NR4A3-deficient immune cells and uses thereof

Abstract

The present disclosure provides a method for promoting the sustained effector function of immune cells, comprising modifying the cells to overexpress c-Jun and to reduce the level of NR4A gene and / or protein. Also provided are modified cells, such as immune cells, modified to overexpress c-Jun and to reduce the expression level of NR4A gene and / or protein. Overexpressing c-Jun and simultaneously reducing the expression level of NR4A gene and / or protein results in exhaustion / dysfunction-resistant cells that are both apoptosis-resistant and immune checkpoint-resistant, and also maintain anti-tumor function in the tumor microenvironment.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 195,956, filed June 2, 2021, and U.S. Provisional Application No. 63 / 365,023, filed May 19, 2022, each of which is incorporated herein by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4385_087PC02_Seqlisting_ST25.txt, Size: 97,218 bytes, Creation Date: June 1, 2022) submitted with this application are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to cell-based (e.g., T cell) cancer immunotherapy comprising the administration of immune cells modified to reduce expression levels of the NR4A gene and / or protein and to overexpress c-JUN. [Background technology]

[0004] Cancer immunotherapy relies on T cells, the immune system's primary killers of infected and abnormal cells, to attack and kill tumor cells. However, immunotherapy faces a significant obstacle: the killing ability of T cells can decline, a phenomenon often referred to as exhaustion. Immune checkpoint blockade, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor-engineered (TCR) T cell therapy use functionally active T cells isolated from patients and require highly functional T cells to be effective. These T cells are engineered to recognize specific antigens on target cancer cells and expanded ex vivo.

[0005] When the immune system is forced to remain active for a long time, for example, due to persistent viral infection or the gradual development of cancer, effector T cells can become exhausted. One characteristic of exhausted T cells is increased expression of immune checkpoint proteins such as PD-1 and CTLA-4, which can cause them to withdraw (i.e., become nonfunctional). Immune checkpoint inhibitors block these checkpoint proteins and thereby enhance the immune response against tumors. Several studies suggest that blocking the activity of checkpoint proteins does not achieve this goal in exhausted T cells. This is important because so-called hot tumors, i.e., tumors that contain high levels of immune cells and therefore should be ideal candidates for responding to immunotherapy, often contain a population composed largely of exhausted T cells. Furthermore, the tumor microenvironment can induce senescence and exhausted cell phenotypes. Therefore, devising strategies to reverse and / or prevent these exhausted states is crucial for improving the efficacy of immunotherapy. Summary of the Invention

[0006] In some aspects, the present disclosure provides cell compositions comprising a population of modified immune cells that express (i) a reduced expression level of a nuclear receptor subfamily 4 group A gene and / or protein selected from the group consisting of the NR4A member 1 (NR4A1) gene and / or protein, the NR4A member 2 (NR4A2) gene and / or protein, and the NR4A member 3 (NR4A3) gene and / or protein, and (ii) an increased expression level of a c-Jun protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A1 gene and / or NR4A1 protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A2 gene and / or NR4A2 protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A3 gene and / or NR4A3 protein. In some embodiments, the NR4A genes and / or NR4A proteins comprise both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.

[0007] In some embodiments, the expression level of the NR4A gene and / or NR4A protein in the population of modified immune 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 composition (e.g., a corresponding cell composition in which the cells have not been modified to reduce the expression level of the NR4A gene and / or NR4A protein).

[0008] In some embodiments, the modified immune cells include lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, and any combination thereof. In some embodiments, the lymphocytes include T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) cells, and any combination thereof. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cells comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), e.g., an engineered TCR. In some embodiments, the modified immune cells comprise a CAR and / or TCR that specifically binds to a tumor antigen. In some embodiments, the CAR and / or the TCR is selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5 , HMWMAA, o-acetyl-GD2, folate receptor beta, 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, MAGEAl, ETV6-AML, sperm protein 17, XAGE1, Tie2, 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 (e.g., KRAS, HRAS, NRAS mutant proteins), hTERT, sarcoma translocation breakpoints, 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 Specifically binds to hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, or any combination thereof.

[0009] In some embodiments, the CAR and / or the TCR specifically binds to ROR1. In some embodiments, the CAR comprises an antigen-binding domain derived from R12, R11, 2A2, or any combination thereof. In some embodiments, the CAR comprises a heavy chain variable domain comprising SEQ ID NO: 17 and a light chain variable domain comprising SEQ ID NO: 21.

[0010] In some embodiments, the population of engineered immune cells has less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% effector T cells. In some embodiments, the population of engineered immune cells has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% naive T (T N ) cells, central memory T cells (T CM cells), stem memory T (T SCM ) cells, or any combination thereof.

[0011] In some embodiments, the modified immune cells are modified with a gene editing tool to reduce the expression of the NR4A gene and / or NR4A protein. In some embodiments, the gene editing tool can reduce the level of (i) the NR4A1 gene and / or protein, (ii) the NR4A2 gene and / or protein, (iii) the NR4A3 gene and / or protein, or (iv) any combination thereof. In some 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 CRISPR. In some embodiments, the gene editing tool comprises a guide RNA comprising, consisting of, or consisting essentially of a sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96.

[0012] In some embodiments, the c-Jun protein comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the c-Jun protein comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0013] In some embodiments, the modified immune cells described herein are modified with a nucleotide sequence encoding a c-Jun protein such that the modified immune cells overexpress the c-Jun protein. In some embodiments, the nucleotide sequence encoding the c-Jun protein is (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8. (c) a nucleic acid sequence having 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 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10; (d) a nucleic acid sequence having at least about 10%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, (e) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12; (f) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13;(g) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14; (h) a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 95% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15; (i) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16.

