NRF-2-deficient cells and uses thereof

Abstract

The present disclosure relates to T cell anti-cancer immunotherapy based on the modulation of Nrf2 expression, i.e., anti-cancer therapy using Nrf2-targeted immune cells, e.g., T cells. The present disclosure solves the problem of immune tolerance exhibited by cancer cells to T cells by deeply interfering with Nrf2 expression in T cells. In other words, targeting Nrf2 can improve the efficacy of anti-cancer immunotherapy. The present disclosure may provide a new T cell anti-cancer immunotherapy targeting Nrf2, as well as T cells for second-generation anti-cancer immunotherapy. This technology is applicable to the preparation of CAR-T cells, engineered T cells, and TIL T cells, as well as the treatment of various solid cancers, including lymphoma. This represents a new anti-cancer therapy that effectively improves therapeutic efficacy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to Korean Patent Application No. 10-2018-0158428, filed December 10, 2018, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the Sequence Listing submitted with this application, submitted electronically as an ASCII text file (Name: 4241_010PC01_Seqlisting_ST25.txt, Size: 26,683 bytes, Created: December 9, 2019), are hereby incorporated by reference in their entirety.

[0003] The present disclosure relates to cell-based (e.g., T cell) anti-cancer immunotherapy based on Nrf2 expression. In particular, the cells provided herein are engineered to express reduced levels of Nrf2. [Background technology]

[0004] Myeloid-derived suppressor cells (MDSCs) in solid tumors generate an immunosuppressive tumor microenvironment (TME), which can then inhibit the antitumor response of effector T cells by producing high levels of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). This environment allows tumor cells to evade anticancer immune responses and promotes cancer cell growth and metastasis, one of the hallmarks of tumorigenesis. While this environment is known to affect the activity of tumor-infiltrating lymphocytes (TILs), no studies have been conducted on T cells to correlate with response factors. Nuclear factor E2-related factor 2 (Nrf2) is one of the classic factors responsible for oxidative stress (OS). While the role of Nrf2 in tumor cell responses has been extensively evaluated and accumulated, the impact of OS on the antitumor response of TILs, particularly cytotoxic CD8+ T cells, and the role of Nrf2 in causing OS have not been studied.

[0005] The role of Nrf2 in tumorigenesis has been controversial. Many previous studies have suggested that activation of Nrf2 can prevent carcinogenesis, while other studies have shown that carcinogenesis can be induced by abnormal or continuous activation of Nrf2 in vivo. In fact, overexpression of Nrf2 can reduce 5-year survival rates and induce resistance to anticancer drugs. The current problem is that no studies have been conducted on Nrf2 in TILs, and there is an urgent need to conduct relevant research. In particular, it is necessary to develop anticancer immunotherapy that involves regulating Nrf2 expression in cytotoxic CD8+ T cells. Despite advances in cancer immunotherapy, patients with certain malignancies (e.g., metastatic or refractory solid tumors) still have a very poor prognosis. Only a small proportion of such patients actually experience long-term cancer remission, and many either do not respond to antibodies or initially respond but eventually develop resistance. (Sharma, P., et al., Cell 168(4):707-723(2017)) Therefore, there remains a need for new treatment options with acceptable safety profiles and high efficacy in cancer patients. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sharma,P.,et al.,Cell 168(4):707-723(2017) Summary of the Invention

[0007] The present inventors identified a mechanism that regulates nuclear factor E2-related factor 2 (Nrf2) in cytotoxic CD8+ T cells, inhibited Nrf2 expression, and developed T cells whose cytotoxic function persists in the immunosuppressive TME. Furthermore, Nrf2-deficient human CAR-T cells outperform conventional T cells in producing the antitumor cytokines IFNg and granzyme B.

[0008] An object of the present disclosure is to provide a pharmaceutical composition for preventing and treating cancer. In some embodiments, the present disclosure provides T cells in which Nrf2 expression is reduced or inhibited.

[0009] The above-mentioned problems can be solved by the present disclosure, namely, by providing a pharmaceutical composition for preventing and treating canceration, which involves, for example, reducing or inhibiting Nrf2 expression in T cells.

[0010] The present disclosure relates to anti-cancer immunotherapy based on targeting Nrf2 expression in cells (e.g., T cells). Cancer cells can evade immune system surveillance, and one of the main mechanisms is by exhausting T cells. The present disclosure makes it possible to solve the problem of T cell exhaustion exhibited by cancer cells by deeply interfering with Nrf2 expression in T cells (i.e., reducing the expression of the NFE2L2 gene and / or Nrf2 protein in the cells). In other words, according to the present disclosure, the effectiveness of anti-cancer immunotherapy against solid tumors can be improved by targeting Nrf2 expression in cells (e.g., reducing or inhibiting the expression of the NFE2L2 gene and / or Nrf2 protein), which is the greatest feature of the present disclosure. The present disclosure may provide new T cell anti-cancer immunotherapy based on regulating Nrf2 expression in cells, and T cells for second-generation anti-cancer immunotherapy. This technology is applicable to the preparation of CAR-T cells, engineered T cells, and TIL T cells, as well as the treatment of various solid tumors, including leukemia. This can be said to be a new anti-cancer therapy that effectively improves treatment efficacy.

[0011] Provided herein are methods of treating a tumor in a subject in need thereof, comprising administering to the subject modified cells that express reduced levels of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, the expression level of the NFE2L2 gene and / or Nrf2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein).

[0012] In some embodiments, administration of the modified cells disclosed herein (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) reduces tumor volume in a subject compared to a reference tumor volume (e.g., the tumor volume in the subject before administration and / or the tumor volume in a subject that did not receive administration). In certain embodiments, the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor volume.

[0013] In some embodiments, administration of the modified cells disclosed herein (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) reduces tumor weight in a subject compared to a reference tumor weight (e.g., tumor weight in the subject before administration and / or tumor weight in a subject that does not receive administration). In certain embodiments, tumor weight is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor weight.

[0014] In some embodiments, administration of the modified cells disclosed herein (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) improves one or more characteristics of tumor-infiltrating lymphocytes (TILs) in a subject. In certain embodiments, the TILs, upon stimulation with a cognate antigen, produce increased amounts of IFN-γ compared to reference TILs (e.g., TILs from a subject that did not receive the treatment). In some embodiments, the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference TILs. In some embodiments, the TILs are CD8+ TILs. In some embodiments, the TILs are CD4+ TILs.

[0015] In some embodiments, the modified cells (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) exhibit increased resistance to oxidative stress compared to reference cells (e.g., corresponding cells not modified to express relatively low levels of Nrf2 protein). In certain embodiments, oxidative stress resistance is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cells. In some embodiments, increased oxidative stress resistance includes the ability to proliferate in the presence of elevated concentrations of reactive oxygen species, produce IFN-γ, and / or express granzyme B. In some embodiments, the reactive oxygen species includes hydrogen peroxide (HO).

[0016] In some embodiments, the modified cells that can be administered in the methods disclosed herein are immune cells. In particular embodiments, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or a combination thereof. In particular embodiments, the lymphocytes are T cells. In further embodiments, the T cells include a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR.

[0017] In some embodiments, tumors that can be treated with the methods disclosed herein are derived from cancers including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In certain embodiments, the cancer is colon cancer, skin cancer, lymphoma, lung cancer, or a combination thereof.

[0018] In some embodiments, the methods of treating a tumor disclosed herein comprise administering to the subject an additional therapeutic agent. In certain embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of costimulatory molecules, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

[0019] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM3 antibody, or any combination thereof.

[0020] In some embodiments, the additional therapeutic agent and the modified cells are administered simultaneously, hi other embodiments, the additional therapeutic agent and the modified cells are administered sequentially.

[0021] In some embodiments, the modified cells (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) that can be used to treat tumors using the methods disclosed herein are administered supplementally, intramuscularly, subcutaneously, by eye drop, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, intratumorally, or any combination thereof.

[0022] The present disclosure further provides methods for improving the anti-tumor immune response of chimeric antigen receptor (CAR)-expressing cells, the methods comprising modifying the cells to express reduced levels of the NFE2L2 gene and / or Nrf2 protein, wherein the relatively low expression of the NFE2L2 gene and / or Nrf2 protein improves the anti-tumor immune response of the cells. In some embodiments, the expression level of the NFE2L2 gene and / or Nrf2 protein in the modified cells is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein).

[0023] In some embodiments, the modified cells, upon stimulation with a cognate antigen, produce an increased amount of IFN-γ compared to a reference cell (e.g., a corresponding cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cell.

[0024] In some embodiments, the modified cells exhibit increased oxidative stress resistance compared to reference cells (e.g., corresponding cells that have not been modified to express relatively low levels of Nrf2 protein). In certain embodiments, oxidative stress resistance is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cells. In some embodiments, increased oxidative stress resistance includes the ability to proliferate in the presence of high concentrations of reactive oxygen species, produce IFN-γ, and / or express granzyme B. In certain embodiments, the reactive oxygen species includes hydrogen peroxide (HO).

[0025] In some embodiments, modifying a cell in the method disclosed herein comprises contacting the cell with a gene editing tool that can reduce the expression level of the NFE2L2 gene and / or Nrf2 protein in the cell. In certain embodiments, the gene editing tool comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. In some embodiments, the gene editing tool is shRNA.

[0026] In some embodiments, the modified cells (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) are immune cells. In particular embodiments, the immune cells comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the lymphocytes comprise T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or a combination thereof. In particular embodiments, the lymphocytes are T cells.

[0027] Also provided herein are methods of preparing immune cells for chimeric antigen receptor engineering, comprising contacting the immune cells with a gene editing tool to reduce the expression level of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, the expression level of the NFE2L2 gene and / or Nrf2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein).

[0028] In some embodiments, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or any combination thereof. In certain embodiments, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or any combination thereof. In some embodiments, the lymphocytes are T cells.

[0029] In some embodiments, the gene editing tool that can be used in the method for preparing immune cells for chimeric antigen receptor engineering includes shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. In certain embodiments, the gene editing tool is shRNA.

[0030] In some embodiments, the methods of preparing immune cells for chimeric antigen receptor engineering disclosed herein further comprise modifying the immune cells to express a chimeric antigen receptor (CAR). In certain embodiments, modifying the immune cells to express a CAR comprises contacting the immune cells with a nucleic acid sequence encoding the CAR.

[0031] In some embodiments, the nucleic acid encoding the gene editing tool is expressed from an expression vector. In particular embodiments, the nucleic acid sequence encoding the CAR is expressed from an expression vector. In some embodiments, the gene editing tool and the CAR are encoded on separate expression vectors. In other embodiments, the gene editing tool and the CAR are encoded on the same expression vector.

[0032] Provided herein is a pharmaceutical composition for preventing or treating cancer, comprising a T cell with reduced Nrf2 expression. In some embodiments, the T cell comprises a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR.

