successfully achieved using CRISPR-Cas9.

[0191] Figure 13 shows the enhancement of CART cell amplification by suppressing CTLA-4 and TGF-β. CTLA-4 and TGF-β were knocked down in CD19CART-Foxp3 cells using shRNA. Both knockout CD19CART-Foxp3 cells showed cell amplification superior to that of normal CART cells.

[0192] Figure 14 shows TGFb signaling blockade by the dominant-negative TGFb receptor (dnTGFRbR). Introducing dnTGFRbR into CD19CART-Foxp3 cells blocked TGFb signaling, resulting in increased IFN-γ production.

[0193] (Example 2: Example of a mutation in FOXP3) In this example, the structure of FOXP3 and the mutation site were examined, as shown in Figure 15.

[0194] (a) The three-dimensional structure of FOXP3 predicted using Alphafold3 is shown. It is suggested that FOXP3 forms a leucine zipper structure with dimers 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.

[0195] (b) (c) Structure of FOXP3 and mutation site. We hypothesized that introducing a FOXP3 mutation from the leucine zipper region to the forkhead region into CAR would inhibit complex formation with RUNX1 and NFAT1, allowing CAR-T cells to acquire the tumor-local survival function of regulatory T cells while reducing the immunosuppressive function impairment caused by FOXP3, thereby improving the antitumor effect. We also investigated the possibility of immunosuppressive function impairment by introducing FOXP3 mutations observed in IPEX syndrome, a fatal autoimmune disease associated with FOXP3 mutations.

[0196] (Example 3: Antitumor effect of mutant FOXP3-expressing CAR-T cells (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 cells (FOXP3-T cells containing CAR) using an NSG mouse model was demonstrated.

[0197] (a) Human pancreatic cancer cell line Aspc-1 (tumor burden 90-725 mm3) was infused into NSG mice, and the antitumor effects of Untruncated T cells (UTD), cCAR, WT, F325D, F331D, F331D, K332D, H334D, R337Q, F340D, Y342F, W348Q, M370I, A372P, R386H, R397W, E399R / E401A, and D409A mutations were examined. The R397W mutation showed a significant tumor reduction effect compared to all other groups. The K332D, R337Q, M370I, A372P, and R386H mutations showed antitumor effects equivalent to those of cCAR.

[0198] (b) Human pancreatic cancer cell line Aspc-1 (tumor burden 20-180 mm3) was infused into NSG mice, and the antitumor effects of the UTD, cCAR, WT, K332D, R337Q, Y342F, R356E, F367L, M370I, A372P, R386H, V396E, R397W, V398E, and V408E mutations were evaluated. Similar antitumor effects were observed for the cCAR, R356E, F367L, M370I, A372P, R386H, and R397W mutations.

[0199] (c) Intractable tumors (tumor volume 180-400 mm3) in which the oncogene MYC, which is involved in enhanced glycolysis and increased cell proliferation, was forcibly expressed in the human pancreatic cancer cell line Aspc-1 were infused into NSG mice, and the antitumor effects of cCAR, K332D, M370I, and R397W mutations were confirmed. It has been reported that the MYC expression model induces a low-sugar, high-lactate state in the tumor microenvironment by enhancing glycolysis. In this model, the R397W mutation showed a higher tumor reduction effect than all other groups.

[0200] (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 was measured as follows.

[0201] (a) The established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 for 72 hours in a CAR-T:Tumor ratio of 10:1 in a low-sugar, high-fatty acid environment (Glucose 0 mM, Palmitic acid 2 mM).

[0202] (b) Changes in CAR-T cell counts in the culture environment were evaluated in three donors. WT, K356E, F367L, A372P, and R397W mutations tended to have significantly higher viable cell counts compared to cCAR.

[0203] (c) CPT1A expression was evaluated by FCM in three donors at 72 hours of co-culture. CPT1A expression tended to be higher than that of cCAR in K356E, F367L, M370I, A372P, R386H, and R397W.

[0204] (d) Palmitic acid was added to Glucose 0 mM RPMI (0.2 mM, 0.5 mM, 1.0 mM), and the human leukemia cell line Nalm6 was reacted with CAR-T:Tumor = 10:1. The cells were then co-cultured for 72 hours, and CPT1A expression was confirmed. WT, F367L, and R397W mutations showed a tendency for CPT1A expression to increase in a fatty acid concentration-dependent manner, but this tendency was not observed in cCAR.