[0014] In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12.In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the modified immune cells are modified with a transcriptional activator capable of increasing endogenous expression of c-Jun.

[0015] In some embodiments, the population of modified immune cells exhibits one or more improved properties in a subject compared to reference immune cells (e.g., corresponding cells that have not been modified to increase c-Jun expression and / or decrease expression of NR4A gene(s) and / or NR4A protein(s)). In some embodiments, the improved one or more properties of the modified immune cells include (i) improved proliferation, (ii) improved cytotoxicity, (iii) improved cytokine expression, (iv) improved persistence, or (v) any combination thereof.

[0016] In some embodiments, the modified immune cells exhibit improved cytokine expression. In some embodiments, the cytokine is interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or any combination thereof. In some embodiments, the expression level of the IL-2 is increased by at least about 2-fold to at least about 10-fold compared to the expression level of IL-2 in a reference population of immune cells. In some embodiments, the expression level of the IFN-γ is increased by at least about 2-fold to at least about 10-fold compared to the expression level of IFN-γ in a reference population of immune cells. In some embodiments, the expression level of the TNF-α is increased by at least about 2-fold to at least about 10-fold compared to the expression level of TNF-α in a reference population of immune cells.

[0017] In some aspects, the modified immune cells exhibit reduced exhaustion or dysfunction compared to reference immune cells (e.g., corresponding cells that have not been modified to increase c-Jun expression and / or decrease expression of NR4A gene(s) and / or NR4A protein(s)). In some aspects, the modified immune cells exhibit reduced or no apoptosis (apoptosis resistant). In some aspects, the modified immune cells express reduced immune checkpoint markers (immune checkpoint resistant). In some aspects, the modified immune cells maintain anti-tumor function in the tumor microenvironment (TME). In some aspects, the modified immune cells exhibit (i) improved activity in a hypoxic environment, (ii) improved activity in a low nutrient (i.e., glucose) environment, (iii) improved activity in the presence of inhibitory metabolites / cytokines (e.g., adenosine, TGF-β, ROS, etc.), (iv) improved activity upon exposure to inhibitory cells (e.g., MDSCs, Tregs, etc.), or (v) any combination thereof.

[0018] In some aspects, the present disclosure provides pharmaceutical compositions comprising a population of modified immune cells described herein and a pharmaceutically acceptable carrier.

[0019] In some embodiments, the present disclosure provides methods of treating a tumor in a subject in need thereof, the methods comprising administering to the subject a cell composition or pharmaceutical composition described herein. In some embodiments, 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 not receiving the administration). In some embodiments, the tumor volume after the administration reduces 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 tumor volume (e.g., the tumor volume in the subject before the administration and / or the tumor volume in a subject not receiving the administration). In some embodiments, the administration reduces tumor weight in the subject compared to a reference tumor weight (e.g., the tumor weight in the subject before the administration and / or the tumor weight in a subject not receiving the administration). 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 the administration compared to a reference tumor weight (e.g., the tumor weight in the subject before the administration and / or the tumor weight in a subject that did not receive the administration).

[0020] In some embodiments, the administration improves one or more properties of immune cells in the subject. In some embodiments, the improved properties of the immune cells include (i) improved proliferation, (ii) improved cytotoxicity, (iii) improved cytokine expression, (iv) improved persistence, or (v) any combination thereof. In some embodiments, the administration improves cytokine expression. In some embodiments, the cytokine includes IL-2, IFN-γ, TNF-α, or any combination thereof. In some embodiments, the administration reduces or prevents exhaustion or dysfunction of the immune cells. In some embodiments, the immune cells exhibit reduced or no apoptosis (apoptosis resistant). In some embodiments, the immune cells exhibit reduced or no immune checkpoint markers (immune checkpoint resistant). In some embodiments, the immune cells maintain anti-tumor function in the tumor microenvironment (TME). In some embodiments, the immune cells exhibit (i) increased activity in a hypoxic environment, (ii) increased activity in a low nutrient (i.e., glucose) environment, (iii) increased activity in the presence of inhibitory metabolites / cytokine resistance (adenosine, TGF-β, ROS, etc.), (iv) increased activity upon exposure to inhibitory cells (MDSCs, Tregs, etc.), or any combination thereof.

[0021] In some embodiments, the tumor is derived from cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

[0022] In some embodiments, the method includes administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent includes a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune-based therapy, a cytokine, surgery, radiation therapy, an activator of costimulatory molecules, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor includes 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, and any combination thereof.

[0023] In some embodiments, the additional therapeutic agent and the cell composition are administered simultaneously. In some embodiments, the additional therapeutic agent and the cell composition are administered sequentially. In some embodiments, the cell composition is administered parenterally, intramuscularly, subcutaneously, ophthalmically, intravenously, intraperitoneally, intradermally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intrathecally, intracisternally, intracapsularly, intratumorally, or any combination thereof.