[0033] Also provided herein are cells prepared by the methods disclosed herein. In certain embodiments, the cells comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR. In some embodiments, the cells are T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, NK cells, or any combination thereof. In certain embodiments, for example, the following are provided: (Item 1) A method of treating a tumor in a subject in need thereof, comprising administering to the subject modified cells that express reduced levels of the NFE2L2 gene and / or Nrf2 protein. (Item 2) 2. The method of item 1, wherein the expression level of the NFE2L2 gene and / or Nrf2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). (Item 3) 3. The method of item 1 or 2, wherein the administration reduces tumor volume in the subject compared to a reference tumor volume (e.g., the tumor volume in the subject before the administration and / or the tumor volume in a subject that did not receive the administration). (Item 4) 4. The method of item 3, wherein the tumor volume is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after said administration compared to the reference tumor volume. (Item 5) 5. The method of any one of items 1 to 4, wherein the administration reduces tumor weight in the subject compared to a reference tumor weight (e.g., tumor weight in the subject before the administration and / or tumor weight in a subject that did not receive the administration). (Item 6) 6. The method of item 5, wherein the tumor weight is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after said administration compared to the reference tumor weight. (Item 7) 7. The method of any one of items 1 to 6, wherein the administration improves one or more characteristics of tumor-infiltrating lymphocytes (TILs) in the subject. (Item 8) 8. The method of claim 7, wherein the TILs, when stimulated with a cognate antigen, produce increased amounts of IFN-γ compared to a reference TIL (e.g., a TIL from a subject that has not received the administration). (Item 9) 9. The method of item 8, wherein the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference TILs. (Item 10) 10. The method of any one of items 7 to 9, wherein the TILs are CD8+ TILs. (Item 11) 10. The method of any one of items 7 to 9, wherein the TILs are CD4+ TILs. (Item 12) 12. The method of any one of items 1 to 11, wherein the modified cells exhibit increased resistance to oxidative stress compared to reference cells (e.g., corresponding cells that have not been modified to express relatively low levels of Nrf2 protein). (Item 13) 13. The method of claim 12, wherein the oxidative stress resistance is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cell. (Item 14) 14. The method of item 12 or 13, wherein the increased resistance to oxidative stress comprises the ability to proliferate in the presence of high concentrations of reactive oxygen species, to produce IFN-γ, and / or to express granzyme B. (Item 15) The reactive oxygen species is hydrogen peroxide (H 2 O 2 Item 15. The method according to item 14, comprising: (Item 16) 16. The method according to any one of items 1 to 15, wherein the modified cells are immune cells. (Item 17) 17. The method of claim 16, wherein the immune cells comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. (Item 18) 18. The method of item 17, wherein the lymphocytes comprise T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or any combination thereof. (Item 19) 19. The method of claim 18, wherein the lymphocytes are T cells. (Item 20) 20. The method of claim 19, wherein the T cells comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR. (Item 21) 21. The method of any one of items 1 to 20, wherein the tumor is derived from cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. (Item 22) 22. The method of claim 21, wherein the cancer is colon cancer, skin cancer, lymphoma, lung cancer, or a combination thereof. (Item 23) 23. The method of any one of items 1 to 22, comprising administering to the subject an additional therapeutic agent. (Item 24) 24. The method of item 23, wherein the additional therapeutic agent comprises a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation procedure, an activator of costimulatory molecules, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. (Item 25) 25. The method of item 24, wherein the additional therapeutic agent is an immune checkpoint inhibitor. (Item 26) 26. The method of item 24 or 25, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM3 antibody, or any combination thereof. (Item 27) 27. The method of any one of items 23 to 26, wherein the additional therapeutic agent and the modified cells are administered simultaneously. (Item 28) 27. The method of any one of items 23 to 26, wherein the additional therapeutic agent and the modified cells are administered sequentially. (Item 29) 29. The method of any one of items 1 to 28, wherein the modified cells are administered by supplemental administration, intramuscular administration, subcutaneous administration, eye drop administration, intravenous administration, intraperitoneal administration, intradermal administration, intraorbital administration, intracerebral administration, intracranial administration, intraspinal administration, intraventricular administration, intrathecal administration, intracisternal administration, intracapsular administration, intratumoral administration, or any combination thereof. (Item 30) 1. A method for improving an anti-tumor immune response of a chimeric antigen receptor (CAR)-expressing cell, comprising modifying the cell to express reduced levels of the NFE2L2 gene and / or Nrf2 protein, wherein the relatively lower expression of the NFE2L2 gene and / or Nrf2 protein improves the anti-tumor immune response of the cell. (Item 31) 31. The method of claim 30, wherein the expression level of the NFE2L2 gene and / or Nrf2 protein in the modified cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). (Item 32) 32. The method of paragraph 30 or 31, wherein the modified cells, when stimulated with a cognate antigen, produce an increased amount of IFN-γ compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). (Item 33) 33. The method of claim 32, wherein the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cell. (Item 34) 34. The method of any one of items 30 to 33, wherein the modified cells exhibit increased resistance to oxidative stress compared to reference cells (e.g., corresponding cells that have not been modified to express relatively low levels of Nrf2 protein). (Item 35) 35. The method of item 34, wherein the oxidative stress resistance is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cell. (Item 36) 36. The method of item 34 or 35, wherein the increased resistance to oxidative stress comprises the ability to proliferate in the presence of high concentrations of reactive oxygen species, to produce IFN-γ, and / or to express granzyme B. (Item 37) The reactive oxygen species is hydrogen peroxide (H 2 O 2 37. The method according to item 36, comprising: (Item 38) 38. The method of any one of items 30 to 37, wherein modifying the cell comprises contacting the cell with a gene editing tool capable of reducing the expression level of the NFE2L2 gene and / or Nrf2 protein in the cell. (Item 39) 39. The method of claim 38, wherein the gene editing tool comprises an shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. (Item 40) 40. The method of claim 38 or 39, wherein the gene editing tool is an shRNA. (Item 41) 41. The method according to any one of items 30 to 40, wherein the modified cells are immune cells. (Item 42) 42. The method of claim 41, wherein the immune cells comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. (Item 43) 43. The method of claim 42, wherein the lymphocytes comprise T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or a combination thereof. (Item 44) 44. The method of claim 43, wherein the lymphocytes are T cells. (Item 45) 1. A method of preparing immune cells for chimeric antigen receptor engineering, the method comprising contacting the immune cells with a gene editing tool to reduce expression levels of the NFE2L2 gene and / or Nrf2 protein. (Item 46) 46. ​​The method of claim 45, wherein the expression level of the NFE2L2 gene and / or Nrf2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). (Item 47) 47. The method of item 45 or 46, wherein the immune cells comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or any combination thereof. (Item 48) 48. The method of item 47, wherein the lymphocytes comprise T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or any combination thereof. (Item 49) 49. The method of claim 48, wherein the lymphocytes are T cells. (Item 50) 50. The method of any one of items 45 to 49, wherein the gene editing tool comprises shRNA, siRNA, miRNA, antisense oligonucleotide, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. (Item 51) 51. The method of claim 50, wherein the gene editing tool is an shRNA. (Item 52) 52. The method of any one of items 45 to 51, wherein the nucleic acid encoding the gene editing tool is expressed from an expression vector. (Item 53) 53. The method of any one of items 45 to 52, further comprising modifying the immune cells to express a chimeric antigen receptor (CAR). (Item 54) 54. The method of claim 53, wherein modifying the immune cell to express the CAR comprises contacting the immune cell with a nucleic acid sequence encoding the CAR. (Item 55) 55. The method of claim 54, wherein the nucleic acid sequence encoding the CAR is expressed from an expression vector. (Item 56) 56. The method of claim 55, wherein the gene editing tool and the CAR are encoded by separate expression vectors. (Item 57) 56. The method of claim 55, wherein the gene editing tool and the CAR are encoded in the same expression vector. (Item 58) A pharmaceutical composition for preventing or treating cancer, comprising T cells with reduced Nrf2 expression. (Item 59) 59. The pharmaceutical composition of item 58, wherein the T cells comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR. (Item 60) 58. Cells prepared by the method according to any one of items 30 to 57. (Item 61) 61. The cell of paragraph 60, further comprising a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR. (Item 62) 62. The cell of item 60 or 61, which is a T cell, a tumor-infiltrating lymphocyte (TIL), a lymphokine-activated killer cell, a NK cell, or any combination thereof. [Brief explanation of the drawings]

[0034] [Figure 1]Figure 1 shows changes in Nrf2 mRNA expression in cytotoxic CD8+ T cells with increasing oxidative stress (represented by elevated H2O2 concentrations). [Figure 2] Nrf2 mRNA expression in various cell populations isolated from tissues of tumor-bearing and tumor-free mice is shown. The cells shown (from left to right) include: (i) tumor-infiltrating T cells (from tumor-bearing mice), (ii) tumor-infiltrating B cells (from tumor-bearing mice), (iii) non-T and non-B TILs (DNs) (from tumor-bearing mice), (iv) draining lymph node T cells ("dLN") (from tumor-bearing mice), (v) non-draining lymph node T cells (from tumor-bearing mice), and (vi) non-draining lymph node T cells (from tumor-free mice). [Figure 3] Figure 1 shows the growth rate of melanoma in Nrf2-deficient and wild-type (WT) mice. Specifically, a comparison of tumor growth at various time points after tumor (i.e., melanoma) inoculation is presented in Nrf2- / - and wild-type mice (i.e., those expressing normal levels of Nrf2). [Figure 4] Figure 1 shows images of melanoma proliferation (i.e., growth) that varied with Nrf2 expression. Specifically, images of melanoma tumors isolated from wild-type mice (left) and Nrf2- / - mice (right) are presented. [Figure 5] Figure 1 shows lymphoma growth that varied with Nrf2 expression. Specifically, a comparison of tumor volume at various time points after tumor (i.e., lymphoma) inoculation in Nrf2- / - and wild-type mice (i.e., those expressing normal levels of Nrf2) is presented. [Figure 6] Images of lymphoma proliferation (i.e., growth) that changed with Nrf2 expression are shown. Specifically, images of lymphoma tumors isolated from wild-type mice (left) and Nrf2- / - mice (right) are presented. [Figure 7]Images of cancer cell metastasis in the lungs of wild-type and Nrf2- / - mice are shown, along with changes in Nrf2 expression. The images on the left are photographic representations of lung tissue isolated from representative wild-type (top) and Nrf2- / - animals (bottom). The images on the right are immunohistochemical images of lung tissue from wild-type (top) and Nrf2- / - animals (bottom). Tumors are identified by arrows in the images. [Figure 8] Panels A and B show that T cell-mediated antitumor immune responses changed with Nrf2 expression. Specifically, panels A and B demonstrate that the improved antitumor immune responses observed in Nrf2- / - animals are T cell-mediated. Comparisons of tumor growth (A) and tumor weight (B) are presented for the following groups: (i) tumor-bearing wild-type animals given a control IgG antibody ("WT + IgG" or "open circles"), (ii) tumor-bearing wild-type animals given a T cell-depleting anti-CD3 antibody ("WT + α-CD3" or "shaded circles"), (iii) tumor-bearing Nrf2- / - animals given a control IgG antibody ("Nrf2- / - + IgG" or "open squares"), and (iv) tumor-bearing Nrf2- / - animals given a T cell-depleting anti-CD3 antibody ("Nrf2- / - + α-CD3" or "filled squares"). [Figure 9] Panels A and B demonstrate that the improved antitumor immune response observed in Nrf2- / - animals is independent of B cells. Comparisons of tumor growth (A) and tumor weight (B) are presented for the following groups: (i) tumor-bearing wild-type animals given a control IgG antibody ("WT + IgG" or "open circles"), (ii) tumor-bearing wild-type animals given a B cell-depleting anti-B220 antibody ("WT + α-B220" or "shaded circles"), (iii) tumor-bearing Nrf2- / - animals given a control IgG antibody ("Nrf2- / - + IgG" or "open squares"), and (iv) tumor-bearing Nrf2- / - animals given a B cell-depleting anti-B220 antibody ("Nrf2- / - + α-B220" or "filled squares"). [Figure 10]Figure 1 shows the altered T cell activity with Nrf2 expression. Specifically, a comparison of IL-17 (y-axis) and IFN-γ (x-axis) production by draining lymph node T cells ("draining LNT") and TILs isolated from tumor-bearing wild-type (top) and Nrf2- / - animals (bottom) is presented. Two distinct classes of T cells are further divided based on their CD4 and CD8 expression. [Figure 11] We demonstrate the anti-cancer efficacy of Nrf2-deficient toxic CD8+ T cells in an adoptive transfer model. Specifically, OVA-specific CD8+ T cells were isolated from wild-type, Nrf2+ / -, or Nrf2- / - mice and adoptively transferred into wild-type animals inoculated with EG7-OVA tumor cells. Treatment groups were as follows: (1) WT mice with WT cells, (2) WT mice with Nrf2+ / - cells, and (3) WT mice with Nrf2- / - cells. Comparisons of tumor volume within each animal at various time points after tumor inoculation are presented. [Figure 12] FIG. 1 is a schematic diagram of the process of the present disclosure, i.e., tumor immunotherapy involving modulation of Nrf2 expression in cytotoxic CD8+ T cells. [Figure 13] (A) and (B) show a comparison of antitumor immune responses in wild-type mice (open circles), Nrf2- / - mice (filled squares), and Nrf2 transgenic mice (filled triangles) inoculated with MC38 (colon) tumor cells. (A) shows the tumor volume of each treatment group at various time points after tumor inoculation. (B) shows a comparison of tumor volume 28 days after tumor inoculation. [Figure 14A]Figure 14 shows the ability of T cells isolated from wild-type animals (left column, "WT"), Nrf2 transgenic animals (middle column, "Nrf2Tg"), and Nrf2- / - animals (right column, "Nrf2KO") to resist oxidative stress (OS) induced by the presence of hydrogen peroxide (HO). In Figure 14A, OS resistance is measured by the amount of IFN-γ produced by T cells from each animal upon TCR stimulation in the presence or absence of HO. Figure 14B is a graphical representation of the flow cytometry data presented in Figure 14A. In Figure 14C, OS resistance is measured by granzyme B expression. Figure 14D is a graphical representation of the flow cytometry data presented in Figure 14C. In both Figures 14A and 14C, (i) the cells shown in the top row were not TCR-stimulated or exposed to HO, (ii) the cells shown in the middle row were TCR-stimulated but not exposed to HO, and (iii) the cells shown in the bottom row were both TCR-stimulated and exposed to HO. In each flow plot, the boxed area on the left represents activated T cells (i.e., those that proliferated upon TCR stimulation), and the boxed area on the right represents T cells that did not proliferate upon TCR stimulation. Proliferation is indicated based on the CFSE profile. [Figure 14B]Figure 14 shows the ability of T cells isolated from wild-type animals (left column, "WT"), Nrf2 transgenic animals (middle column, "Nrf2Tg"), and Nrf2- / - animals (right column, "Nrf2KO") to resist oxidative stress (OS) induced by the presence of hydrogen peroxide (HO). In Figure 14A, OS resistance is measured by the amount of IFN-γ produced by T cells from each animal upon TCR stimulation in the presence or absence of HO. Figure 14B is a graphical representation of the flow cytometry data presented in Figure 14A. In Figure 14C, OS resistance is measured by granzyme B expression. Figure 14D is a graphical representation of the flow cytometry data presented in Figure 14C. In both Figures 14A and 14C, (i) the cells shown in the top row were not TCR-stimulated or exposed to HO, (ii) the cells shown in the middle row were TCR-stimulated but not exposed to HO, and (iii) the cells shown in the bottom row were both TCR-stimulated and exposed to HO. In each flow plot, the boxed area on the left represents activated T cells (i.e., those that proliferated upon TCR stimulation), and the boxed area on the right represents T cells that did not proliferate upon TCR stimulation. Proliferation is indicated based on the CFSE profile. [Figure 14C]Figure 14 shows the ability of T cells isolated from wild-type animals (left column, "WT"), Nrf2 transgenic animals (middle column, "Nrf2Tg"), and Nrf2- / - animals (right column, "Nrf2KO") to resist oxidative stress (OS) induced by the presence of hydrogen peroxide (HO). In Figure 14A, OS resistance is measured by the amount of IFN-γ produced by T cells from each animal upon TCR stimulation in the presence or absence of HO. Figure 14B is a graphical representation of the flow cytometry data presented in Figure 14A. In Figure 14C, OS resistance is measured by granzyme B expression. Figure 14D is a graphical representation of the flow cytometry data presented in Figure 14C. In both Figures 14A and 14C, (i) the cells shown in the top row were not TCR-stimulated or exposed to HO, (ii) the cells shown in the middle row were TCR-stimulated but not exposed to HO, and (iii) the cells shown in the bottom row were both TCR-stimulated and exposed to HO. In each flow plot, the boxed area on the left represents activated T cells (i.e., those that proliferated upon TCR stimulation), and the boxed area on the right represents T cells that did not proliferate upon TCR stimulation. Proliferation is indicated based on the CFSE profile. [Figure 14D]Figure 14 shows the ability of T cells isolated from wild-type animals (left column, "WT"), Nrf2 transgenic animals (middle column, "Nrf2Tg"), and Nrf2- / - animals (right column, "Nrf2KO") to resist oxidative stress (OS) induced by the presence of hydrogen peroxide (HO). In Figure 14A, OS resistance is measured by the amount of IFN-γ produced by T cells from each animal upon TCR stimulation in the presence or absence of HO. Figure 14B is a graphical representation of the flow cytometry data presented in Figure 14A. In Figure 14C, OS resistance is measured by granzyme B expression. Figure 14D is a graphical representation of the flow cytometry data presented in Figure 14C. In both Figures 14A and 14C, (i) the cells shown in the top row were not TCR-stimulated or exposed to HO, (ii) the cells shown in the middle row were TCR-stimulated but not exposed to HO, and (iii) the cells shown in the bottom row were both TCR-stimulated and exposed to HO. In each flow plot, the boxed area on the left represents activated T cells (i.e., those that proliferated upon TCR stimulation), and the boxed area on the right represents T cells that did not proliferate upon TCR stimulation. Proliferation is indicated based on the CFSE profile. [Figure 15A] 1 shows generation of CD19-specific CAR T cells lacking Nrf2 expression. Schematic of the CD19 CAR construct. [Figure 15B] Figure 1 shows generation of CD19-specific CAR T cells lacking Nrf2 expression. Figure 2 shows knockdown of Nrf2 mRNA expression in transduced CD19 CAR T cells using shRNA specific for Nrf2 mRNA. [Figure 15C] Figure 1 shows the generation of CD19-specific CAR T cells lacking Nrf2 expression. Figure 2 shows the activity of CD19-specific Nrf2-deficient CAR T cells in resisting OS, as measured by the amount of IFN-γ produced after anti-CD3 stimulation in the presence of H2O2. Data shown at the top are for control CAR T cells (i.e., those expressing normal levels of Nrf2). [Figure 16]Panels A, B, and C show the results of Nrf2 protein expression in human T cells using the CRISPR / Cas9 system. Panels A, B, and C present the results using three different guide RNAs (gRNAs) targeting the Nrf2 gene and the Cas9 protein. T7E1 analysis was used to detect on-target CRISPR / Cas9 editing events in the Nrf2 gene. The percentages shown represent the indel mutation rates of individual gRNAs in the Nrf2 gene after modification by the Cas9 / gRNA complex. DETAILED DESCRIPTION OF THE INVENTION

[0035] Cancer cells can escape immune system surveillance, and one of the main mechanisms is to exhaust T cells. The present disclosure solves the problem of TME-induced immune exhaustion against TILs by deeply interfering with Nrf2 expression in T cells. In other words, targeting Nrf2 can improve the efficacy of anti-cancer immunotherapy, which is the greatest feature of the present disclosure. Therefore, the present disclosure relates to a method for treating tumors in a subject in need thereof, comprising administering modified cells with reduced expression of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, the present disclosure relates to a method for improving the anti-tumor efficacy of CAR or TCR-engineered cells. In some embodiments, the present disclosure relates to a method for preventing or inhibiting immune tolerance of immune cells to tumors.