[0205] (e) Celltrace violet 2.5 μM was used for staining before Nalm6 stimulation, and expression was evaluated after 72 hours. The F367L, M370I, A372P, and R397W mutations showed higher mitotic activity compared to cCAR.

[0206] (f) The established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 for 48 hours in a CAR-T:Tumor ratio of 10:1 and a low-sugar, high-fatty acid environment (Glucose 0 mM, Palmitic acid 2 mM). The CAR-T cells were then transferred to Glu 0 mM RPMI, and their metabolic function was analyzed in two donors using an extracellular flux analyzer (Tcell Metabolic Profiling Kit). Donor 2 showed a tendency for increased OCR after BAM15 stimulation in WT, K356E, and R397W mutations, while Donor 3 showed a tendency for increased OCR after BAM15 stimulation in WT, F367L, M370I, A372P, and R386H mutations.

[0207] (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.

[0208] Figure 18 shows an example demonstrating metabolic activity in a high-lactic acid environment.

[0209] Established CAR-T cells were co-cultured with the human leukemia cell line Nalm6 for 48 hours in a CAR-T:Tumor ratio of 10:1 and a low-sugar, high-fatty acid environment (Glucose 0 mM, Palmitic acid 2 mM). After co-culture, the CAR-T cells were transferred to Glu 0 mM RPMI, 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). Donor 2 showed a tendency for increased OCR after BAM15 stimulation in WT, K356E, and R397W mutations, while Donor 3 showed a tendency for increased OCR after BAM15 stimulation in WT, F367L, M370I, A372P, and R386H mutations.

[0210] (Example 6: Oxidative phosphorylation activity in a high-lactic acid environment) This example demonstrates the oxidative phosphorylation activity of the cells of this disclosure in a high-lactic acid environment. Figure 19 shows another example of oxidative phosphorylation activity in a high-lactic acid environment. (a) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc1 for 96 hours in a CAR-T:Tumor = 10:1, low-sugar, high-lactic acid environment (Glucose 0 mM, Lactate 40 mM). (b) Changes in the number of CAR-T cells in the culture environment were evaluated in one donor. WT, F367L, M370I A372P, and R397W mutations tended to have a higher number of viable cells compared to cCAR. (C) Established CAR-T cells were co-cultured with the human pancreatic cancer cell line Aspc-1 for 96 hours in a CAR-T:Tumor = 10:1 low-sugar, high-fatty acid environment (Glucose 0 mM, Lactate 40 mM). After that, the CAR-T cells were transferred to Glu 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). The WT, K356E, F367L, M370I, A372P, R386H, and R397W mutations were suggested to exhibit aerobic respiration-dominant metabolic activity compared to cCAR when stimulated with FCCP.

[0211] (Example 7: Proliferative ability and phenotypic changes of CAR-T cells by repeated stimulation) In this example, the proliferative ability and phenotypic changes of CAR-T cells by repeated stimulation were demonstrated.

[0212] Figure 20 shows the proliferative capacity and phenotypic changes of CAR-T cells after repeated stimulation. (a) Established CAR-T cells were co-cultured with the pancreatic cancer cell line Aspc-1 in a CAR-T:Tumor ratio of 1:1, and stimulation with Aspc-1 was repeated every 96 hours. (b) The change in the number of CAR-T cells (doubling) after repeated stimulation was evaluated. F367L and R397W mutations tended to show higher cell proliferation compared to cCAR. (c) Cellrace violet staining was performed with 2.5 μM before Aspc-1 stimulation, and expression was evaluated on Day 12 (after 3 stimulations). WT, F367L, and A372P mutations showed higher proliferative capacity compared to cCAR. (d) Ki-67 expression after one 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 mutations compared to cCAR. (e)-(f) Tim-3 and TOX expression levels and the percentage of PD-1+ and Tim-3+ cells after three stimulations 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 mutations compared to cCAR. TOX expression tended to be lower in WT, R356E, F367L, and R397W mutations compared to cCAR. Furthermore, the proportion of PD-1+ and Tim-3+ cells tended to be lower in those with WT, K356E, F367L, A372P, R386H, and R397W mutations.

[0213] (Example 8: Changes in traits in a tumor environment) This example shows changes in traits in a tumor environment.