[0024] In some aspects, the disclosure provides methods for preparing an immune cell (or cell composition) described herein, the method comprising modifying the cell with a gene editing tool, wherein the gene editing tool reduces expression of any one of the NR4A gene and / or NR4A protein, and modifying the immune cell to overexpress c-Jun. In some aspects, modifying the immune cell to overexpress c-Jun comprises contacting the immune cell with a nucleotide sequence encoding a c-Jun protein. In some aspects, modifying the immune cell to overexpress c-Jun comprises contacting the immune cell with a transcriptional activator capable of increasing expression of endogenous c-Jun protein. In some aspects, the transcriptional activator is coupled to a Cas protein modified to lack endonuclease activity.

[0025] Also provided herein is a method for producing a cell that overexpresses c-Jun protein and has reduced levels of the NR4A gene and / or NR4A protein, the method comprising modifying the cell with (i) a nucleotide sequence encoding c-Jun protein and (ii) a gene editing tool, wherein the gene editing tool comprises a guide RNA (gRNA) and is capable of reducing expression of the NR4A gene and / or NR4A protein, the gRNA comprising, consisting essentially of, or consisting of a sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96.

[0026] In some aspects, the present disclosure provides methods of reducing or inhibiting exhaustion of cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), the methods comprising modifying the cells to reduce expression levels of the NR4A gene and / or protein, and modifying the cells to overexpress a c-Jun protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A1 gene and / or NR4A1 protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A2 gene and / or NR4A2 protein. In some aspects, the NR4A gene and / or NR4A protein comprises the NR4A3 gene and / or NR4A3 protein. In some aspects, reduced expression of the NR4A gene and / or protein reduces or inhibits exhaustion of the cells.

[0027] In some embodiments, the cells are immune cells. In some embodiments, the expression level of the NR4A gene and / or protein in the 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 composition (e.g., a corresponding cell composition in which the cells have not been modified to reduce the expression level of the NR4A gene and / or NR4A protein).

[0028] In some embodiments, modifying the cell comprises contacting the cell with a gene editing tool capable of reducing the expression level of the NR4A gene and / or protein in the cell. In some embodiments, modifying the cell to overexpress c-Jun protein comprises contacting the immune cell with a nucleotide sequence encoding a c-Jun protein. In some embodiments, modifying the immune cell to overexpress c-Jun comprises contacting the immune cell with a transcriptional activator capable of increasing expression of endogenous c-Jun protein. In some embodiments, the transcriptional activator is linked to a Cas protein modified to lack endonuclease activity.

[0029] In some aspects, the present disclosure further provides a method of increasing cytokine production by a cell expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR) in response to antigenic stimulation, the method comprising: (i) modifying the cell with a nucleotide sequence encoding a c-Jun protein, such that the cell overexpresses the c-Jun protein after modification; and (ii) modifying the cell with a gene editing tool, wherein the gene editing tool comprises a guide RNA (gRNA) that reduces expression of the NR4A gene and / or NR4A protein. and the gRNA can be lowered, wherein the gRNA comprises, consists essentially of, or consists of the sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96.

[0030] In some embodiments, the cytokine comprises IFN-γ, IL-2, TNF-α, or a combination thereof. In some embodiments, after the modification, production of the cytokine in response to the antigenic stimulus is increased by at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold compared to a reference cell (e.g., a corresponding cell that has not been modified with the c-Jun nucleotide sequence and / or gene editing tool).

[0031] The present disclosure also provides a method of increasing effector function of a cell expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR) in response to sustained antigen stimulation, the method comprising: (i) modifying the cell with a nucleotide sequence encoding a c-Jun protein, such that the cell overexpresses the c-Jun protein after modification; and (ii) modifying the cell with a gene editing tool, wherein the gene editing tool comprises a guide RNA (gRNA) capable of reducing expression of the NR4A gene and / or NR4A protein, wherein the gRNA comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96.

[0032] In some embodiments, after the modification, the cells retain effector function upon at least one, at least two, or at least three additional antigen stimulation assays compared to a reference cell (e.g., a corresponding cell that was not modified with the c-Jun nucleotide sequence and / or gene editing tool). In some embodiments, the effector function includes (i) the ability to kill target cells (e.g., tumor cells), (ii) the ability to produce cytokines upon additional antigen stimulation, or (iii) both (i) and (ii).

[0033] In some aspects, the present disclosure provides a cell composition prepared by a method described herein. In some aspects, provided herein are cell compositions comprising cells that (a) express a ligand binding protein (e.g., a CAR or TCR); (b) have increased levels of c-Jun protein; and (b) have decreased levels of expression of (i) the NR4A1 gene and / or NR4A1 protein, (ii) the NR4A2 gene and / or NR4A2 protein, (iii) the NR4A3 gene and / or NR4A3 protein, or (iv) any combination of (i)-(iii), wherein the cells have been modified with a gRNA comprising, consisting of, or consisting essentially of a sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96. In some embodiments, the cell composition is an in vivo cell. In some embodiments, the cell is an ex vivo cell or an in vitro cell. In some embodiments, a pharmaceutical composition comprises the cell.

[0034] In some aspects, the present disclosure provides kits comprising (i) a gene editing tool for reducing expression of the NR4A gene and / or protein, (ii) a vector comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), (iii) a nucleotide sequence encoding a c-Jun protein, and instructions for treating a tumor according to the methods described herein. In some aspects, the disclosure provides kits comprising (i) a gene editing tool for reducing expression of the NR4A gene and / or protein, (ii) a vector comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), (iii) a nucleotide sequence encoding a c-Jun protein, and instructions for preparing a cell composition according to the methods described herein.

[0035] In some aspects, the present disclosure provides for the use of a cellular composition or pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a tumor in a subject, including administration to the subject in need thereof.