[0036] I. Definition So that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise specified herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this specification.

[0037] Throughout this disclosure, the terms "a" or "an" applied to an entity refers to one or more of that entity. For example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0038] Furthermore, when used herein, "and / or" shall be construed as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0039] Whenever an embodiment is described herein using the word "comprising," it should be understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0041] Units, prefixes, and symbols are shown in their respective formats recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0042] The term "about" is used herein to mean approximately, roughly, roughly, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify a numerical value to be above or below the stated value, for example, by a variance of up to or below 10 percent (higher or lower).

[0043] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Various routes of administration of the therapeutic agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, intrapulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the therapeutic agents described herein can be administered by a non-parenteral route, such as topical, epidermal, or mucosal administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically, and can be administered, e.g., once, multiple times, and / or over one or more extended periods of time.

[0044] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "cognate antigen" refers to an antigen that is recognized by an immune cell (e.g., a T cell) and thereby induces immune cell activation (e.g., inducing effector functions such as cytokine production and / or triggering intracellular signals for cell proliferation).

[0045] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, including, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides. Unless otherwise specified, the terms "protein" and "polypeptide" may be used interchangeably.

[0046] The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0047] As used herein, the term "vector" is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0048] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth result in the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancers, metastatic cancers, and recurrent cancers.

[0049] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign agents that protects the organism from these agents and the diseases they cause. An immune response is mediated by the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues in the vertebrate body. Immune responses include, for example, T cells, e.g., CD4 + or CD8 + This includes activation or inhibition of effector T cells, such as T cells, or Th cells, or inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are synonymous and refer to any lymphocyte produced or processed by the thymus. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a NKT cell.

[0050] As used herein, the term "anti-tumor immune response" refers to an immune response against tumor antigens.

[0051] As used herein, the term "tumor-infiltrating lymphocytes" or "TILs" refers to lymphocytes (e.g., effector T cells) that have migrated from the periphery (e.g., from the blood) into a tumor. In some embodiments, tumor-infiltrating lymphocytes are CD4+ TILs. In other embodiments, tumor-infiltrating lymphocytes are CD8+ TILs.

[0052] The increased ability to stimulate an immune response or the immune system may be due to improved agonist activity of T cell costimulatory receptors and / or improved antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be reflected in a fold increase in EC50 or maximal activity level in assays that measure immune responses, such as cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by detecting CD107a or granzymes), and changes in proliferation. The ability to stimulate an immune response or immune system activity may be improved by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0053] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0054] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to that amount of an agent that produces a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, palliation, attenuation, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or slow other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or slow tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, slow to some extent, or even stop cancer cell invasion into peripheral organs, (iv) inhibit (i.e., slow to some extent, or even stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In some embodiments, a "therapeutically effective amount" is the amount of modified cells herein that has been clinically proven to result in a significant reduction in cancer, such as advanced solid tumors, or a slowing (regression) of cancer progression. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, for example, by assaying the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0055] As used herein, the term "standard of care" refers to a treatment that is accepted by medical professionals as appropriate for a particular type of disease and is widely used by medical professionals. This term may be used interchangeably with any of the following terms: "best practice," "standard of care," and "standard therapy."

[0056] By way of example, an "anti-cancer drug" promotes the regression of a cancer in a subject or prevents further tumor growth. In certain embodiments, a therapeutically effective amount of a drug promotes the regression of a cancer to the point of eliminating the cancer. "Promoting cancer regression" means that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of functional impairment or disability due to disease affliction. Furthermore, the terms "effective" and "efficacy" with respect to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote the regression of a cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from the administration of a drug.

[0057] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins control the activation or function of T cells. Many checkpoint proteins are known, including CTLA-4 and its ligands CD80 and CD86, and PD-1 and its ligands PD-L1 and PD-L2. Pardoll, DM, Nat Rev Cancer 12(4):252-64 (2012). These proteins are responsible for costimulatory or inhibitory interactions in T cell responses. Immune checkpoint proteins control and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0058] As used herein, the term "oxidative stress" refers to a condition characterized by an excess of oxidants and / or reduced antioxidant levels. Cellular oxidants can include, but are not limited to, oxygen radicals (superoxide anion, hydroxyl radical, and / or peroxyl radical); reactive non-reactive oxygen species such as hydrogen peroxide and singlet oxygen; carbon radicals; nitrogen radicals; sulfur radicals; and combinations thereof. In some embodiments, a state of oxidative stress can result, for example, in cell damage, cell dysfunction, and / or cell death.

[0059] As used herein, the term "modified cell" refers to a cell that differs from its unmodified counterpart. As is apparent from the present disclosure, the modified cells disclosed herein express reduced levels of the NFE2L2 gene and / or Nrf2 protein compared to a reference cell (e.g., its unmodified counterpart). In some embodiments, the modified cell is generated by introducing a foreign or exogenous nucleic acid into a cell. In certain embodiments, the foreign or exogenous nucleic acid may encode a gene editing tool disclosed herein. In other embodiments, the foreign or exogenous nucleic acid may encode a chimeric antigen receptor (such as those described herein). Nucleic acids can be introduced by, for example, electroporation (see, e.g., Heiser WC Transscription Factor Protocols: Methods in Molecular Biology™ 2000;130:117-134), chemical (e.g., calcium phosphate or lipid) transfection (see, e.g., Lewis WH, et al., Somatic Cell Genet. 1980 May;6(3):333-47; Chen C., et al., Mol Cell Biol. 1987 August;7(8):2745-2752), fusion with bacterial protoplasts containing recombinant plasmids (see, e.g., Schaffner W. Proc Natl Acad Sci USA. 1980 April;77(4):2163-7), or by direct microinjection of purified DNA into cell nuclei (see, e.g., Capecchi MR Cell. 1980 November;22(2 Pt The vector can be introduced into cells by methods known in the art, such as those described in U.S. Pat. No. 6,279,479-88.

[0060] As used herein, the term "high concentration" means a higher than normal level of a substance (eg, reactive oxygen species) compared to an appropriate control (eg, healthy tissue or cells).

[0061] As used herein, the term "reactive oxygen species" refers to highly reactive chemicals containing oxygen that readily react with other molecules, resulting in potentially damaging modifications. Reactive oxygen species include, for example, oxygen ions, inorganic and organic free radicals and peroxides, such as hydrogen peroxide, superoxide, hydroxyl radicals, lipid hydroperoxidase, and singlet oxygen. These are typically very small molecules that are highly reactive due to the presence of unpaired valence shell electrons. Nearly all cancers are associated with high concentrations of reactive oxygen species. Liou, G., et al., Free Radic Res 44(5):1-31(2010).

[0062] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a recombinant fusion protein having an antigen-specific extracellular domain linked to an intracellular domain that instructs a cell to perform a specialized function upon binding of an antigen to the extracellular domain. The terms "artificial T cell receptor," "chimeric T cell receptor," and "chimeric immune receptor" may each be used interchangeably herein with the term "chimeric antigen receptor." Chimeric antigen receptors are distinguished from other antigen-binding agents by their ability to bind MHC-independent antigens and transmit activation signals via their intracellular domains.

[0063] The antigen-specific extracellular domain of the chimeric antigen receptor recognizes and specifically binds to an antigen, typically a surface-expressed antigen of a malignant disease. The antigen-specific extracellular domain specifically binds to the antigen with an affinity constant or affinity of interaction (K) of, for example, about 0.1 pM to about 10 μM, e.g., about 0.1 pM to about 1 μM, or about 0.1 pM to about 100 nM. D) binds to the antigen. Methods for determining the affinity of the interaction are known in the art. Antigen-specific extracellular domains suitable for use in the CARs of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some embodiments, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains are suitable for use, including cAb VHH (camelid antibody variable domains) and humanized versions thereof, lgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single-domain antibody variable domains), and "camelized" antibody variable domains. In some embodiments, T-cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTvs, single-chain two-domain TCRs comprising V alpha V beta), are also suitable for use.

[0064] The chimeric antigen receptors disclosed herein may comprise an intracellular domain that provides an intracellular signal to a cell (expressing the CAR) upon binding of an antigen to the antigen-specific extracellular domain. In some embodiments, the intracellular signaling domain of the CAR is responsible for activating at least one of the effector functions of the T cell in which the chimeric receptor is expressed.

[0065] The term "intracellular domain" refers to the portion of a CAR that transmits an effector function signal and instructs a T cell to perform a specialized function upon antigen binding to the extracellular domain. Non-limiting examples of suitable intracellular domains include the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB1 chain, 829, Fc RIII, Fc RI, and combinations of signaling molecules such as CD3 zeta and CD28, CD27, 4-1BB, DAP-10, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the activation protein family, such as FcγRIII and FcεRI, may also be used. While the entire intracellular domain is typically used, it is often not necessary to use the entire intracellular polypeptide. In cases where a truncated portion of the intracellular signaling domain can be used, such a truncated portion may be used in place of the intact chain, so long as it still transmits the effector function signal. Thus, the term intracellular domain is intended to include any truncated portion of the intracellular domain sufficient to transmit the effector function signal. Typically, the antigen-specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane domain. The transmembrane domain traverses the cell membrane, anchoring the CAR to the T cell surface and connecting the extracellular domain to the intracellular signaling domain, thus influencing the expression of the CAR on the T cell surface. The chimeric antigen receptor may further comprise one or more costimulatory domains and / or one or more spacers. The costimulatory domain is derived from the intracellular signaling domain of a costimulatory protein, which enhances cytokine production, proliferation, cytotoxicity, and / or persistence in vivo. A "peptide hinge" connects the antigen-specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, and optionally, the costimulatory domain is fused to a second costimulatory domain, which is fused to a signaling domain, including but not limited to CD3ζ.For example, the inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain, and in the case of tandem CARs, between multiple scFvs, can affect the flexibility of the antigen-binding domain(s) and thus CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art.

[0066] As used herein, the term "gene editing" refers to the process of changing the genetic information present in the genome of a cell. This gene editing can be performed by manipulating genomic DNA, resulting in the modification of genetic information. In some embodiments, such gene editing can affect the expression of the edited DNA. In other embodiments, such gene editing does not affect the expression of the edited DNA. In some embodiments, the gene editing of the modified cells disclosed herein can be performed using the gene editing tools described herein. Non-limiting examples of gene editing tools include RNA interference molecules (e.g., shRNA, siRNA, miRNA), antisense oligonucleotides, CRISPR, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, restriction endonucleases, or any combination thereof.

[0067] As used herein, the terms "ug" and "uM" are used synonymously with "μg" and "μM", respectively.

[0068] The various aspects described herein are described in further detail in the subsections below.

[0069] II. Methods of the Disclosure IIa. Treatment method The present disclosure relates to methods of treating a tumor (or cancer) in a subject in need thereof, comprising administering to the subject modified cells that express reduced levels of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, "reduced levels of the NFE2L2 gene and / or Nrf2 protein" comprises no expression of the NFE2L2 gene and / or Nrf2 protein.

[0070] The term "Nrf2" or "nuclear factor erythroid 2-related factor 2" refers to a protein that belongs to the family of basic leucine zipper protein transcription factors. Nrf2 is also known as "nuclear factor erythroid 2-like 2," "nuclear factor erythroid-derived 2-like 2," "HEBP1," and "IMDDHH."

[0071] In humans, Nrf2 is encoded by the NFE2L2 gene located on chromosome 2. Three human Nrf2 isoforms are known. Isoform 1 (UniProt: Q16236-1) consists of 605 amino acids and is designated as the canonical sequence. Isoform 2 (UniProt: Q16236-2) consists of 589 amino acids and differs from the canonical sequence by the deletion of amino acids 1 to 16. Isoform 3 (UniProt: Q16236-3) consists of 582 amino acids and differs from the canonical sequence by the deletion of amino acids 1 to 16 and amino acids 135 to 141. Table 1 (below) presents the amino acid sequences of the three known Nrf2 isoforms. [Table 1-1] [Table 1-2]

[0072] As used herein, the term "Nrf2" includes any variant or isoform of Nrf2 naturally expressed by a cell. The following naturally occurring variants of Nrf2 are known in the art: (i) amino acid position 31: G→R; (ii) amino acid position 43: R→Q; (iii) amino acid position 79: E→K; (iv) amino acid position 80: T→K; (v) amino acid position 81: G→S; (vi) amino acid position 99: S→P; and (vii) amino acid position 268: V→M. Thus, in some embodiments, the modified cells disclosed herein express reduced levels of the NFE2L2 gene and / or Nrf2 protein associated with isoform 1. In certain embodiments, the modified cells disclosed herein express reduced levels of the NFE2L2 gene and / or Nrf2 protein associated with isoform 2. In a further aspect, the modified cells disclosed herein express reduced levels of the NFE2L2 gene and / or Nrf2 protein associated with isoform 3. In yet a further aspect, the modified cells disclosed herein express reduced levels of the NFE2L2 gene and / or Nrf2 protein associated with all isoforms.