[0214] Figure 21 shows the changes in traits in the tumor environment.

[0215] After engraftment of the pancreatic cancer cell line Aspc1 into NSG mice, CAR-T cells were administered, and the tumors were excised 10 days later. The characteristics of the CAR-T cells contained within the tumors were then evaluated.

[0216] Regarding the proportion of PD-1+ and Tim-3+ cells, a decrease in the proportion of both positive cells was observed in the Y342F, R386H, and R397W mutations (a). Furthermore, TOX expression tended to decrease in the Y342F, R386H, and R397W mutations (b), while CCR8 expression tended to increase in the M370I and A372P mutations (c).

[0217] (Example 9: Changes in gene expression profiles due to RNA sequencing) This example shows changes in gene expression profiles due to RNA sequencing.

[0218] As shown in Figure 22, the changes in gene expression profiles due to RNA sequencing are illustrated.

[0219] RNA extraction was performed 12 days after CAR-T cell establishment, and RNA sequencing was carried out. (a) Heatmaps revealed three groups: one showing gene expression patterns similar to Th2 cells, such as IL13 (A372P, R337Q, R397W), one showing gene expression patterns similar to WT (F367L, Y342F, R386H, K332D, K356E), and one showing gene expression patterns similar to cCAR (M370I). (b) The gene expression patterns of WT and each mutant FOXP3-CART were compared with cCAR, and a volcanoplot was created. The R397W mutation showed characteristic gene expression patterns, including upexpression of IL13, as well as high expression of TNFRSF8 (CD30) and BATF3. (c) The gene expression patterns of each mutant FOXP3-CART were compared with those of the WT, and volcanoplots were created. Compared to the WT, the Y342F, F367L, and R386H mutants showed decreased expression of genes involved in the suppressive function of regulatory T cells, such as MYB and CTLA4. The Y342F, R386H, and R397W mutants showed decreased expression of genes involved in CAR-T cell dysfunction, such as ID3 and SOX4. The R337Q, A372P, and R397W mutants showed gene expression profiles similar to those of Th2 cells, such as IL13 and IL4, and the R397W mutant in particular showed characteristic increases in the expression of BATF3 and TNFRSF8.

[0220] (Example 10: Geneontology Analysis) This example shows a Geneontology analysis.

[0221] As shown in Figure 23, gene ontology analysis was performed. Gene ontology analysis (MF) was conducted, and changes in molecular function due to RNA sequencing were compared with cCAR. WT, K356E, and F367L mutations showed increased expression of genes involved in cell invasion, such as chemokine receptor activity. On the other hand, R337Q, A372P, and R397W mutations showed increased expression of genes involved in ATP hydrolysis activity and protein kinase activity.

[0222] (Example 11: Changes in the binding pattern of FOXP3 dimer and RUNX1 by Alphafold3) This example shows the analysis of various mutants. The method and materials are as follows.

[0223] (Methods and Materials) Using Alphafold3, the complex structure of the FOXP3 dimer and RUNX1 was predicted for each FOXP3 mutation, and the change in the binding pattern between the FOXP3 dimer and RUNX1 by Alphafold3 was demonstrated. (Results) The results are shown in Figure 24. In the amino acid sequence shown in Sequence ID No. 27, changes in binding with RUNX1 were observed in the F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A mutations, and in particular, the F367L mutation resulted in the loss of binding between RUNX1 and the FOXP3 Forkhead region.

[0224] 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 between the FOXP3 dimer and RUNX1 by Alphafold3 is shown.

[0225] Alphafold3 was used to predict the complex structure of the FOXP3 dimer and RUNX1 for each FOXP3 mutation. Changes in binding with RUNX1 were observed in the F331D, R337Q, K356E, T359W / N361W / E399R / E401R, F367L, M370I, F371L, A372P, R386H, R397W, V398E, E399R / E401R, and D409A mutations, with the F367L mutation being particularly characterized by a loss of binding between RUNX1 and the FOXP3 Forkhead region.

[0226] (Example 12: Enhancement of Foxp3 Expression) Endogenous Foxp3 expression is induced and / or enhanced using the CRISPR-dCas Activation system. Cells with enhanced Foxp3 expression are 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 immunoeffector function, and modified metabolic capacity as described in Example 1.