[0036] In some aspects, the present disclosure provides a cellular composition of the pharmaceutical composition described herein for the treatment of a tumor in a subject in need thereof, comprising administering to the subject a composition of the cell of the pharmaceutical composition described herein for the treatment of a tumor in a subject in need thereof. [Brief explanation of the drawings]

[0037] [Figure 1] Shown are the percentages of NR4A3 expression in NR4A3-edited ("NR4A3 KO") and control non-edited CD4+ (left) and CD8+ (right) ROR1 CAR T cells overexpressing ("+c-Jun") or not overexpressing ("-c-Jun") c-Jun on day 7 of CAR T cell generation after 2 hours of CD3 / CD28 Dynabead stimulation in four independent donors (stimulation, filled circles). Unstimulated cells (open circles, no Dynabeads) served as a negative control. An unpaired t-test of stimulation conditions was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 2] Shown are the percentages of expression of EGFR+R12+ ROR1 CAR in NR4A1-edited ("NR4A1 KO"), NR4A2-edited ("NR4A2 KO"), NR4A3-edited ("NR4A3 KO"), and control unedited CD4+ (open circles) and CD8+ (filled circles) ROR1 CAR T cells with or without overexpressing c-Jun (left) and the geometric mean fluorescence of ROR1 CAR in EGFR+R12+ T cells (right) from four donors on day 7 of CAR T cell generation. Statistical analysis using an unpaired t-test of stimulation conditions was not significant. [Figure 3]Shows sequential anti-ROR1 lysis of H1975-NLR NSCLC cells in a serial stimulation assay in four independent donors by NR4A-edited, control non-edited ROR1 CAR T cells with or without c-Jun overexpression, and mock-untransduced T cells. The different groups shown include: (a) NR4A1 knockout without c-Jun overexpression (triangle), (b) NR4A2 knockout without c-Jun overexpression (star), (c) NR4A3 knockout without c-Jun overexpression (filled circle), (d) control non-edited ROR1 CAR T cells without c-Jun overexpression (x symbol), (e) NR4A1 knockout with c-Jun overexpression (diamond), (f) NR4A2 knockout with c-Jun overexpression (asterisk), (g) NR4A3 knockout with c-Jun overexpression (open circle), (h) control non-edited ROR1 CAR T cells with c-Jun overexpression (vertical line), and (i) non-transduced mock T cells (square). Lysis of H1975-NLR target cells was quantified by measuring total NLR intensity. NLR intensity was normalized to the starting intensity after re-plating for each stimulation round. NLR-NucLight Red. Each graph shows data from four independent donors. [Figure 4A] Figure 1 shows interferon-gamma (IFN-γ) secretion resulting from NR4A-edited, control unedited ROR1 CAR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an H1975 sequential stimulation assay corresponding to Figure 3. The different groups shown are the same as in Figure 3. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The graph shows data from four independent donors. Error bars represent the mean + / - SD of triplicate wells. [Figure 4B]Figure 3 shows interleukin-2 (IL-2) secretion resulting from NR4A-edited, control non-edited ROR1 CAR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an H1975 sequential stimulation assay corresponding to Figure 3. The different groups shown are the same as in Figure 3. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The graph shows data from four independent donors. Error bars represent the mean + / - SD of triplicate wells. [Figure 4C] Figure 1 shows tumor necrosis factor-alpha (TNF-α) secretion resulting from NR4A-edited, control non-edited ROR1 CAR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an H1975 sequential stimulation assay corresponding to Figure 3. The different groups shown are the same as in Figure 3. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The graph shows data from four independent donors. Error bars represent the mean + / - SD of triplicate wells. [Figure 5A] Shown are the percentages of ROR1 CAR expression in EGFR+R12+CD4+ (top) and CD8+ (bottom) T cells from NR4A-edited and control non-edited ROR1 CAR T cells with or without overexpressing c-Jun after each re-plating during an H1975 sequential stimulation assay corresponding to Figure 3 . The different groups shown include: (a) NR4A1 knockout with no c-Jun overexpression (triangle), (b) NR4A2 knockout with no c-Jun overexpression (star), (c) NR4A3 knockout with no c-Jun overexpression (filled circle), (d) control non-edited ROR1 CAR T cells with no c-Jun overexpression (x symbol), (e) NR4A1 knockout with c-Jun overexpression (diamond), (f) NR4A2 knockout with c-Jun overexpression (asterisk), (g) NR4A3 knockout with c-Jun overexpression (open circle), and (h) control non-edited ROR1 CAR T cells with c-Jun overexpression (vertical line). [Figure 5B]Figure 1 shows the fold change in predicted CD3+ROR1 CAR+T cell numbers from NR4A-edited and control non-edited ROR1 CAR T cells with or without c-Jun overexpression during the H1975 sequential stimulation assay corresponding to Figure 3. The predicted cell numbers were calculated assuming 25% of the cells transferred to the next stimulation. The fold change was calculated as (predicted cell numbers from stimulation / predicted cell numbers from the previous stimulation). The graph shows data from four independent donors. Groups shown are the same as in Figure 3. [Figure 6] Expression of inhibitory receptors (TIM3, CD39, and PD1) on ROR1 CAR+CD4+ (top) and CD8+ (bottom) T cells from NR4A3-edited ("NR4A3 KO") and control non-edited ROR1 CAR T cells overexpressing ("+cJun") or not overexpressing ("-cJun") c-Jun from an H1975 serial stimulation assay corresponding to the second stimulation in Figure 3 is shown. Paired t-test was used for statistical analysis. *p<0.05, **p<0.005. n=4 independent donors. [Figure 7] Panels A and B show the sequential lysis of A549-NLR and H1975-NLR cells, respectively, by anti-ROR1 CAR T cells overexpressing c-Jun and engineered to reduce levels of NR4A1, NR4A2, and NR4A3 (triple KO, NR4A TKO). Non-edited anti-ROR1 CAR T cells overexpressing c-Jun ("Control ROR1 CAR" containing endogenous levels of NR4A1, NR4A2, and NR4A3) and untreated target cells ("Target Alone") are shown as controls. Target cell lysis was quantified by measuring total NLR intensity. NLR intensity was normalized to the starting intensity after replating for each stimulation round. NLR-NucLight Red. [Figure 8]A and B show the levels of IFN-γ produced by anti-ROR1 CAR T cells that overexpress c-Jun and were engineered to reduce the levels of NR4A1, NR4A2, and NR4A3 (triple knockout (NR4A TKO, black bars)) in a sequential stimulation assay (see Figures 7A and 7B) using A549 and H1975 target cells, respectively. Supernatants were collected 24 hours after each replating (i.e., Stim 1, Stim 2, Stim 3, Stim 4, and Stim 5), and cytokines were quantified by MSD. The levels of IFN-γ produced by anti-ROR1 CAR T cells that overexpress c-Jun and were unedited ("control ROR1" CAR T cells containing endogenous levels of NR4A1, NR4A2, and NR4A3) were measured. CAR) is shown as a control (gray bar). In each of A and B, results from three independent donors are shown. An unpaired t-test was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 9] Panels A and B show the levels of IL-2 produced by anti-ROR1 CAR T cells that overexpressed c-Jun and were engineered to reduce the levels of NR4A1, NR4A2, and NR4A3 (triple knockout (NR4A TKO), black bars) in a sequential