[0073] In some embodiments, Nrf2, or any variants and isoforms thereof, may be isolated from cells or tissues that naturally express them, or may be recombinantly produced. The nucleic acid sequences of polynucleotides encoding the above human Nrf2 isoforms are provided in Table 2 (below). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0074] In some embodiments, the expression level of the NFE2L2 gene is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene). In some embodiments, the expression level of the Nrf2 protein is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, the expression levels of both the NFE2L2 gene and the Nrf2 protein are reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene).

[0075] In some embodiments, treating a tumor comprises reducing tumor volume in a subject. Accordingly, in certain embodiments, administering modified cells of the present disclosure (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) to a subject reduces tumor volume in the subject compared to a reference tumor volume. In some embodiments, the reference tumor volume is the tumor volume in the subject prior to administration of the modified cells. In further embodiments, the reference tumor volume is the tumor volume in a corresponding subject that did not receive the administration. In some embodiments, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor volume.

[0076] In some embodiments, treating a tumor comprises reducing tumor weight in a subject. In certain embodiments, the modified cells disclosed herein, when administered to a subject, can reduce tumor weight in the subject. In some embodiments, the tumor weight is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after administration compared to the reference tumor weight. In some embodiments, the reference tumor weight is the tumor weight in the subject before administration of the modified cells. In further embodiments, the reference tumor weight is the tumor weight in a corresponding subject that does not receive the administration.

[0077] In some embodiments, administration of the modified cells of the present disclosure increases TIL (e.g., CD4 + or CD8 +In certain embodiments, the number and / or proportion of TILs in the tumor is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference (e.g., a subject that did not receive the modified cells or a corresponding value in the same subject prior to administration of the modified cells).

[0078] In some embodiments, administration of modified cells of the present disclosure (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) can reduce the number and / or proportion of regulatory T cells (Tregs) in a subject's tumor. In some embodiments, regulatory T cells are CD4 + In some embodiments, the regulatory T cells are Foxp3 + In certain embodiments, the number and / or proportion of regulatory T cells in the tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., the corresponding number and / or proportion in a subject who did not receive the modified cells).

[0079] In some embodiments, administration of the modified cells disclosed herein increases the CD8 + The ratio of TILs to Tregs may be increased. In certain embodiments, CD8 +The ratio of TILs to Tregs is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% after administration compared to a reference (e.g., the number and / or proportion of TILs in a tumor in a subject that did not receive the modified cells).

[0080] In some embodiments, administration of the modified cells disclosed herein can reduce the number and / or proportion of myeloid-derived suppressor cells (MDSCs) in a subject's tumor. As used herein, the term "myeloid-derived suppressor cells" (MDSCs) refers to a heterogeneous immune cell population defined by their myeloid origin, immature state, and ability to potently suppress T cell responses. They are known to proliferate in certain pathological conditions, such as chronic infection and cancer. In certain embodiments, the MDSCs are monocytic MDSCs (M-MDSCs). In other embodiments, the MDSCs are polymorphonuclear MDSCs (PMN-MDSCs). In some embodiments, the number and / or proportion of MDSCs in a tumor is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to a reference (e.g., a value in a corresponding subject not receiving the modified cells).

[0081] In some embodiments, administration of the modified cells disclosed herein increases the CD8 + The ratio of TILs to MDSCs can be increased. +After administration, the ratio of TILs to MDSCs increases by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to a reference (e.g., the value of a corresponding subject that did not receive the modified cells).

[0082] In some embodiments, the modified cells disclosed herein (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) comprise immune cells. In particular embodiments, the immune cells comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In further embodiments, the modified immune cells disclosed herein are lymphocytes. In some embodiments, the lymphocytes comprise T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or any combination thereof. In further embodiments, the modified immune cells may further comprise a chimeric antigen receptor (CAR). In some embodiments, the modified immune cells may further comprise a T cell receptor, e.g., an engineered TCR. Thus, in particular embodiments, the modified cells disclosed herein are T cells. In some embodiments, the modified cells disclosed herein are TILs. In some embodiments, the modified cells disclosed herein are NK cells. In some embodiments, the modified cells disclosed herein are lymphokine-activated killer cells. In some embodiments, T cells comprise a CAR. In other embodiments, the modified cells disclosed herein are NK cells. In certain embodiments, NK cells comprise a CAR. In further embodiments, the modified cells of the present disclosure comprise both T cells and NK cells. In certain embodiments, both T cells and NK cells comprise a CAR.

[0083] In some embodiments, the methods of the present invention can be used to modify any immune cell type. In other embodiments, the methods of the present invention can be used to modify cells for any adoptive cell transfer (ACT) therapy (also known as adoptive cell therapy). ACT therapy can be autologous or allogeneic therapy. In some embodiments, ACT therapy includes, but is not limited to, CAR T therapy, tumor-infiltrating lymphocyte (TIL) therapy, NK cell therapy, or any combination thereof.

[0084] In some embodiments, the methods of the present invention can be used to modify TILs for TIL therapy. The use of TILs as adoptive cell transfer therapy to treat cancer has been studied for over 20 years, using TIL adoptive cell therapy for melanoma. Rosenberg SA et al. (July 2011). Clinical Cancer Research 17(13):4550-7 (July 2011). In adoptive T cell transfer therapy, TILs are expanded ex vivo from small fragments of surgically resected tumors or from single-cell suspensions isolated from tumor fragments. Multiple individual cultures are established, grown separately, and assayed for specific tumor recognition. TILs are expanded for several weeks. The specific TIL line that shows the best tumor response is then further expanded using a "rapid expansion protocol" (REP), typically using anti-CD3 activation for two weeks. TILs expanded in culture can be modified at any time during the ex vivo process to reduce expression of the NFE2L2 gene and / or Nrf2 protein. The final TILs after REP are infused back into the patient, a process that may include a preparatory chemotherapy regimen to deplete endogenous lymphocytes to allow the adoptively transferred TILs to sufficiently approach and surround the tumor site.

[0085] In some embodiments, the methods of the invention can be used to modify T cells that contain a T cell receptor, e.g., an engineered TCR. As used herein, the term "engineered TCR" or "engineered T cell receptor" refers to a T cell receptor (TCR) that has been selected, cloned, and / or engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen that is then introduced into a T cell population.

[0086] In some embodiments, the modified cells disclosed herein (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) may have one or more improved properties. In certain embodiments, improving one or more properties of a cell (e.g., an immune cell, e.g., a tumor-infiltrating lymphocyte) may be useful in treating a tumor (e.g., reducing tumor volume and / or tumor weight). One or more properties that may be improved with the present disclosure include any property of a cell (e.g., a TIL) that may be useful in treating cancer. For example, in certain embodiments, such properties include: (i) increased expansion and / or proliferation; (ii) increased persistence and / or survival (e.g., reduced exhaustion / anergy); (iii) increased anti-tumor activity (e.g., the ability to target and kill tumor cells); and (iv) combinations thereof.

[0087] In some embodiments, the expansion and / or proliferation of the modified cells disclosed herein is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference (e.g., a subject that did not receive the modified cells or a corresponding value in the same subject prior to administration of the modified cells). In some embodiments, the persistence and / or viability of the modified cells disclosed herein is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference (e.g., a subject that did not receive the modified cells or a corresponding value in the same subject prior to administration of the modified cells). In further aspects, the anti-tumor activity of the modified cells disclosed herein is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference (e.g., a subject that did not receive the modified cells or a corresponding value in the same subject prior to administration of the modified cells).

[0088] In some embodiments, the one or more characteristics may include the ability of the cells (e.g., TILs) to produce effector molecules useful for treating tumors. In certain embodiments, the effector molecules include cytokines. Non-limiting examples of effector molecules include IFN-γ, TNF-α, IL-2, granzyme B, perforin, MIP-1β, CD107a, or combinations thereof. Thus, in some embodiments, the modified cells disclosed herein can produce increased amounts of IFN-γ when stimulated with an antigen, such as a cognate antigen (e.g., a tumor antigen). In certain embodiments, the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference cell (e.g., a corresponding cell that has not been modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein).

[0089] As described herein, Nrf2 has been reported in the art to be important in protecting cells from oxidative stress, which may be caused by agents such as reactive oxygen species and reactive nitrogen species. Applicant has discovered that the modified cells disclosed herein (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) exhibit increased resistance to oxidative stress compared to reference cells. In some embodiments, the reference cells are corresponding cells that have not been modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein. In certain embodiments, the oxidative stress resistance of the modified cells disclosed herein is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% or more compared to the reference cells.

[0090] In some embodiments, the modified cells disclosed herein can be modified using gene editing tools.In some embodiments, the modified cells disclosed herein can be modified using RNAi.In some embodiments, the modified cells disclosed herein can be modified using antisense oligonucleotides.Further description of the useful gene editing tools of the present disclosure is provided elsewhere herein.

[0091] In some embodiments, the modified cells described herein may be further modified to express a chimeric antigen receptor. In some embodiments, the modified CAR-expressing cells may have improved anti-cancer properties.

[0092] Without being bound by any theory, the modified cells of the present disclosure can reduce or prevent T cell exhaustion.

[0093] Whether a cell exhibits increased resistance to oxidative stress can be measured by any method available in the art. In some embodiments, the increased resistance of a cell to oxidative stress can result in the cell exhibiting improved function. For example, in certain embodiments, the modified cells disclosed herein can proliferate in the presence of high concentrations of reactive oxygen species. In some embodiments, the modified cells of the present disclosure can express a cytolytic molecule in the presence of high concentrations of reactive oxygen species. In certain embodiments, the cytolytic molecule comprises granzyme B. In further embodiments, the modified cells of the present disclosure can produce a cytokine in the presence of high concentrations of reactive oxygen species. In some embodiments, the cytokine comprises IFN-γ. In some embodiments, the reactive oxygen species comprises hydrogen peroxide (H2O2). In certain embodiments, the modified cells disclosed herein can (i) proliferate, (ii) express a cytolytic molecule, and (iii) produce a cytokine in the presence of high concentrations of reactive oxygen species.

[0094] As described herein, the modified cells of the present disclosure (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) can be used to treat various types of cancer. Non-limiting examples of cancers (or tumors) that can be treated with the methods disclosed herein include squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colon cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, pancreatic cancer, cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or or carcinoma), gastric cancer, germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer (e.g., esophagogastric junction cancer), small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, ureteral cancer, renal pelvic cancer, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, ascites Environmentally induced cancers, including those induced by vests, virus-associated cancers or cancers of viral origin (e.g., tumors associated with or derived from human papillomavirus (HPV)), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid (producing B cells, T cells, NK cells, and plasma cells), e.g., all types of leukemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic, and / or myelomas. myeloid leukemias, such as acute myeloid leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematologic malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic lymphoma (e.g., Ki1; + ) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary somatic Liquid lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphoid system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and Waldenström syndrome. Lymphoplasmacytic lymphoma (LPL) with leukemia-associated macroglobulinemia; myelomas, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratoma, fibrosarcoma, rhabdomyosarcoma, and thyroid cancer; Tumors of mesenchymal origin, including rhabdomyosarcoma and osteosarcoma; and other tumors, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratoma, hematopoietic tumors of the lymphoid system, such as T-cell tumors and B-cell tumors, including, but not limited to, T-prolymphocytic leukemia (T-PLL) including small cell and cerebriform cell types, T-cell large granular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head and neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination thereof.

[0095] In some embodiments, cancers (or tumors) that can be treated with the modified cells disclosed herein include breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In certain embodiments, the cancer (or tumor) that can be treated with the methods of the present disclosure is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer (or tumor) that can be treated is brain cancer. In certain embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer (or tumor) that can be treated with the methods of the present disclosure is skin cancer. In some embodiments, the skin cancer is basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), melanoma, Merkel cell carcinoma (MCC), or a combination thereof. In certain embodiments, the head and neck cancer is head and neck squamous cell carcinoma. In further embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the esophageal cancer is gastroesophageal junction cancer. In certain embodiments, the kidney cancer is renal cell carcinoma. In some embodiments, the liver cancer is hepatocellular carcinoma. In certain embodiments, cancers that can be treated with the present disclosure include colon cancer, skin cancer, lymphoma, lung cancer, or a combination thereof.

[0096] In some embodiments, the modified cells disclosed herein can be used in combination with other therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Accordingly, in certain embodiments, the methods of treating tumors disclosed herein include administering the modified cells of the present disclosure in combination with one or more additional therapeutic agents. Such agents may include, for example, chemotherapeutic agents, targeted anti-cancer therapies, oncolytic agents, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation procedures, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some embodiments, the modified cells disclosed herein (i.e., those expressing reduced levels of the NFE2L2 gene and / or Nrf2 protein) can be used in combination with standard of care treatment (e.g., surgery, radiation, and chemotherapy). The methods described herein can also be used as maintenance therapy, e.g., therapy aimed at preventing tumor development or recurrence.

[0097] In some embodiments, the modified cells of the present disclosure can be used in combination with one or more anti-cancer drugs to target multiple components of the immune pathway. Non-limiting examples of such combinations include therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF-secreting cellular vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example, by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or depleting or blocking Tregs or other immune suppressor cells (e.g., myeloid-derived suppressor cells); therapies that stimulate positive immune regulation, for example, by stimulating the CD-137, OX-40, and / or CD40 or GITR pathways and / or agonists that stimulate T cell effector function; therapies that systemically increase the frequency of anti-tumor T cells, for example, using CD25 antagonists (e.g., daclizumab) or ex These include therapies that deplete or inhibit Tregs, such as Tregs in tumors, by in vivo anti-CD25 bead depletion; therapies that affect the function of suppressor myeloid cells in tumors; therapies that improve the immunogenicity of tumor cells (e.g., anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, e.g., cells modified with chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; therapies that reverse / prevent T cell anergy or exhaustion; therapies that induce innate immune activation and / or inflammation at the tumor site; administration of immune-stimulating cytokines; blockade of immune-suppressing cytokines; or any combination thereof.