[0227] (Example 13: Example of using other CARs) We will investigate whether T cell enhancement similar to that observed in CD19CART-FOXP3mut. cells can be induced by substituting various structures of the CD19CAR (CD19CAR-FOXP3mut.) having the FOXP3 derivative shown in Example 1. Specifically, we will substitute single-chain antibodies that react to other antigens such as CD19, 20, 22, mesotherin, EGFRvIII, GD2, claudin6, and claudin9, or zetakin CARs such as IL15Ra, or CARs with other hinges such as CD8 and IgG4, or CARs with other intracellular activation domains such as 4-1BB, CD28, CD27, and ICOS, and establish CAR-T cells using the plasmids. Furthermore, the above procedure involves replacing the CAR from a lentiviral vector system to a retroviral vector system or a non-viral vector plasmid system to establish various CART-FOXP3 cells.

[0228] The T cell phenotype, chemokine expression, and fatigue / activation marker expression of each CART-FOXP3 cell line will be analyzed by flow cytometry and RNA sequencing. Furthermore, the cells will be co-cultured with various antigen-positive cell lines (e.g., luciferase-expressing AsPC1, Nalm-6, Raji, and U87d cell lines) to evaluate cytotoxic activity using luciferase activity, intracellular cytokine production using intracellular staining and ELISA, and CAR-T cell amplification and apoptosis through repeated co-culture. Metabolic analysis will be performed using a flux analyzer.

[0229] The CAR-T cells described above, regardless of their CAR structure or gene transfer method, exhibit the same tendencies as CD19CART-FOXP3mut., showing increased effector function, enhanced memory function, altered chemokine receptor profile, and metabolic changes.

[0230] Using a different CAR than in Example 1, we will evaluate the induction of Foxp3 expression and the modification of its metabolic capacity.

[0231] The following CAR configurations can be used: - Single-chain antibodies that react to other antigens such as CD19, 20, 22, mesotherin, EGFRvIII, GD2, claudin6, claudin9, etc., or zetakin CARs such as IL15Ra - CARs with other hinges such as CD8, IgG4, etc. - CARs with other intracellular activation domains such as 4-1BB, CD28, CD27, ICOS - Combinations of the above These CARs are expressed in T cells using lentiviruses, retroviruses, or nonviral vectors. The resulting CART cells are evaluated in the same manner as in Example 1.

[0232] (Example 14: Suppression of Immunosuppressive Gene Expression) The expression of immunosuppressive genes is suppressed.

[0233] The following are examples of immunosuppressive genes whose expression is suppressed:

[0234]

[0235] Cells obtained through expression suppression will be compared to CART cells that have not undergone such suppression, and superior cell amplification will be confirmed.

[0236] (Example 15: Antitumor effect of mutant FOXP3-expressing CAR-T cells (FOXP3-T cells containing CAR)) In this example, the antitumor effect of mutant FOXP3-expressing CAR-T cells (FOXP3-T cells containing CAR) was further investigated.

[0237] In detail, this example shows an instance in which the antitumor effect of mutant FOXP3-expressing CAR-T cells (FOXP3-T cells containing CAR) was demonstrated using an NSG mouse model. (a) Human CD19-expressing human pancreatic cancer cell line Aspc-1 was infused into NSG mice, and the antitumor effects of Transduced T cells (UTD), R397W mutant FOXP3-introduced CAR-T cells 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 showed a significant tumor reduction effect compared to all other groups. The K332D, R337Q, M370I, A372P, and R386H mutations showed antitumor effects equivalent to those of cCAR. (b) Human pancreatic cancer cell line Aspc-1 (tumor burden 20-180 mm3) was infused into NSG mice, and the antitumor effects of the 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 showed equivalent antitumor effects. (c) Intractable tumors (tumor volume 180-400 mm3) in which the oncogene MYC, which is involved in enhanced glycolysis and increased cell proliferation, was forcibly expressed in the human pancreatic cancer cell line Aspc-1 were infused into NSG mice, and the antitumor effects of cCAR, K332D, M370I, and R397W mutations were confirmed. It has been reported that the MYC expression model induces a low-sugar, high-lactate state in the tumor microenvironment by enhancing glycolysis. In this model, the R397W mutation showed a higher tumor reduction effect than all other groups.