stimulation assay using A549 and H1975 target cells, respectively (see, e.g., Figures 7A and 7B). Supernatants were collected 24 hours after each replating (i.e., Stim 1, Stim 2, Stim 3, Stim 4, and Stim 5), and cytokines were quantified by MSD. Anti-ROR1 CAR T cells that overexpressed c-Jun and had no editing ("control ROR1 CAR" containing endogenous levels of NR4A1, NR4A2, and NR4A3) are shown as a control (gray bars). In each of panels A and B, results from three independent donors are shown. An unpaired t-test was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 10]A and B show the levels of TNF-α produced by anti-ROR1 CAR T cells (triple knockout (NR4A TKO, black bars)) that overexpress c-Jun and were engineered to reduce levels of NR4A1, NR4A2, and NR4A3 in a sequential stimulation assay (see, e.g., Figures 7A and 7B) using A549 and H1975 target cells, respectively. Supernatants were collected 24 hours after each replating (i.e., Stim 1, Stim 2, Stim 3, Stim 4, and Stim 5), and cytokines were quantified by MSD. Anti-ROR1 CAR T cells overexpressing c-Jun and without editing ("control ROR1" containing endogenous levels of NR4A1, NR4A2, and NR4A3) were used in a sequential stimulation assay (see, e.g., Figures 7A and 7B). CAR) is shown as a control (gray bar). In each of A and B, results from three independent donors are shown. An unpaired t-test was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 11] Figure 7 shows the persistence of anti-ROR1 CAR T cells engineered to overexpress c-Jun and reduce levels of NR4A1, NR4A2, and NR4A3 (triple knockout (NR4A TKO), triangles) in a sequential stimulation assay (see, e.g., Figures 7A and 7B) using H1975 target cells. The NR4A-edited anti-ROR1 CAR T cells and control groups are the same as those described in Figures 7A and 7B. Persistence was measured by quantifying the number of cParp(-)CD3+EGFR+ROR1 CAR T cells by flow cytometry after each sequential stimulation (i.e., stimulation-1, stimulation-2, stimulation-3, stimulation-4). [Figure 12]Percentages of NR4A3 expression in NR4A3-edited (KO) and control non-edited CD4+ (left graph) and CD8+ (right graph) NY-ESO-1 TCR T cells overexpressing or not overexpressing c-Jun are shown on day 7 of TCR T cell generation after 2 hours of PMA + ionomycin stimulation from three independent donors (stimulation, filled circles). Unstimulated cells (open circles, no PMA + ionomycin stimulation) served as a negative control. An unpaired t-test of stimulation conditions was used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001). [Figure 13] Percentages of TCRv13.1+NY-ESO-1 TCR expression on NR4A1-edited, NR4A2-edited, NR4A3-edited (KO), and control unedited CD4+ (open circles) and CD8+ (filled circles) NY-ESO-1 TCR T cells with or without c-Jun overexpression (left graph) and geometric mean fluorescence of NY-ESO-1 TCR on TCRv13.1+ T cells (right graph) are shown from three donors on day 7 of TCR T cell generation. An unpaired t-test was used for statistical analysis. ****p<0.0001. [Figure 14]Figure 1 shows sequential lysis of NY-ESO-1+A375-NLR melanoma cells in a serial stimulation assay by NR4A-edited (KO), control unedited NY-ESO-1 TCR T cells with or without c-Jun overexpression, and mock-untransduced T cells from three independent donors. Specifically, different NY-ESO-1 TCR T cells are shown, including: (a) NR4A1 knockout with no c-Jun overexpression (triangle), (b) NR4A2 knockout with no c-Jun overexpression (star), (c) NR4A3 knockout with no c-Jun overexpression (filled circle), (d) control non-edited ROR1 CAR T cells with no c-Jun overexpression (x symbol), (e) NR4A1 knockout with c-Jun overexpression (diamond), (f) NR4A2 knockout with c-Jun overexpression (asterisk), (g) NR4A3 knockout with c-Jun overexpression (open circle), (h) control non-edited ROR1 CAR T cells with c-Jun overexpression (vertical line), and (i) untransduced mock T cells (square). Lysis of A375-NLR target cells was quantified by measuring total NLR counts. NLR counts were normalized to starting counts after re-plating at each stimulation round. NLR-NucLight Red. Each graph represents data from an independent donor. [Figure 15A] Figure 14 shows interferon-gamma (IFN-γ) secretion from NR4A-edited, control unedited NY-ESO-1 TCR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an A375 continuous stimulation assay corresponding to Figure 14. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The various groups are the same as those described in Figure 14. The graph shows data from three independent donors. [Figure 15B]Figure 14 shows interleukin-2 (IL-2) secretion from NR4A-edited, control unedited NY-ESO-1 TCR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an A375 continuous stimulation assay corresponding to Figure 14. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The various groups are the same as those described in Figure 14. The graph shows data from three independent donors. [Figure 15C] Figure 14 shows tumor necrosis factor-alpha (TNF-α) secretion resulting from NR4A-edited, control unedited NY-ESO-1 TCR T cells with or without c-Jun overexpression, and mock-untransduced T cells in an A375 continuous stimulation assay corresponding to Figure 14. Supernatants were collected 24 hours after each replating, and cytokines were quantified by MSD. The various groups are the same as those described in Figure 14. The graph shows data from three independent donors. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present disclosure is directed to compositions comprising a population of modified immune cells having (i) reduced expression levels of the Nuclear Receptor Subfamily 4 Group A (NR4A) member 1 (NR4A1), member 2 (NR4A2), or member 3 (NR4A3) gene and / or NR4A1, NR4A2, or NR4A3 protein, and (ii) increased expression levels of the transcription factor c-Jun. As further described herein, in some embodiments, immune cells useful in the present disclosure are modified to reduce the expression level of a single member of the NR4A family (a "single knockout"). For example, in some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of the NR4A1 gene and / or NR4A1 protein. In some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) decreased levels of the NR4A3 gene and / or NR4A3 protein. In some embodiments, the immune cells useful in the present disclosure are modified to reduce the expression levels of two members of the NR4A family ("double knockout"). For example, in some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of both the NR4A1 gene and / or NR4A1 protein and the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of both the NR4A1 gene and / or NR4A1 protein and the NR4A3 gene and / or NR4A3 protein. In some aspects, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein.In some embodiments, immune cells useful in the present disclosure are modified to reduce the expression levels of all members of the NR4A family ("triple knockout"). Thus, in some embodiments, the modified immune cells described herein have (i) increased levels of c-Jun and (ii) reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein. The reduction in the level of the NR4A gene can be achieved using gene editing techniques, such as CRISPR. As is apparent from the present disclosure, unless otherwise specified, the term "NR4A gene and / or protein" includes any of the NR4A single knockout, double knockout, and triple knockout described herein.