[0098] In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling by a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the methods of the present invention include a CTLA-4 antagonist (e.g., an anti-CTLA-4 antibody), a PD-1 antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a combination thereof. Non-limiting examples of such immune checkpoint inhibitors include anti-PD1 antibodies (e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof; anti-PD-L1 antibodies (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and anti-CTLA-4 antibodies (e.g., ipilimumab (YERVOY®), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof).

[0099] In some embodiments, the anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through a particular immune checkpoint pathway). In certain embodiments, the immune checkpoint activator comprises an OX40 agonist (e.g., an anti-OX40 antibody), a LAG-3 agonist (e.g., an anti-LAG-3 antibody), a 4-1BB (CD137) agonist (e.g., an anti-CD137 antibody), a GITR agonist (e.g., an anti-GITR antibody), or a combination thereof.

[0100] In some embodiments, the modified cells disclosed herein are administered to a subject before or after administration of an additional therapeutic agent. In other embodiments, the modified cells are administered to a subject simultaneously with the additional therapeutic agent. In certain embodiments, the modified cells and the additional therapeutic agent may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In other embodiments, the modified cells and the additional therapeutic agent are administered simultaneously as separate compositions.

[0101] In some embodiments, subjects that can be treated with the present disclosure are non-human animals, such as rats or mice. In some embodiments, subjects that can be treated are humans.

[0102] IIb. Methods for improving immune responses In some embodiments, the present disclosure relates to methods for improving a subject's immune response. In particular, the methods disclosed herein can be used to improve (e.g., increase) a subject's immune response, e.g., to prevent, reduce, or inhibit immune tolerance to immune cell therapy, e.g., chimeric antigen receptor (CAR)-expressing cells or engineered T cell receptor (TCR)-expressing cells. In certain embodiments, the immune response is an anti-tumor immune response. In certain embodiments, improving the immune response includes preventing, reducing, or inhibiting immune tolerance. Thus, provided herein are methods for improving the anti-tumor immune response of CAR-expressing cells or TCR-expressing cells, comprising modifying the cells to express reduced levels of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, reducing the expression of the NFE2L2 gene and / or Nrf2 protein improves the anti-tumor immune response of CAR-expressing cells or TCR-expressing cells. In some embodiments, reducing the expression of the NFE2L2 gene and / or Nrf2 protein reduces or inhibits immune tolerance of CAR-expressing cells or TCR-expressing cells.

[0103] In some embodiments, the expression level of the NFE2L2 gene and / or Nrf2 protein in the CAR- or TCR-expressing cell is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, expression of the NFE2L2 gene and / or Nrf2 protein in the CAR- or TCR-expressing cell is completely inhibited following modification.

[0104] In some embodiments, the anti-tumor immune response of an engineered (i.e., engineered to express reduced levels of the NFE2L2 gene and / or Nrf2 protein) CAR- or TCR-expressing cell is increased by at least about at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% or more compared to a reference anti-tumor immune response (e.g., the anti-tumor immune response of a cell that has not been engineered to express reduced levels of the NFE2L2 gene and / or Nrf2 protein).

[0105] The anti-tumor immune response of CAR-expressing cells or TCR-expressing cells can be measured using various methods known in the art. For example, in some embodiments, the anti-tumor immune response of CAR-expressing cells or TCR-expressing cells can be observed by measuring the amount of effector molecules produced by the cells when stimulated with a cognate antigen (e.g., by ELISA or flow cytometry). In certain embodiments, the effector molecule comprises a cytokine, such as one useful for treating tumors. Non-limiting examples of effector molecules include IFN-γ, TNF-α, IL-2, granzyme B, perforin, MIP-1β, CD107a, or a combination thereof. In certain embodiments, the cytokine is IFN-γ. Thus, in some embodiments, improving the anti-tumor response of cells comprises increasing the amount of IFN-γ produced by the cells. In some embodiments, the modified CAR-expressing cells or modified TCR-expressing cells (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) produce an increased amount of IFN-γ when stimulated with a cognate antigen (e.g., a tumor antigen) compared to a reference cell (e.g., a corresponding cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to the reference cell.

[0106] In some embodiments, the anti-tumor immune response of a CAR- or TCR-expressing cell can be measured by assessing the cell's ability to proliferate upon stimulation with a cognate antigen (e.g., a tumor antigen). In certain embodiments, modified CAR- or TCR-expressing cells (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) exhibit increased proliferation upon stimulation compared to reference cells (e.g., corresponding cells that have not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In some embodiments, the proliferation of modified CAR- or TCR-expressing cells is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to the reference cells.

[0107] In some embodiments, the anti-tumor immune response of a CAR- or TCR-expressing cell can be assessed by observing the expression of different phenotypic markers on the cell surface (e.g., using flow cytometry). For example, in certain embodiments, improving the anti-tumor immune response of a CAR- or TCR-expressing cell (i.e., by reducing expression of the NFE2L2 gene and / or Nrf2 protein) comprises reducing the expression of one or more immune checkpoint inhibitor molecules (e.g., PD-1) in the cell. Thus, in some embodiments, the modified CAR- or TCR-expressing cell disclosed herein expresses reduced levels of one or more immune checkpoint inhibitors. In certain embodiments, the expression level of one or more immune checkpoint inhibitor molecules is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% as compared to a reference cell (e.g., a corresponding cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein).

[0108] In some embodiments, the improved anti-tumor immune response of the CAR- or TCR-expressing cells comprises increased expression of a marker associated with effector activity (e.g., anti-tumor activity). Non-limiting examples of markers associated with effector activity include Ki-67, granzyme B, T-bet, Eomes, CXCR3, or a combination thereof. As will be apparent to one of skill in the art, in some embodiments, the marker associated with effector activity may be a cytokine, such as those described above (e.g., IFN-γ, TNF-α, IL-2). In particular embodiments, the marker associated with effector activity is granzyme B. In some embodiments, the modified CAR- or TCR-expressing cells disclosed herein express elevated levels of granzyme B compared to reference cells (e.g., corresponding cells that have not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, the expression level of Granzyme B in the modified CAR-expressing or TCR-expressing cell is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to the reference cell.

[0109] In some embodiments, reducing the expression of the NFE2L2 gene and / or Nrf2 protein in a CAR- or TCR-expressing cell can increase the resistance of the CAR- or TCR-expressing cell to oxidative stress, such as that caused by reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). In some embodiments, oxidative stress resistance is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference cell (e.g., a corresponding cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein).

[0110] As described herein, oxidative stress resistance of cells can be measured by various methods known in the art. In some embodiments, oxidative stress resistance can be observed by assessing whether cells maintain functionality and / or viability in the presence of oxidative stress (e.g., high concentrations of reactive oxygen species). In certain embodiments, modified CAR-expressing cells disclosed herein can proliferate in the presence of high concentrations of reactive oxygen species. In some embodiments, modified CAR-expressing cells disclosed herein can express cytolytic molecules in the presence of high concentrations of reactive oxygen species. In certain embodiments, the cytolytic molecules comprise granzyme B. In further embodiments, modified CAR-expressing cells or TCR-expressing cells of the present disclosure can produce cytokines in the presence of high concentrations of reactive oxygen species. In some embodiments, the cytokines comprise IFN-γ. In some embodiments, the reactive oxygen species comprise hydrogen peroxide (H2O2). In certain embodiments, modified CAR-expressing cells disclosed herein can (i) proliferate, (ii) express cytolytic molecules, and (iii) produce cytokines in the presence of high concentrations of reactive oxygen species.

[0111] To reduce the expression of the NFE2L2 gene and / or Nrf2 protein in a CAR-expressing cell or a TCR-expressing cell, any method known in the art for reducing the expression of a gene and / or protein in a cell can be used. For example, in some embodiments, the expression of the NFE2L2 gene and its encoded Nrf2 protein in a CAR-expressing cell or a TCR-expressing cell can be reduced by contacting the cell with a gene editing tool that can reduce the expression level of the NFE2L2 gene and its encoded Nrf2 protein. Non-limiting examples of gene editing tools are provided elsewhere herein.

[0112] While the above methods for reducing expression of the NFE2L2 gene and / or Nrf2 protein are provided in the context of CAR-expressing cells, one of skill in the art will recognize that the methods disclosed herein can be used with any cell in which it is desired to reduce expression of the NFE2L2 gene and / or Nrf2 protein. In some embodiments, the cell to be modified (i.e., in which expression of the NFE2L2 gene and / or Nrf2 protein is to be reduced) is an immune cell. In certain embodiments, the immune cell comprises a lymphocyte, a neutrophil, a monocyte, a macrophage, a dendritic cell, or a combination thereof. In some embodiments, the lymphocyte comprises a T cell, a tumor-infiltrating lymphocyte (TIL), a lymphokine-activated killer cell, a natural killer (NK) cell, or a combination thereof. In certain embodiments, the lymphocyte is a T cell, e.g., a CD4+ T cell or a CD8+ T cell. In further embodiments, the lymphocyte is a tumor-infiltrating lymphocyte (TIL). In certain embodiments, the TIL is a CD8+ TIL. In other embodiments, the TILs are CD4+ TILs. Thus, in some embodiments, the CAR-expressing cells that can be modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein are immune cells. In particular embodiments, the CAR-expressing cells that can be modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein are T cells (i.e., CAR T cells). In some embodiments, the CAR-expressing or TCR-expressing cells that can be modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein are NK cells (i.e., CAR NK cells).

[0113] In some embodiments, contacting the cell to be modified with the gene editing tool can be performed in vivo, in vitro, ex vivo, or a combination thereof. In certain embodiments, the contacting is performed in vivo (e.g., gene therapy). In other embodiments, the contacting is performed in vitro. In further embodiments, the contacting is performed ex vivo.

[0114] IIc. Cells containing reduced levels of the NFE2L2 gene and / or Nrf2 protein In some embodiments, the present disclosure provides cells, e.g., immune cells, e.g., CAR-expressing cells or TCR-expressing cells, that express reduced levels of the NFE2L2 gene and / or Nrf2 protein. In certain embodiments, the expression of the NFE2L2 gene and / or Nrf2 protein in immune cells, e.g., CAR-expressing cells or TCR-expressing cells, generated according to the present disclosure is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to a reference cell (e.g., a corresponding cell that has not been modified to express a relatively low level of the NFE2L2 gene and / or Nrf2 protein). In some embodiments, the expression of the NFE2L2 gene and / or Nrf2 protein in the CAR-expressing cells or TCR-expressing cells is completely inhibited.

[0115] In some embodiments, the present disclosure relates to a method of preparing cells for chimeric antigen receptor or T cell receptor engineering, the method comprising contacting the cells with a gene editing tool to reduce the expression level of the NFE2L2 gene and / or Nrf2 protein in the cells. As disclosed herein, the expression level of the NFE2L2 gene and / or Nrf2 protein in a CAR-expressing cell or a TCR-expressing cell can be reduced using various methods. In certain embodiments, these methods comprise one or more gene editing tools described elsewhere herein. In some embodiments, the expression of the NFE2L2 gene and its encoded Nrf2 protein is reduced in a CAR-expressing cell or a TCR-expressing cell by contacting the cell with an shRNA (e.g., specific for the NFE2L2 gene). In a further embodiment, the expression of the NFE2L2 gene and its encoded Nrf2 protein is reduced by contacting the cell with a CRISPR (e.g., a CRISPR-Cas9 system) (e.g., specific for the NFE2L2 gene).

[0116] In some embodiments, contact between the gene editing tool and the cell includes various delivery routes. Generally, for the gene editing tool disclosed herein to reduce the expression of the NFE2L2 gene and / or Nrf2 protein in the cell, the gene editing tool must be able to enter the cell and bind to the gene of interest. In some embodiments, any delivery vehicle known in the art for delivering a molecule of interest to a cell can be used. See U.S. Patent No. 10,047,355 B2, which is incorporated herein by reference in its entirety. Further disclosure regarding vectors that can be used is provided elsewhere in this disclosure.

[0117] In some embodiments, the method of preparing cells for chimeric antigen receptor engineering further comprises modifying the cells to express a CAR or TCR. In certain embodiments, modifying the cells to express a CAR or TCR comprises contacting the cells with a nucleic acid sequence encoding a CAR. In some embodiments, the nucleic acid sequence encoding the CAR is expressed from a vector (e.g., an expression vector).

[0118] In some embodiments, the CAR or TCR that can be expressed in the modified cells disclosed herein targets one or more antigens expressed on tumor cells, such as malignant B cells, malignant T cells, or malignant plasma cells. In certain embodiments, the CAR can target an antigen selected from CD2, CD3ε, CD4, CD5, CD7, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, the extracellular portion of the APRIL protein, or a combination thereof.Other non-limiting examples of antigens to which a CAR may bind include TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bc r-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma metastatic breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof.

[0119] In certain embodiments, the modified cells of the present disclosure may express a T cell receptor (TCR) that targets a tumor antigen. T cell receptors are heterodimers composed of two distinct transmembrane polypeptide chains, an α chain and a β chain, each of which consists of a constant region that anchors the chain within the T cell surface membrane and a variable region that recognizes and binds to antigens presented by the MHC. The TCR complex is associated with two heterodimers, CD3γε and CD3δε, and six polypeptides that form one homodimer, CD3ζ, which together form the CD3 complex. T cell receptor-engineered T cell therapy utilizes the modification of T cells bearing these complexes to specifically target antigens expressed by specific tumor cells.

[0120] In some embodiments, the modified TCR-engineered cells may target major types of common tumor-associated antigens (common TAAs) and unique tumor-associated antigens (unique TAAs), or tumor-specific antigens. The former may include, but are not limited to, cancer-testis (CT) antigens, overexpressed antigens, and differentiation antigens, while the latter may include, but are not limited to, neoantigens and oncovirus antigens. Human papillomavirus (HPV) E6 and E7 proteins belong to the oncovirus antigen category.

[0121] In some embodiments, modified TCR-engineered cells may target melanoma-associated antigens (MAGEs), including, but not limited to, CT antigens such as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12. In some embodiments, modified TCR-engineered cells may target glycoprotein (gp100) found primarily in melanoma and normal melanocytes, melanoma antigen recognized by T cells (MART-1), and / or tyrosinase. In some embodiments, modified TCR-engineered cells may target Wilms' tumor 1 (WT1), an overexpressed antigen highly expressed in most acute myeloid leukemias (AMLs), acute lymphoblastic leukemias, almost all types of solid tumors, and several critical tissues, such as cardiac tissue. In some embodiments, the modified TCR-engineered cells can target mesothelin, another overexpressed antigen that is highly expressed in mesothelioma but is also present in mesothelial cells of several tissues, including the trachea.