[0238] The results are shown in Figure 26. In this example, the human CD19-expressing human pancreatic cancer cell line AsPC-1 was subcutaneously infused into NSG mice, and after engraftment, 1 x 10⁻¹⁵ cells were introduced. ６Untransformed T cells (UTDs), CAR-positive cells, 1 x 10⁶ ６ Unsorted R397W mutant FOXP3-introduced CAR-T cells (bulk), 1 x 10⁶ ６ CAR-T cells (CD4) purified to be CD4-positive using magnetic beads of cells, or 1 × 10⁶ of the same. ６ The antitumor effect was evaluated by administering purified R397W mutant TOXP3-transduced CAR-T cells (CD8) into CD8-positive cells via tail vein. Figure 26(A) shows the change in tumor volume, (B) shows the survival curve, (C) shows the tumor volume 22 days after treatment, and (D) shows the complete remission rate for each group at 56 days after treatment.

[0239] As shown, FOXP3 CAR-T cells were demonstrated to exert antitumor effects in all cell forms: CD4, CD8, and whole cells. AsPC1-tCD19 cell subcutaneous tumor-bearing NSG mice were administered via tail vein with non-genetically modified T cells (UTD), whole R397W mutant FOXP3 enhanced CAR-T cells (Bulk), CD4-positive cells (CD4) selected with magnetic beads, and CD8-positive cells (CD8), and tumor volume was measured. While no antitumor effect was observed with UTD, R397W mutant CAR-T cells showed effective antitumor activity in all groups: Bulk, CD4, and CD8. Compared to UTD, effective tumor reduction and survival effects were observed in all groups: Bulk, CD4, and CD8. On the other hand, compared to the CD8 monotherapy group, the CD4 and Bulk groups tended to show higher antitumor effects and remission rates. In both the Bulk and CD4 groups, the survival rate at 56 days after T-cell administration, the end date of the experiment, was 100%.

[0240] As described above, compared to UTD, effective tumor reduction and survival effects were observed in all three groups: Bulk, CD4, and CD8. Furthermore, significant antitumor effects and survival improvement effects were observed in all three groups: Bulk, CD4, and CD8.

[0241] Next, Figure 27 shows the behavior of IL-2, a factor necessary for T cell effector function, and CTLA-4, TGF-β, and ICOS, factors that can negatively regulate T cell effector function. These factors are known to be induced by FOXP3 in conventional CAR-T cells (cCAR), wild-type FOXP3 (WT), and CAR-T cells with each FOXP3 mutant, and are known to negatively regulate T cell effector function (CTLA-4, TGF-β, ICOS). Furthermore, their expression is known to be suppressed by FOXP3, and they are known to be factors necessary for or directly involved in T cell effector function (IL-2). Therefore, their expression was evaluated by flow cytometry (FCM) and ELISA.

[0242] In CAR-T (WT) cells introduced with wild-type FOXP3, compared to conventional CAR-T cells (cCAR), increased expression of CTLA-4, TGF-β, and ICOS (factors that negatively regulate T cell effector function), and decreased expression of IL-2 (a factor necessary for or directly involved in T cell effector function) were observed. On the other hand, in mutant FOXP3-introduced CAR-T cells, the increased expression of CTLA-4, TGF-β, and ICOS observed in wild-type FOXP3-introduced CAR-T cells was canceled, and IL-2 expression was also restored.

[0243] Figure 28 shows the results of investigations into annexin V, OXPHOS dependence, and CPT-1A expression. These were evaluated using CD95 stimulation to increase annexin-v expression, flux analyzer, and FCM, respectively. These factors are known to be characteristic of regulatory T cells that are induced by FOXP3 and use FOXP3 as the master regulator in conventional CAR-T cells (cCAR), wild-type FOXP3 (WT), and CAR-T cells with each FOXP3 mutant, and are also advantageous factors for T cells to exert effector functions. These factors include the anti-apoptotic effect after CD95 stimulation (a), oxidative phosphorylation intensity (b), and fatty acid transporter (CPT-1A) expression (c).

[0244] After CD95 stimulation, apoptosis was suppressed in wild-type FOXP3-introduced CAR-T cells compared to conventional CAR-T cells (cCAR). The suppression of apoptosis observed in the wt (wt) group was also observed in mutant FOXP3 (F397L, R397W) cells (a). Furthermore, wild-type FOXP3-introduced CAR-T cells showed increased oxidative phosphorylation dependence (b) and increased fatty acid transporter expression (c) compared to conventional CAR-T cells (cCAR). These changes observed in the wt group were maintained in mutant FOXP3 (F397L, R397W) cells.