[0039] Both a decrease in the level of NR4A gene and / or NR4A protein expression and an increase in the expression level of the transcription factor c-Jun can lead to one or more sustained effector functions in immune cells, such as, for example, the immune cells described herein. One aspect of sustained effector function is improved T cell activation (e.g., increased proliferation, increased cytotoxicity, increased cytokine expression). A decrease in the level of the NR4A1, NR4A2, or NR4A3 gene and / or protein (or a combination thereof) and an increase in the expression level of the transcription factor c-Jun can result in exhausted / dysfunction-resistant cells. Furthermore, a decrease in the level of the NR4A3 gene and / or NR4A3 protein and an increase in the expression level of the transcription factor c-Jun can result in the maintenance of anti-tumor function in the TME environment.

[0040] The present disclosure also provides a method for treating, for example, a tumor in a subject in need of such treatment, the method comprising administering to the subject a cell composition described herein (e.g., a composition comprising immune cells that have been modified to (i) reduce the level of one or more members of the NR4A family and (ii) increase the level of the c-Jun protein).

[0041] The present disclosure also provides, for example, methods of generating modified immune cells, methods of using the modified immune cells, pharmaceutical compositions comprising the modified immune cells, or kits comprising the modified immune cells, having (i) reduced expression levels of one of the Nuclear Receptor Subfamily 4 Group A member (NR4A1, NR4A2, or NR4A3) genes and / or proteins and the endogenous expression levels of two other NR4A members (e.g., the NR4A1 and NR4A2 genes and proteins, the NR4A1 and NR4A3 genes and proteins, or the NR4A2 and NR4A3 genes and proteins), and (ii) increased expression levels of the transcription factor c-Jun. As further described herein, in some aspects, the present disclosure also provides methods of generating modified immune cells having increased levels of c-Jun protein and reduced levels of two or all three members of the NR4A family.

[0042] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to the particular compositions or process steps described, as such may, of course, vary. As will be apparent to one of ordinary skill in the art after reading this disclosure, each of the individual aspects described and illustrated herein has separate components and features that may be readily separated from or combined with the features of any of the other aspects without departing from the scope or spirit of the present disclosure. Any recited method may be carried out in the order of events recited, or in any other order that is logically possible.