[0122] In some embodiments, the modified TCR engineered cells can target any neoantigen that can be formed by random somatic mutations specific to an individual tumor.

[0123] In some embodiments, the modified immune cells of the present disclosure, such as CAR T cells or NK cells or TCR-engineered T cells, may target any one of tumor antigens. Non-limiting examples of antigens that the modified TCR-engineered cells may target include CD2, CD3ε, CD4, CD5, CD7, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, the extracellular portion of the APRIL protein, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2(Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gplOO, bc r-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma metastatic breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and any combination thereof.

[0124] In some embodiments, the cells that can be prepared to express CAR or TCR include immune cells. In certain embodiments, the immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the immune cells are lymphocytes. In certain embodiments, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, or a combination thereof. In some embodiments, the immune cells that can be prepared to express CAR are T cells (CAR T cells), for example, CD8+ T cells or CD4+ T cells. In certain embodiments, the T cells are natural killer T cells (NKT cells). In a further embodiment, the immune cells are NK cells (CAR NK cells).

[0125] In some embodiments, the CAR-expressing cells disclosed herein are CAR T cells. In particular embodiments, the CAR T cells are mono-CAR T cells. In further embodiments, the CAR T cells are genome-edited CAR T cells. In particular embodiments, the CAR T cells are dual-CAR T cells. In some embodiments, the CAR T cells are tandem-CAR T cells. In some embodiments, the CAR-expressing cells disclosed herein are CAR NKT cells. In particular embodiments, the CAR NKT cells are mono-CAR NKT cells. In further embodiments, the CAR NKT cells are dual-CAR NKT cells. In some embodiments, the CAR NKT cells are tandem-CAR NKT cells. Examples of such CAR T cells and CAR NKT cells are provided in International Application No. PCT / US2019 / 044195.

[0126] In some embodiments, the gene editing tool disclosed herein is expressed from a vector comprising the nucleic acid sequence encoding the gene editing tool.In certain embodiments, the nucleic acid sequence encoding the gene editing tool and the nucleic acid sequence encoding CAR or TCR are on separate vectors.In further embodiments, the nucleic acid sequence encoding the gene editing tool and the nucleic acid sequence encoding CAR or TCR are on the same vector.

[0127] As described herein, CAR- or TCR-expressing cells (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) generated by the methods disclosed herein may exhibit improved properties. For example, in certain embodiments, CAR- or TCR-expressing cells generated herein may exhibit increased effector activity compared to reference cells (e.g., CAR- or TCR-expressing cells that have not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In some embodiments, CAR- or TCR-expressing cells disclosed herein (i.e., those that express reduced levels of the NFE2L2 gene and / or Nrf2 protein) produce increased amounts of IFN-γ when stimulated with an antigen, such as a cognate antigen (e.g., a tumor antigen). In some embodiments, the amount of IFN-γ produced is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% or more compared to a reference cell (e.g., a CAR-expressing cell or a TCR-expressing cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein).

[0128] In some embodiments, the CAR-expressing or TCR-expressing cells of the present disclosure exhibit increased resistance to oxidative stress compared to a reference cell (e.g., a CAR-expressing or TCR-expressing cell that has not been modified to express relatively low levels of the NFE2L2 gene and / or Nrf2 protein). In certain embodiments, the CAR-expressing or TCR-expressing cells described herein can proliferate in the presence of high concentrations of reactive oxygen species. In further embodiments, the CAR-expressing or TCR-expressing cells disclosed herein can express a cytolytic molecule in the presence of high concentrations of reactive oxygen species. In certain embodiments, the cytolytic molecule comprises granzyme B. In some embodiments, the CAR-expressing or TCR-expressing cells generated by the methods described herein can produce cytokines (e.g., IFN-γ) in the presence of high concentrations of reactive oxygen species. In some embodiments, the reactive oxygen species comprises hydrogen peroxide (H2O2).

[0129] IId. Gene editing tools One or more gene editing tools can be used to modify the cells of the present disclosure, non-limiting examples of which are disclosed below.

[0130] CRISPR / Cas system In some embodiments, gene editing tools that can be used in the present disclosure include CRISPR / Cas systems. Such systems can use, for example, Cas9 nuclease, which is optionally codon-optimized for the desired cell type in which it is to be expressed (e.g., T cells, e.g., CAR-expressing T cells). Such systems can also use guide RNAs (gRNAs) that comprise two separate molecules. In certain embodiments, the bimolecular gRNA comprises a crRNA-like ("CRISPR RNA" or "targeter RNA" or "crRNA" or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA" or "activator RNA" or "tracrRNA" or "scaffold") molecule.

[0131] The crRNA contains both the DNA targeting segment (single strand) of the gRNA and a stretch of nucleotides that forms one half of the double-stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA. The corresponding tracrRNA (activator RNA) contains a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. Thus, the stretch of nucleotides in the crRNA is complementary to the stretch of nucleotides in the tracrRNA and hybridizes to form the dsRNA duplex of the protein-binding domain of the gRNA. Thus, each crRNA has a corresponding tracrRNA. The crRNA also contains a single-stranded DNA targeting segment. Thus, the gRNA contains a sequence that hybridizes to the target sequence (e.g., Nrf2 mRNA) and the tracrRNA. Thus, the crRNA and tracrRNA hybridize (as a matching pair) to form the gRNA. When used for intracellular modification, the exact sequence and / or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species (e.g., human) in which the RNA molecule is to be used.

[0132] Naturally occurring genes encoding the three elements (Cas9, tracrRNA, and crRNA) are typically organized as an operon(s). Naturally occurring CRISPR RNAs vary depending on the Cas9 system and organism, but often contain a 21-72 nucleotide targeting segment flanked by two 21-46 nucleotide direct repeats (DRs) (see, e.g., WO2014 / 131833). In S. pyogenes, the DRs are 36 nucleotides long, and the targeting segment is 30 nucleotides long. The 3'-located DRs are complementary to the corresponding tracrRNA, hybridize to the corresponding tracrRNA, and then bind to the Cas9 protein.

[0133] Alternatively, the CRISPR system used herein can also employ a fusion crRNA-tracrRNA construct (i.e., a single transcript) that functions with a codon-optimized Cas9. This single RNA is often referred to as the guide RNA or gRNA. Within the gRNA, the crRNA portion is identified as the "target sequence" for a given recognition site, and the tracrRNA is often referred to as the "scaffold." Briefly, a short DNA fragment containing the target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid contains the target sequence (approximately 20 nucleotides in some embodiments), some form of tracrRNA sequence (scaffold), as well as a suitable promoter active in the cell and elements necessary for proper processing in eukaryotic cells. Many of these systems rely on special complementary oligos that anneal to form double-stranded DNA and are then cloned into the gRNA expression plasmid.

[0134] Then, the gRNA expression cassette and the Cas9 expression cassette are introduced into the cell. See, for example, Mali P et al., (2013) Science 2013 Feb.15;339(6121):823-6, Jinek M et al., Science 2012 Aug.17;337(6096):816-21, Hwang WY et al., Nat Biotechnol 2013 March;31(3):227-9, Jiang W et al., Nat Biotechnol 2013 March;31(3):233-9, and Cong L et al., Science 2013 Feb.15;339(6121):819-23. Each of these is incorporated herein by reference in its entirety. See also, e.g., WO / 2013 / 176772A1, WO / 2014 / 065596A1, WO / 2014 / 089290A1, WO / 2014 / 093622A2, WO / 2014 / 099750A2, and WO / 2013142578A1, each of which is incorporated by reference herein in its entirety.

[0135] In some embodiments, the Cas9 nuclease may be provided in the form of a protein. In some embodiments, the Cas9 protein may be provided in the form of a complex with a gRNA. In other embodiments, the Cas9 nuclease may be provided in the form of a nucleic acid encoding the protein. The nucleic acid encoding the Cas9 nuclease may be RNA (e.g., messenger RNA (mRNA)) or DNA. In some embodiments, the gRNA may be provided in the form of RNA. In other embodiments, the gRNA may be provided in the form of DNA encoding the RNA. In some embodiments, the gRNA may be provided in the form of separate crRNA and tracrRNA molecules, or separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0136] In some embodiments, the gRNA comprises a third nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). In certain embodiments, the Cas protein is a type I Cas protein. In further embodiments, the Cas protein is a type II Cas protein. In certain embodiments, the type II Cas protein is Cas9. In some embodiments, the type II Cas, e.g., Cas9, is a human codon-optimized Cas.

[0137] In some embodiments, the Cas protein is a "nickase" that can create a single-strand break (i.e., a "nick") within a target nucleic acid sequence without cleaving both strands of double-stranded DNA (dsDNA). For example, Cas9 contains two nuclease domains (a RuvC-like nuclease domain and an HNH-like nuclease domain) that are responsible for cleaving opposite DNA strands. Mutations in either of these domains can result in nickases. Examples of mutations that result in nickases can be found, for example, in WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference.

[0138] In certain embodiments, two distinct Cas proteins (e.g., nickases) specific for target sites on each strand of dsDNA can create overhanging sequences complementary to overhanging sequences on another nucleic acid or distinct regions on the same nucleic acid. The overhanging ends created by contacting a nucleic acid with two nickases specific for target sites on both strands of dsDNA can be either 5' or 3' overhanging ends. For example, an overhanging sequence can be created by a first nickase creating a single-strand break in the first strand of dsDNA and a second nickase creating a single-strand break in the second strand of dsDNA. The target sites of each nickase that create the single-strand break can be selected so that the resulting overhanging end sequence is complementary to an overhanging end sequence on a different nucleic acid molecule. Complementary overhanging ends of two different nucleic acid molecules can be annealed by the methods disclosed herein. In some embodiments, the target site for the nickase on the first strand is different from the target site for the nickase on the second strand.

[0139] TALEN In some embodiments, the gene editing tool that can be used to edit (e.g., reduce or inhibit) the expression of the NFE2L2 gene and / or Nrf2 protein is a nuclease agent, such as a transcription activator-like effector nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to create double-strand breaks at specific target sequences in the genomes of prokaryotes or eukaryotes. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or a functional portion thereof, to the catalytic domain of an endonuclease, such as FokI.

[0140] The unique modular TAL effector DNA-binding domain allows for the design of proteins with potentially arbitrary DNA recognition specificity. Thus, the DNA-binding domain of a TAL effector nuclease can be engineered to recognize a specific DNA target site and thus used to create a double-strand break at the desired target sequence. See WO2010 / 079430, Morbitzer et al., (2010) PNAS 10.1073 / pnas.1013133107, Scholze & Boch (2010) Virulence 1:428-432, Christian et al., Genetics (2010) 186:757-761, Li et al., (2010) Nuc. Acids Res. (2010) doi:10.1093 / nar / gkq704, and Miller et al., (2011) Nature Biotechnology 29:143-148, all of which are incorporated herein by reference in their entireties.

[0141] Non-limiting examples of suitable TAL nucleases and methods for preparing suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Nos. 2011 / 0239315 A1, 2011 / 0269234 A1, 2011 / 0145940 A1, 2003 / 0232410 A1, 2005 / 0208489 A1, 2005 / 0026157 A1, 2005 / 0064474 A1, 2006 / 0188987 A1, and 2006 / 0063231 A1 (each of which is incorporated by reference herein).

[0142] In various embodiments, for example, a TAL effector nuclease is engineered that cleaves at or near a target nucleic acid sequence at a genomic locus of interest, where the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. TAL nucleases suitable for use in the various methods and compositions provided herein include those specifically designed to bind at or near a target nucleic acid sequence to be modified by a targeting vector described herein.

[0143] In some embodiments, each monomer of the TALEN comprises 12-25 TAL repeats, and each TAL repeat binds to a 1-bp subsite. In certain embodiments, the nuclease agent is a chimeric protein comprising a TAL repeat-based DNA-binding domain operably linked to an independent nuclease. In further embodiments, the independent nuclease is a FokI endonuclease. In some embodiments, the nuclease agent comprises a first TAL repeat-based DNA-binding domain and a second TAL repeat-based DNA-binding domain, each of the first and second TAL repeat-based DNA-binding domains being operably linked to a FokI nuclease, wherein the first and second TAL repeat-based DNA-binding domains recognize two adjacent target DNA sequences in each strand of the target DNA sequence separated by a cleavage site of about 6 bp to about 40 bp, and the FokI nuclease dimerizes and makes a double-stranded break in the target sequence.

[0144] In some embodiments, the nuclease agent comprises a first TAL repeat-based DNA binding domain and a second TAL repeat-based DNA binding domain, each of the first and second TAL repeat-based DNA binding domains being operably linked to a FokI nuclease, wherein the first and second TAL repeat-based DNA binding domains recognize two adjacent target DNA sequences in each strand of the target DNA sequence separated by a 5 bp or 6 bp cleavage site, and the FokI nuclease dimerizes and makes a double-stranded cleavage.

[0145] Zinc finger nucleases (ZFNs) In some embodiments, gene editing tools useful in the present disclosure include nuclease agents, such as zinc finger nuclease (ZFN) systems. Zinc finger-based systems include fusion proteins containing two protein domains: a zinc finger DNA-binding domain and an enzymatic domain. A "zinc finger DNA-binding domain," "zinc finger protein," or "ZFP" is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. By binding to a target DNA sequence (e.g., NFE2L2), the zinc finger domain directs the activity of the enzymatic domain to the vicinity of the sequence, thus inducing modification of endogenous target genes near the target sequence. Zinc finger domains can be engineered to bind to virtually any desired sequence. As disclosed herein, in some embodiments, the zinc finger domain binds to a DNA sequence encoding the Nrf2 protein. Thus, after identifying a target locus (e.g., a target locus among the target genes listed in Table 1) containing a target DNA sequence for which cleavage or recombination is desired, one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences within the target locus. Expression of a fusion protein containing the zinc finger binding domain and the enzymatic domain in a cell results in modification of the target locus.