[0245] From the above, it is understood that all of these mutations confer the functions intended for in this disclosure.

[0246] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted solely by the claims. This application claims priority to Patent No. 2025-010184 filed with the Japan Patent Office on 23 January 2025, and it is understood that the contents of that application, as well as the patents, patent applications and other documents cited herein, should be incorporated herein by reference as if their contents were specifically described herein.

[0247] According to this disclosure, it is possible to provide cells that are competitive against cancer cells without starving or becoming exhausted, even in glucose-depleted environments such as the tumor microenvironment (TME). Therefore, it is possible to develop strategies to improve the effectiveness of cell-based immunotherapy, and applications in the medical field are expected.

[0248] Sequence IDs 1-6: Sequence of shCTLA-4 Sequence IDs 7-10: Sequence of shTGFb1 Sequence IDs 11-13: CTLA-4 CRISPR-Cas9 guide RNA sequence Sequence IDs 14-16: TGFb CRISPR-Cas9 guide RNA sequence Sequence IDs 17-22: Primer sequences used in the example Sequence ID 23: Target sequence of shCTLA-4_#4 Sequence ID 24: Target sequence of shTGF-β1_#1 Sequence ID 25: Target sequence of sgCTLA-4_#1 Sequence ID 26: Target sequence of sgTGF-β1_#3 Sequence ID 27: Amino acid sequence of human FOXP3 sequence (NP_054728.2)

Claims

1. T cells (FOXP3-T cells) that (exogenously) express Foxp3, its variants, or any part thereof, and / or have enhanced (endogenous) Foxp3 expression and possess immune effector function.

2. The FOXP3-T cells according to claim 1, wherein the FOXP3-T cells are modified such that the function and / or expression of factors that negatively regulate immune effector function induced by FOXP3 (hereinafter referred to as "negative regulators") is reduced or eliminated.

3. The FOXP3-T cell according to claim 1 or 2, wherein the Foxp3 is modified to confer immune effector function to the Foxp3-T cell, or to maintain or enhance the immune effector function of the T cell.

4. The FOXP3-T cell according to any one of claims 1 to 3, wherein the Foxp3 is modified such that the expression and / or function of factors that negatively regulate immune effector function (negative regulators) is reduced or eliminated, and effector function is conferred to the T cell, or the effector function of the T cell is maintained or enhanced.

5. The FOXP3-T cell according to any one of claims 1 to 4, wherein the FOXP3-T cell is a cell with modified metabolic capacity.

6. The FOXP3-T cells according to any one of claims 1 to 5, wherein the FOXP3-T cells are cells whose chemokine receptor expression and / or migration to, infiltration into and survival in the local environment have been modified.

7. A T cell (FOXP3-T cell) having effector function, modified to express Foxp3, a variant thereof, or a part thereof, and / or having enhanced expression of Foxp3, wherein the FOXP3-T cell is modified such that the inhibitory function of the negative regulator is reduced or eliminated, according to any one of claims 1 to 6.

8. The FOXP3-T cells according to any one of claims 1 to 7, wherein the expression of chemokine receptors related to migration to, infiltration into, and survival in the local environment is enhanced in the FOXP3-T cells.

9. FOXP3-T cells according to any one of claims 1 to 8, satisfying at least one predetermined condition.

10. The FOXP3-T cell according to any one of claims 1 to 9, wherein the T cell includes an externally inserted antigen receptor such as a chimeric antigen receptor (CAR).

11. The FOXP3-T cell according to claim 10, wherein the CAR is expressed in the T cell.

12. FOXP3-T cells according to any one of claims 1 to 11, wherein the expression of at least one immunosuppressive gene is reduced or substantially absent.

13. The FOXP3-T cell according to claim 12, 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.

14. A human effector T cell, the FOXP3-T cell according to any one of claims 1 to 13.

15. A method for improving the tumor environment or anti-tumor immune response in a subject, comprising: A) a step of diagnosing the local environment of a subject, wherein the diagnosis is achieved by determining the status of Treg cells or gene mutations of environmental factors of target cells in the subject; and C) a step of providing T cells having immune effector function and / or factors that modify the tumor environment based on the local environment.