[0043] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects may be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, since the scope of the disclosure will be limited only by the appended claims.

[0044] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0045] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "an immune cell" is understood to refer to one or more immune cells. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0046] Furthermore, as used herein, "and / or" should be considered as a specific disclosure of each of the two specified properties or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0047] Whenever an embodiment is described herein in conjunction with the phrase "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0048] 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.

[0049] Units, prefixes, and symbols are shown in their Systeme International de Unites (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limiting of the various aspects of the disclosure, which aspects may 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.

[0050] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0051] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within or more than 1 standard deviation, per practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean a value that is up to an order of magnitude or up to 5 times greater. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0052] As used herein, the term "approximately" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In some embodiments, unless otherwise specified or clear from the context, the term "approximately" refers to a range of values ​​within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value (above or below the reference value) in either direction (except when such number exceeds 100% of the possible values).

[0053] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless otherwise indicated.

[0054] 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 for the therapeutic agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, transpulmonary, 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 a topical, epidermal, or mucosal route of administration, e.g., intranasally, orally, intravaginally, rectally, sublingually, or topically, and can be administered, for example, once, multiple times, and / or over one or more extended periods of time.

[0055] 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., induces effector functions such as cytokine production and / or induces intracellular signals for cell proliferation).

[0056] Nucleotides are represented by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction. Nucleotides are represented herein by the commonly known single-letter symbols of nucleotides recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0057] It should be understood that T and U in the disclosed sequences are interchangeable depending on whether the sequence is DNA or RNA. For example, the spacer sequence of a gRNA is presented in this disclosure as DNA (A / T / C / G), while the chimeric frame of the gRNA is presented as RNA (A / U / C / G).

[0058] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0059] 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, such as, 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.

[0060] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0061] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes polynucleotides modified, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), whether spliced ​​or unspliced, polyribonucleotides (containing D-ribose), including mRNA and gRNA, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers in which the polymer contains nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA.

[0062] As used herein, the term "vector" is intended to refer to 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 operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, also included are other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0063] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors, which can spread to adjacent tissues and metastasize to distant sites in the body via the lymphatic system or bloodstream. As used herein, "cancer" refers to primary cancer, metastatic cancer, and recurrent cancer.

[0064] 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. Immune responses are mediated by the action of cells of the immune system (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 that selectively target, bind to, damage, destroy, and / or eliminate from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, T cells, e.g., effector T cells or Th cells, e.g., CD4 + or CD8 + T cell activation or inhibition, or T reg 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, a T cell is a CD4 + In some embodiments, the T cells are CD8 + In some embodiments, the T cells are NKT cells.

[0065] As used herein, the term "anti-tumor immune response" refers to an immune response against tumor antigens. An increased immune response or ability to stimulate the immune system can be due to improved agonistic activity of T cell costimulatory receptors and / or improved antagonistic activity of inhibitory receptors. An increased immune response or ability to stimulate the immune system can be due to increased EC activity in assays that measure immune responses, such as cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly by detecting CD107a or granzymes), and changes in proliferation. 50or may be reflected in a fold increase in maximum activity level. In some embodiments, the ability to stimulate an immune response or activity of the immune system may be improved by, for example, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the ability to stimulate an immune response or activity of the immune system may be improved, for example, by at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more.

[0066] 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.

[0067] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an agent (e.g., an engineered immune cell disclosed herein) that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, alleviation, reduction, 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 cause tumor shrinkage 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 slow tumor growth. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of the composition can, for example, (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) relieve to some extent one or more symptoms associated with cancer.

[0068] 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 an advanced solid tumor, or a slowing of cancer progression (regression). 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.

[0069] As used herein, the term "standard of care" refers to a treatment that is accepted by medical professionals as an appropriate treatment for a particular type of disease and is widely used by medical professionals. The term may be used interchangeably with any of the following terms: "best practice," "standard of care," and "standard of care."

[0070] By way of example, an "anti-cancer drug" promotes regression of a cancer in a subject or prevents further tumor growth. In some embodiments, a therapeutically effective amount of the drug promotes regression of the cancer to the point of eliminating the cancer.

[0071] By "promoting cancer regression" is meant that administration of an effective amount of the drug, alone or in combination with an anti-tumor drug, reduces the growth or size of the tumor, causes necrosis of the tumor, reduces the severity of at least one disease symptom, increases the frequency and duration of symptom-free periods of the disease, or prevents functional impairment or disability resulting from the affliction of the disease.

[0072] The terms "effective" and "efficacy" in reference to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (side effects) at the cellular, organ, and / or organismal level resulting from the administration of the drug.

[0073] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents, or modulates one or more immune checkpoint proteins. Checkpoint proteins regulate T cell activation or function. Many checkpoint proteins are known, such as 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 regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0074] As used herein, the term "oxidative stress" refers to a condition characterized by excess 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-radical 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.