[0146] In some embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al., (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific American February:56-65; U.S. Patent No. 6,453,242. Typically, a zinc finger domain is about 30 amino acids long. Each zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence that may overlap by one nucleotide with the four-nucleotide binding site of an adjacent zinc finger). Therefore, the length of the sequence (e.g., target sequence) to which the zinc finger binding domain is engineered determines the number of zinc fingers in the engineered zinc finger binding domain. For example, in a ZFP whose finger motifs do not bind to overlapping subsites, a two-finger binding domain binds to a six-nucleotide target sequence, a three-finger binding domain binds to a nine-nucleotide target sequence, and so on. The binding sites (i.e., subsites) of individual zinc fingers in a target site need not be contiguous, but may be separated by one or several nucleotides, depending on the length and nature of the amino acid sequences between zinc fingers (i.e., inter-finger linkers) in a multi-finger binding domain. In some embodiments, the DNA binding domain of an individual ZFN comprises 3-6 individual zinc finger repeats, each capable of recognizing 9-18 base pairs.

[0147] Zinc finger binding domains can be engineered to bind optimal sequences. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. Engineered zinc finger binding domains can have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection.

[0148] Selection of a target DNA sequence to which a zinc finger domain binds can be performed, for example, according to the method disclosed in U.S. Patent No. 6,453,242. It will be apparent to those skilled in the art that simple visual inspection of the nucleotide sequence may be used to select a target DNA sequence. Therefore, any means can be used to select a target DNA sequence in the methods described herein. Target sites typically have a length of at least 9 nucleotides, and thus are bound by zinc finger binding domains containing at least three zinc fingers. However, for example, a four-finger binding domain can bind to a 12-nucleotide target site, a five-finger binding domain can bind to a 15-nucleotide target site, or a six-finger binding domain can bind to an 18-nucleotide target site. Obviously, larger binding domains (e.g., seven-finger, eight-finger, nine-finger, and more) can also bind to longer target sites.

[0149] The enzyme domain portion of the zinc finger fusion protein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the enzyme domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, e.g., 2002-2003 Catalogue, New England Biolabs, Beverly, Mass., and Belfort et al., (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (e.g., 51 nuclease, mung bean nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease; see also Linn et al., (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as the source of the cleavage domain.

[0150] Exemplary restriction endonucleases (restriction enzymes) suitable for use as the enzymatic domain of the ZFPs described herein are present in many species and can bind to DNA (at a recognition site) in a sequence-specific manner and cleave the DNA at or near the binding site. Certain restriction enzymes (e.g., type IIS) cleave DNA at a site removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme FokI catalyzes double-stranded cleavage of DNA at the 9th nucleotide from its recognition site on one strand and the 13th nucleotide from its recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994, as well as Li et al., (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279, Li et al., (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768, Kim et al., (1994a) Proc. Natl. Acad. Sci. USA 91:883-887, Kim et al., (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in some embodiments, the fusion protein comprises at least one type IIS restriction enzyme enzymatic domain and one or more zinc finger binding domains.

[0151] An exemplary type IIS restriction enzyme whose cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer. Bitinaite et al., (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Thus, to perform targeted double-stranded DNA cleavage using a zinc finger-FokI fusion, two fusion proteins, each containing a FokI enzymatic domain, can be used to reconstitute a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI enzymatic domains can be used. An exemplary ZFP containing a FokI enzymatic domain is described in U.S. Patent No. 9,782,437.

[0152] Meganuclease In some embodiments, gene editing tools that can be used to control Nrf2 expression in cells include nuclease agents, such as meganuclease systems. Meganucleases are classified into four families based on conserved sequence motifs: the "LAGLIDADG," "GIY-YIG," "HNH," and "His-Cys box" families. These motifs are involved in metal ion coordination and phosphodiester bond hydrolysis.

[0153] HEases are notable for their long recognition sites and tolerance of some sequence polymorphisms in DNA substrates. The domains, structures, and functions of meganucleases are known. See, e.g., Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al. (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard (1999) Cell Mol Life Sci 55:1304-26; Stoddard (2006) Q Rev Biophys 38:49-95; and Moure et al. (2002) Nat Struct Biol 9:764.

[0154] In some instances, naturally occurring variant and / or engineered derivative meganucleases are used. Methods for modifying kinetics, cofactor interactions, expression, optimal conditions, and / or recognition site specificity, as well as screening for activity, are known. For example, Epinat et al., (2003) Nucleic Acids Res 31:2952-62, Chevalier et al., (2002) Mol Cell 10:895-905, Gimble et al., (2003) Mol Biol 334:993-1008, Seligman et al., (2002) Nucleic Acids Res. 30:3870-9, Sussman et al., (2004) J Mol Biol 342:31-41, Rosen et al., (2006) Nucleic Acids Res 34:4791-800, Chames et al., (2005) Nucleic Acids Res 33:e178, Smith et al., (2006) Nucleic Acids Res 34:e149, Gruen et al. See, e.g., et al., (2002) Nucleic Acids Res 30:e29, Chen and Zhao, (2005) Nucleic Acids Res 33:e154, WO2005105989, WO2003078619, WO2006097854, WO2006097853, WO2006097784, and WO2004031346, each of which is incorporated herein by reference in its entirety.

[0155] In this specification, without limitation, any meganuclease can be used, including I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SecVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP, PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, or any active variant or fragment thereof.

[0156] In some embodiments, the meganuclease recognizes a double-stranded DNA sequence of 12 to 40 base pairs. In some embodiments, the meganuclease recognizes one perfectly matched target sequence within a genome. In some embodiments, the meganuclease is a homing nuclease. In some embodiments, the homing nuclease is a "LAGLIDADG" family homing nuclease. In some embodiments, the "LAGLIDADG" family homing nuclease is selected from I-SceI, I-CreI, I-Dmol, or a combination thereof.

[0157] Restriction endonucleases In some embodiments, gene editing tools useful in the present disclosure include nuclease agents such as restriction endonucleases, including type I, type II, type III, and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sites but typically cleave at various locations from the nuclease binding site, which can be hundreds of base pairs away from the binding site (recognition site). In type II systems, restriction activity is independent of methylase activity, and cleavage typically occurs at specific sites within or near the binding site. Most type II enzymes cleave palindromic sequences, while type IIa enzymes recognize non-palindromic recognition sites and cleave outside the recognition site, type IIb enzymes double-cleave sequences at both sites outside the recognition site, and type IIs enzymes recognize asymmetric recognition sites and cleave on one side of the recognition site at a defined distance of approximately 1 to 20 nucleotides. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example, in the REBASE database (webpage at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press, Washington, DC).

[0158] As described herein, in some embodiments, gene editing tools (e.g., CRISPR, TALEN, meganuclease, restriction endonuclease, RNAi, antisense oligonucleotide) can be introduced into cells by any means known in the art. In certain embodiments, a polypeptide encoding a particular gene editing tool may be directly introduced into cells. Alternatively, a polynucleotide encoding a gene editing tool may be introduced into cells. In some embodiments, when a polynucleotide encoding a gene editing tool is introduced into cells, the gene editing tool may be expressed transiently, conditionally, or constitutively in the cells. Thus, the polynucleotide encoding the gene editing tool may be included in an expression cassette and operably linked to a conditional promoter, an inducible promoter, a constitutive promoter, or a tissue-specific promoter. Alternatively, the gene editing tool is introduced into cells as mRNA encoding or including the gene editing tool.

[0159] RNAi In some embodiments, gene editing tools that can be used to reduce the expression of Nrf2 in cells include RNA interference molecules ("RNAi"). As used herein, RNAi is an RNA polynucleotide that mediates the decreased expression of endogenous target gene products by degrading the target mRNA through endogenous gene silencing pathways (e.g., Dicer and RNA-induced silencing complex (RISC)). Non-limiting examples of RNAi agents include microRNAs (also referred to herein as "miRNAs"), short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), RNA aptamers, or combinations thereof.

[0160] In some embodiments, gene editing tools useful in the present disclosure include one or more miRNAs. "miRNA" refers to a naturally occurring small non-coding RNA molecule approximately 21-25 nucleotides in length. In some embodiments, miRNAs useful in the present disclosure are at least partially complementary to an Nrf2 mRNA molecule. miRNAs can downregulate (e.g., decrease) the expression of an endogenous target gene product (i.e., Nrf2 protein) through translational repression, mRNA cleavage, and / or deadenylation.

[0161] In some embodiments, gene editing tools that can be used in the present disclosure include one or more shRNAs. "shRNA" (or "short hairpin RNA" molecule) refers to an RNA sequence that contains a double-stranded region and a loop region at one end, forming a hairpin loop, and can be used to reduce and / or silence gene expression. The double-stranded region is typically about 19 to about 29 nucleotides long on each side of the stem, and the loop region is typically about 3 to about 10 nucleotides long (single-stranded overhanging nucleotides at the 3' or 5' end are optional). When shRNAs are cloned into a plasmid or non-replicative recombinant viral vector and introduced into cells, the shRNA coding sequence can be integrated into the genome. Thus, shRNAs can result in stable and consistent suppression of endogenous target gene (i.e., Nrf2) translation and expression.

[0162] In some embodiments, the gene editing tools disclosed herein include one or more siRNAs. "siRNA" refers to a double-stranded RNA molecule, typically about 21-23 nucleotides in length. The siRNA associates with a multiprotein complex called the RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cleaved. The antisense "guide" strand contained in the activated RISC then guides the RISC to the corresponding mRNA due to sequence homology, and the same nuclease cleaves the target mRNA (i.e., Nrf2 mRNA), resulting in specific gene silencing. In certain embodiments, the siRNA is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and has a two-base overhang at its 3' end. The siRNA can be introduced into individual cells and / or culture systems to degrade the target mRNA sequence (i.e., Nrf2 mRNA). siRNAs and shRNAs are further described in Fire et al., Nature 391:19, 1998, and U.S. Patent Nos. 7,732,417, 8,202,846, and 8,383,599, each of which is incorporated by reference herein in its entirety.

[0163] antisense oligonucleotides In some embodiments, the gene editing tool that can be used to reduce the expression of Nrf2 in cells comprises antisense oligonucleotide.As used herein, " antisense oligonucleotide " or " ASO " refers to the oligonucleotide that can regulate the expression of target gene (i.e., Nrf2) by hybridizing with target nucleic acid, particularly with the continuous sequence on target nucleic acid.Antisense oligonucleotide is not essentially double-stranded, and therefore is not siRNA or shRNA.

[0164] In some embodiments, the ASO useful in the present disclosure is single-stranded. It is understood that the single-stranded oligonucleotide of the present disclosure can form a hairpin or intermolecular duplex structure (a duplex between two molecules of the same oligonucleotide) as long as the degree of self- or inter-complementarity is less than approximately 50% over the entire length of the oligonucleotide. In some embodiments, the ASO useful in the present disclosure can contain one or more modified nucleosides or nucleotides, such as 2' sugar-modified nucleosides. Additional modifications that can be made to ASO (for example, those that can be used to inhibit or reduce Nrf2 expression) are provided, for example, in US Publication No. 2019 / 0275148 A1.

[0165] In some embodiments, if an ASO can recruit a nuclease, for example, an RNase H such as RNase H1, the ASO can reduce the expression of Nrf2 protein through the nuclease-mediated degradation of Nrf2 transcripts (e.g., mRNA). RNase H is a ubiquitous enzyme that hydrolyzes the RNA strand of an RNA / DNA duplex. Thus, in certain embodiments, when ASO binds to a target sequence (e.g., Nrf2 mRNA), it can induce the degradation of Nrf2 mRNA, thereby reducing the expression of Nrf2 protein.

[0166] In some embodiments, ASO comprises one or more morpholinos.As used herein, "morpholinos" refers to modified nucleic acid oligomers in which standard nucleobases are linked to morpholine rings and linked via phosphorodiamidate bonds.Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences.However, morpholinos do not target and degrade complementary mRNA sequences, but function by steric inhibition of mRNA translation and alteration of mRNA splicing.

[0167] As disclosed herein, the above examples of gene editing tools are not intended to be limiting, and any gene editing tool available in the art can be used to reduce or inhibit expression of the NFE2L2 gene and / or Nrf2 protein.

[0168] III. Nucleic Acids and Vectors Further embodiments described herein relate to one or more nucleic acid molecules encoding chimeric antigen receptors or T cell receptors that can be expressed in the modified cells of the present disclosure, including gene editing tools that reduce expression of the NFE2L2 gene and / or Nrf2 protein in cells. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" if it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA naturally linked to the isolated DNA) or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein can be, for example, DNA or RNA, and can contain or not contain intron sequences.In certain embodiments, nucleic acid is a cDNA molecule.The nucleic acid described herein can be obtained by using standard molecular biology techniques known in the art.

[0169] In some embodiments, the present disclosure provides vectors comprising gene editing tools that reduce expression of the NFE2L2 gene and / or Nrf2 protein in a cell, and / or comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor or T cell receptor that can be expressed in a modified cell of the disclosure. As described herein, such vectors can be used to modify cells (e.g., CAR-expressing cells) to express reduced levels of the NFE2L2 gene and / or Nrf2 protein, and such modified cells can be used to treat diseases or disorders such as cancer.

[0170] Suitable vectors for the present disclosure include expression vectors, viral vectors, and plasmid vectors. In some embodiments, the vector is a viral vector.

[0171] As used herein, an expression vector refers to any nucleic acid construct that, upon introduction into an appropriate host cell, contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the elements necessary for replication and translation. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0172] As used herein, viral vectors include, but are not limited to, retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40 virus; polyomavirus; Epstein-Barr virus; papillomavirus; herpesvirus; vaccinia virus; poliovirus; and nucleic acid sequences derived from RNA viruses such as retroviruses. Other vectors known in the art can also be readily used. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral integration into host cell DNA.

[0173] In some embodiments, the vector is derived from an adeno-associated virus. In other embodiments, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0174] Other vectors include plasmid vectors. Plasmid vectors have been widely reported in the art and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they do not replicate and integrate into the host genome. However, these plasmids with a promoter compatible with the host cell can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40, and pBlueScript. Further examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids are well known to those of skill in the art. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific DNA fragments.

[0175] IV. Pharmaceutical Compositions Further provided herein are compositions comprising cells (e.g., cells described herein) that have been modified to express reduced levels of the NFE2L2 gene and / or Nrf2 protein, and a pharmaceutically acceptable carrier, excipient, or stabilizer. As described herein, such pharmaceutical compositions can be used to prevent and / or treat cancer. As described herein, in some embodiments, the modified cells present in the pharmaceutical compositions disclosed herein are immune cells, such as T cells (e.g., T cells expressing a CAR or TCR) or NK cells (e.g., NK cells expressing a CAR or TCR).