16. A method for improving the immune status of a subject, comprising: A) a step of diagnosing the immune status of a subject; B) a step of diagnosing gene mutations in the subject; and C) a step of providing T cells having immune effector functions according to the immune status based on the immune status and the gene mutations, and providing factors that modify the local environment (e.g., internal infiltration, BATF, chemokines, etc.) based on the gene mutations.

17. A pharmaceutical product comprising T cells having immune effector function, wherein the T cells are modified to express Foxp3, its variants, or a portion thereof, and / or the expression of Foxp3 is enhanced, or the T cells or a population of said T cells.

18. A method for treating or preventing a disease associated with an abnormal immune status, comprising administering an effective amount of T cells or a population of T cells having immune effector function, wherein the T cells are modified to express Foxp3, a variant thereof, or a portion thereof, and / or the expression of Foxp3 is enhanced, to a subject in need.

19. A population of cells including T cells (FOXP3-T cells) that (exogenously) express Foxp3, its variants, or any part thereof, and / or have enhanced (endogenous) Foxp3 expression and possess immune effector function.

20. A cell population of T cells (FOXP3-T cells) that exogenously express a variant of Foxp3, have immune effector function, and contain a chimeric antigen receptor (CAR), wherein the variant has reduced DNA binding ability, according to claim 19.

21. The cell population according to claim 19 or 20, wherein the mutation in the mutant is located between positions 330 and 400 in SEQ ID NO:

1.

22. The cell population according to any one of claims 19 to 21, wherein the mutation in the mutant includes a mutation in one or more amino acids K332, R337, M370, A372, R386, or R397.

23. The cell population according to any one of claims 19 to 22, wherein the mutation in the mutant includes a mutation in the amino acid K332, R337, M370, A372, R386, or R397.

24. The cell population according to any one of claims 19 to 23, wherein the mutant is K332D, R337Q, M370I, A372P, R386H, or R397W.

25. The cell population according to any one of claims 19 to 24, wherein the FOXP3-T cells are modified such that the function and / or expression of factors that negatively regulate immunoeffector function induced by FOXP3 (hereinafter referred to as "negative regulators") is reduced or eliminated.

26. The cell population according to any one of claims 19 to 25, wherein the Foxp3 is modified to impart immune effector function to the Foxp3-T cells, or to maintain or enhance the immune effector function of the T cells.

27. The cell population according to any one of claims 19 to 26, wherein Foxp3 is modified such that the expression and / or function of factors that negatively regulate immune effector function (negative regulators) is reduced or eliminated, and effector function is conferred to the T cells, or the effector function of the T cells is maintained or enhanced.

28. The cell population according to any one of claims 19 to 27, wherein the Foxp3-T cells are cells with modified metabolic capacity.

29. The cell population according to any one of claims 19 to 28, wherein the Foxp3-T cells are cells whose chemokine receptor expression and / or migration to, infiltration into and survival in the local environment have been modified.

30. The cell population according to any one of claims 19 to 29, wherein the FOXP3-T cells have enhanced expression of chemokine receptors related to migration to, infiltration into, and survival in the local environment.

31. The cell population according to any one of claims 19 to 30, wherein the expression of at least one immunosuppressive gene is reduced or substantially eliminated in the FOXP3-T cells.

32. The cell population according to claim 31, 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.

33. The cell population according to any one of claims 19 to 32, wherein the FOXP3-T cells are human effector T cells.

34. The cell population according to any one of claims 19 to 33, wherein the cell population comprises CD4-positive cells and / or CD8-positive cells.

35. The cell population according to any one of claims 19 to 34, wherein the cell population includes CD4-positive cells.

36. The cell population according to any one of claims 19 to 35, wherein the cell population includes CD4-positive cells and CD8-positive cells.

37. The cell population according to any one of claims 19 to 36, wherein the cell population is enriched with CD4-positive cells.

38. A cell population according to any one of claims 19 to 37, comprising one or more of the features described in any one of claims 2 to 14.

39. A pharmaceutical product comprising FOXP3-T cells according to any one of claims 1 to 14, or a cell population according to any one of claims 19 to 38.

40. The pharmaceutical agent according to claim 39 for treating or preventing cancer.

41. A regenerative medicine product comprising FOXP3-T cells according to any one of claims 1 to 14, or a cell population according to any one of claims 19 to 38.

42. A regenerative medicine product according to claim 41 for treating or preventing cancer.

Images