[0176] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0177] The pharmaceutical composition may be formulated for any route of administration to a subject. Specific examples of routes of administration include intramuscular, subcutaneous, ophthalmic, intravenous, intraperitoneal, intradermal, intraorbital, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, or intratumoral. Parenteral administration, characterized by injection either subcutaneously, intramuscularly, or intravenously, is also contemplated herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions may also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the administered pharmaceutical composition may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0178] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multidose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0179] Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products that are combined with a solvent immediately before use, such as lyophilized powders, subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products that are combined with a vehicle immediately before use, and sterile emulsions. The solutions may be either aqueous or non-aqueous.

[0180] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0181] The pharmaceutical compositions provided herein can be formulated to target specific tissues, receptors, or other areas of the body of the subject to be treated.Many of such targeting methods are well known to those skilled in the art.All of such targeting methods are contemplated herein for the use of the compositions of the present invention.Non-limiting examples of targeting methods are described in, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, and 6,072, each of which is incorporated herein by reference in its entirety. See Nos. 1,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

[0182] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0183] All references, patents, or applications, whether U.S. or foreign, cited in this application are hereby incorporated by reference as if set forth herein in their entirety. In the event of any conflict, the textual disclosure herein will control. [Example]

[0184] The present disclosure will now be described in detail with reference to examples. It should be understood that the described examples are intended to merely illustrate the present disclosure and are not intended to be limiting.

[0185] Example 1. Determination of Nrf2 mRNA expression levels in tumor-infiltrating T cells We determined the level of nuclear factor E2-related factor 2 (Nrf2) mRNA expression in tumor-infiltrating T cells. Nrf2 plays an important role in protecting cells from damage caused by oxidative stress. To better understand the relationship between Nrf2 and oxidative stress, T cells were treated with various concentrations of hydrogen peroxide, a known oxidative stress inducer. As shown in Figure 1, increased oxidative stress (i.e., high H2O2 concentrations) led to higher levels of Nrf2 mRNA expression in T cells.

[0018] Nearly all cancers are associated with elevated oxidative stress. To better understand this phenomenon, T cells were isolated from tumors and / or different lymph nodes of tumor-bearing mice. As a control, T cells were also isolated from lymph nodes of healthy mice. As shown in Figure 2, the level of Nrf2 mRNA expression in tumor-infiltrating T cells was significantly higher than that in T cells from the draining lymph nodes (dLN) or any other lymph nodes of tumor-bearing mice. This result indicates the selectivity of Nrf2 expression in tumor-infiltrating T lymphocytes.

[0186] Example 2. Determining Altered Anti-Cancer Responses with Modulation of Nrf2 Expression To begin to understand how modulation of Nrf2 expression may affect antitumor immune responses, we prepared Nrf2-deficient mice and determined altered T cell-mediated anticancer responses in vivo with modulation of Nrf2 expression.

[0187] 2-1. Determining the ability to inhibit cancer cell proliferation To determine the ability to inhibit cancer cell growth in conjunction with changes in the regulation of Nrf2 expression, cancer cells were injected into Nrf2-deficient and wild-type mice, and tumor volume was then measured. Specifically, melanoma (B16) cells were injected into Nrf2-deficient and wild-type mice, and tumor volume was then measured at various time points after tumor inoculation to observe cancer cell growth. The results are shown in Figures 3 and 4.

[0188] As shown in Figures 3 and 4, after injection of melanoma cells, a significant effect in inhibiting cancer cell growth was observed in Nrf2-deficient mice. For example, by approximately 19 days after tumor inoculation, a significant difference in tumor volume was observed between wild-type and Nrf2- / - animals.

[0189] To confirm that the observed antitumor effects were not specific to melanoma, we injected lymphoma cells into Nrf2-deficient and wild-type mice and then monitored cancer cell growth again by measuring tumor volume at various time points after tumor inoculation. The results are shown in Figures 5 and 6.

[0190] As shown in Figures 5 and 6, after injection of lymphoma cells, a significant effect of inhibiting cancer cell growth was observed in Nrf2-deficient mice, and tumor volume was significantly reduced.

[0191] 2-2. Determination of the ability to inhibit cancer cell metastasis Next, to determine the ability to inhibit cancer cell metastasis in conjunction with changes in the regulation of Nrf2 expression, lung cancer cells were injected (intravenously) into Nrf2-deficient and wild-type mice, and then cancer cell metastasis was observed. The results are shown in Figure 7.

[0192] As shown in Figure 7, after injection of lung cancer cells, significant tumor growth was observed in the lungs of wild-type animals, indicating that the administered tumor cells had spread to the lung tissue. In contrast, a significant effect of inhibiting cancer cell metastasis was observed in Nrf2-deficient mice.

[0193] The above results suggest the potential benefit of reducing Nrf2 expression in the treatment of cancer.

[0194] Example 3. Determination of altered T cell-mediated anti-cancer responses with modulation of Nrf2 expression 3-1. Determining T cell-mediated anti-cancer responses Nrf2-deficient mice exhibited a strong anti-cancer response against various cancer cells. This was confirmed in Example 2 above. To determine whether the above phenomenon was a T cell-mediated anti-cancer response, Nrf2-deficient and wild-type mice were treated to render them T cell-deficient. Specifically, anti-CD3 antibodies were administered to deplete T cells. Several tumor-bearing wild-type and Nrf2-deficient animals were given anti-CD3 antibodies (control animals were given an IgG control antibody). Cancer cell growth was then monitored by measuring both tumor volume and tumor weight at various time points after tumor inoculation. The results are shown in Figures 8A and 8B.

[0195] As shown in Figures 8A and 8B, when Nrf2-deficient mice were in a T cell-deficient state, the effect of inhibiting cancer cell growth in mice was lost. The above results confirmed the close relationship between anti-cancer responses and T cells in Nrf2 deficiency.

[0196] To further support the T cell-mediated nature of the enhanced antitumor immune response observed in Nrf2-deficient animals, we depleted B cells using anti-B220 antibody in both tumor-bearing wild-type and Nrf2-deficient animals. As shown in Figures 9A and 9B, B cell depletion did not affect the antitumor immune response. In Nrf2-deficient animals, both tumor volume and tumor weight were comparable in both animals treated with anti-B220 antibody and control IgG antibody.

[0197] The above results confirm that the antitumor immune responses observed in Nrf2-deficient animals were primarily mediated by T cells.

[0198] 3-2. Determining the level of cancer-related CD8 T cell activation in conjunction with changes in Nrf2 expression To understand how Nrf2 deletion affects T cell function, we observed altered oncotoxic CD8 T cell activation levels in conjunction with changes in Nrf2 expression, thereby confirming the relationship between oncotoxic CD8 T cell activation levels and anti-cancer responses. Specifically, CD4+ and CD8+ T cells were isolated from tumor-bearing wild-type and Nrf2-deficient animals. Cells were isolated from either tumors (i.e., TILs) or draining lymph nodes. T cells were then stimulated with TCR and the amounts of IFN-γ and IL-17 produced were measured. The results are shown in Figure 10.

[0199] As shown in Figure 10, a significant increase in the amount of IFNγ secreted by cancer cytotoxic CD8 T cells was observed in Nrf2-deficient mice. No significant difference was observed in CD4+ T cells. Increased IFN-γ production was observed in both CD8+ T cells isolated from draining lymph nodes and tumors. However, this difference was even more dramatic in CD8+ TILs. The above results support the ability of Nrf2 to adversely and profoundly interfere with T cell responsiveness and activity.

[0200] 3-3. Determining the anti-cancer efficacy of cancer-toxic CD8 T cells in an in vivo model Based on the results of Examples 3-1 and 3-2 above, we determined the anticancer efficacy of oncotoxic CD8 T cells in an in vivo mouse model. Specifically, OVA antigen-specific CD8 T cells (OT-I cells) treated with Nrf2 expression deletion were injected into mice bearing tumor cells (to express the OVA antigen), and the effect of tumor cell inhibition was then observed. As a control, T cells from wild-type and Nrf2+ / - animals were isolated and administered to wild-type tumor-bearing animals. The results are shown in Figure 11.

[0201] As shown in Figure 11, a significant tumor cell inhibitory effect was observed in mice injected with Nrf2-deficient specific CD8 T cells (OT-I cells). A moderate effect was observed when T cells from Nrf2+ / - animals were used for adoptive transfer. The above results confirmed that Nrf2 expression can exert a counter-interference effect in inhibiting tumor cell growth, reducing the responsiveness of CD8 T cells.

[0202] Example 4: Further analysis of the relationship between Nrf2 expression and anti-tumor immune responses To further confirm the results observed in the above examples, MC38 (colon) tumor cells were administered to the following animals: (i) wild-type, (ii) Nrf2-deficient, and (iii) transgenic mice overexpressing Nrf2 ("Nrf2Tg"). The anti-tumor immune response in each group was then observed by measuring tumor volume at various time points after tumor inoculation, as described in the preceding examples.

[0203] As shown in Figures 13A and 13B, loss of Nrf2 expression also improved the antitumor immune response against colon tumor cells. This result further supports the idea that reducing Nrf2 expression may have therapeutic utility against many different types of cancer. Furthermore, the above data suggest that the ability of T cells to treat tumors is inversely proportional to Nrf2 expression. For example, tumor volumes were larger in Nrf2 transgenic animals compared to wild-type animals.

[0204] Example 5: Analysis of oxidative stress resistance in Nrf2-deficient T cells To identify potential mechanisms by which T cells from Nrf2-deficient animals might mount more effective antitumor immune responses, we measured the ability of T cells from wild-type, Nrf2-transgenic, and Nrf2-deficient animals to produce IFN-γ and granzyme B upon TCR stimulation, both in the presence and absence of hydrogen peroxide.

[0205] As shown in Figures 14A-14D, only T cells from Nrf2-deficient animals were able to produce IFN-γ and granzyme B in the presence of hydrogen peroxide. This result suggests that T cells from Nrf2-deficient animals remain functional in many types of cancer and are highly resistant to induced oxidative stress.

[0206] Example 6: Construction of CAR T cells with reduced Nrf2 expression The above results support the potential benefit of using Nrf2-deficient T cells to treat cancer. In pursuit of the development of such immunotherapy, CD19-specific CAR T cells with reduced Nrf2 expression were constructed. Figure 15A is a schematic diagram of the construct used to generate the CAR T cells. Figure 15B shows that CD19-specific CAR T cells were generated using the construct shown in Figure 15A, and Nrf2 mRNA expression was reduced by approximately 50%. As shown in Figure 15C, even with only a 50% reduction, the CAR T cells exhibited improved resistance to oxidative stress. Compared with control CAR T cells that expressed normal levels of Nrf2, CAR T cells with reduced Nrf2 expression produced a significantly higher percentage of IFN-γ in the presence of hydrogen peroxide.

[0207] Next, the CRISPR / Cas9 system was used to identify other ways to reduce Nrf2 expression in T cells (e.g., CAR T cells). Figures 16A, 16B, and 16C present results using three different guide RNAs targeting Nrf2. Table 3 (below) presents the number of mutations, insertions, and deletions observed using the different guide RNAs. As shown, Nrf2-deficient human T cells were successfully engineered using the CRISPR / Cas9 system (specifically, the guide RNA identified as "Sg3"), resulting in 83% efficacy. [Table 3]

[0208] Taken together, the above results indicate that CAR-expressing cells, such as CAR T cells, with reduced Nrf2 expression may be a viable treatment option for many different cancers.

Claims

1. A composition comprising modified immune cells that express reduced levels of the NFE2L2 gene and / or Nrf2 protein, said composition being for treating a tumor in a subject in need thereof, wherein the modified immune cells comprise a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR); The composition, wherein the modified immune cells have previously been contacted with a gene editing tool capable of reducing the expression level of the NFE2L2 gene and / or Nrf2 protein in the modified immune cells, and the modified immune cells are selected from CD8+ T cells, tumor-infiltrating lymphocytes (TILs), or a combination thereof.

2. the modified immune cells are (i) is capable of reducing tumor volume when administered to said subject; (ii) is capable of reducing tumor weight when administered to said subject; (iii) capable of improving one or more properties of tumor-infiltrating lymphocytes (TILs) when administered to said subject; or (iv) The composition of claim 1, which is any combination of (i) to (iii).

3. 3. The composition of claim 1 or 2, wherein the modified immune cells exhibit increased resistance to oxidative stress compared to corresponding non-modified immune cells, and the corresponding non-modified immune cells do not express reduced levels of the NFE2L2 gene and / or Nrf2 protein.

4. The composition of any one of claims 1 to 3, wherein the modified immune cells are CD8+ T cells.

5. 5. The composition of any one of claims 1 to 4, wherein the tumor is derived from cancer comprising breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer (gastric cancer), gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

6. the composition is administered to the subject in combination with an additional therapeutic agent. The composition according to any one of claims 1 to 5.

7. 7. The composition of claim 6, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

8. The composition of any one of claims 1 to 7, wherein the reduced levels of the NFE2L2 gene and / or Nrf2 protein are capable of improving the anti-tumor immune response of the modified immune cells.

9. 9. The composition of any one of claims 1 to 8, wherein the gene editing tool comprises an shRNA, an siRNA, an miRNA, an antisense oligonucleotide, a CRISPR, a zinc finger nuclease, a TALEN, a meganuclease, a restriction endonuclease, or a combination thereof.

10. 1. A method for preparing immune cells for chimeric antigen receptor (CAR) and / or T cell receptor (TCR) engineering, said method comprising: (a) contacting the immune cells with a gene editing tool to reduce the expression level of the NFE2L2 gene and / or Nrf2 protein, wherein the contacting is performed ex vivo or in vitro; (b) modifying the immune cells to express a CAR and / or an engineered TCR, wherein the immune cells are selected from CD8+ T cells, tumor infiltrating lymphocytes (TILs), or a combination thereof. A method comprising:

11. 11. The method of claim 10, wherein the gene editing tool comprises an shRNA, an siRNA, an miRNA, an antisense oligonucleotide, a CRISPR, a zinc finger nuclease, a TALEN, a meganuclease, a restriction endonuclease, or a combination thereof.

12. (i) the gene editing tool is encoded by a nucleic acid sequence contained in a vector; (ii) the CAR and / or TCR are encoded by a nucleic acid sequence contained in a vector; or (iii) The method of claim 10 or 11, wherein both (i) and (ii) are true.

13. 13. The method of Claim 12, wherein the nucleic acid sequence encoding the gene editing tool and the nucleic acid encoding the CAR and / or TCR are comprised in separate vectors or in the same vector.

14. The method of any one of claims 10 to 13, wherein the immune cells are CD8+ T cells.

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