[0075] As used herein, the term "modified cell" refers to a cell, e.g., a T cell, that has been engineered in a non-natural way to cause the phenotype of the cell (i.e., expression level of the NR4A gene and / or NR4A protein and expression level of c-Jun protein) to differ from that of an unmodified cell (i.e., a reference cell). As is evident from the present disclosure, the modified cells disclosed herein overexpress c-Jun protein and have reduced expression levels of the NR4A gene and / or NR4A protein compared to a reference cell (e.g., a corresponding unmodified cell). For example, in some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of the NR4A1 gene and / or NR4A1 protein. In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of the NR4A2 gene and / or NR4A2 protein. In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of the NR4A3 gene and / or NR4A3 protein. In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of (i) the NR4A1 gene and / or protein and (ii) the NR4A2 gene and / or protein. In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of (i) the NR4A1 gene and / or protein and (ii) the NR4A3 gene and / or protein. In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of (i) the NR4A2 gene and / or protein and (ii) the NR4A3 gene and / or protein.In some embodiments, the modified cells described herein overexpress c-Jun protein and have reduced expression levels of (i) the NR4A1 gene and / or protein, (ii) the NR4A2 gene and / or protein, and (iii) the NR4A3 gene and / or protein. As used herein, the term "corresponding cells" refers to cells that belong to the same immune cell class as the modified cells. For example, if the modified cells are T cells, the corresponding cells are also T cells.

[0076] As used herein, the terms "endogenous expression" or "endogenous expression level" or "endogenous level" (or grammatical variants thereof) refer to the expression (e.g., amount, kinetics, etc.) of a gene and / or protein that is naturally occurring (e.g., the gene and / or protein has not been directly manipulated by a non-naturally occurring manipulation). For example, in some embodiments, a modified cell disclosed herein (e.g., a CAR or TCR T cell in which NR4A3 has been knocked down and that overexpresses a c-Jun protein) does not express endogenous levels of the NR4A3 gene and / or protein, but because both the NR4A1 and NR4A2 genes have not been knocked down (e.g., by CRISPR, e.g., a non-naturally occurring manipulation), the modified cell endogenously expresses the NR4A1 and NR4A2 genes and / or NR4A1 and NR4A2 proteins.

[0077] In some embodiments, modified cells are produced by introducing foreign or exogenous nucleic acid into a cell, which in some embodiments may encode a gene editing tool disclosed herein. Nucleic acids can be introduced into cells by methods known in the art, such as electroporation (see, e.g., Heiser W. C., Transscription Factor Protocols: Methods in Molecular Biology™ 2000;130:117-134), chemical (e.g., calcium phosphate or lipid) transfection (see, e.g., Lewis W. H., 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 direct microinjection of purified DNA into cell nuclei (see, e.g., Capecchi M. R., Cell. 1980 November;22(2 Pt 2):479-88).

[0078] It should be understood that disclosures regarding "modified cells" or "cells" are equally applicable to a population of such cells, i.e., a plurality of such cells.

[0079] As used herein, the terms "high concentration" or "high level" and their grammatical variants refer to higher than normal levels of a substance (e.g., reactive oxygen species, ROS) compared to an appropriate control (e.g., healthy tissue or cells).

[0080] As used herein, the terms "reactive oxygen species" and "ROS" refer to highly reactive chemicals, including 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).

[0081] As used herein, the terms "chimeric antigen receptor" and "CAR" refer 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 with the term "chimeric antigen receptor" herein. Chimeric antigen receptors are distinguished from other antigen-binding agents by their ability to both bind MHC-independent antigens and transmit activation signals via their intracellular domains.

[0082] The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds to an antigen, usually a surface-expressed antigen of a malignant tumor. The antigen-specific extracellular domain can, for example, determine the affinity constant or affinity of the interaction (K) that it binds to the antigen. D) specifically binds to an antigen when it binds at 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. Methods for determining the affinity of an 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, and a wide variety of such polypeptides 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, such as cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR 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 and V beta), are also suitable for use.

[0083] The chimeric antigen receptors disclosed herein can also include an intracellular domain that provides an intracellular signal to the cell (expressing the CAR) upon antigen binding to the antigen-specific extracellular domain. In some embodiments, the intracellular signaling domain of the CAR is responsible for activating at least one effector function of the T cell expressing the chimeric receptor.

[0084] The term "intracellular domain" refers to the portion of a CAR that transmits effector function signals and instructs T cells to perform specialized functions when an antigen binds 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. Typically, the entire intracellular domain is used, although 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 can be used in place of the intact chain, as 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 an 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 crosses the cell membrane, anchoring the CAR to the T cell surface and connecting the extracellular domain to the intracellular signaling domain, thereby 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.

[0085] A "peptide hinge" or "spacer" connects the antigen-specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, which is optionally fused to a second costimulatory domain, which is fused to a signaling domain, including but not limited to CD3ζ. For example, including a spacer domain between the antigen-specific extracellular domain and the transmembrane domain, and between multiple scFvs in the case of a tandem CAR, 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.

[0086] As used herein, the terms "ug" and "uM" are used synonymously with "μg" and "μM", respectively.

[0087] 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 correction of genetic information. In some embodiments, such gene editing can affect the expression of the edited DNA. In some embodiments, such gene editing does not affect the expression of the edited DNA. In some embodiments, gene editing of the modified cells disclosed herein can be performed using 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.

[0088] As used herein, the term "nuclease" refers to an enzyme with catalytic activity for DNA cleavage. Any nuclease agent that induces a nick or double-stranded break at a desired recognition site can be used in the methods and compositions disclosed herein. Naturally occurring or native nuclease agents can be used, so long as the nuclease agent induces a nick or double-stranded break at the desired recognition site. Alternatively, modified or engineered nuclease agents can be used. An "engineered nuclease agent" includes a nuclease that has been engineered (modified or derived) from its native form to specifically recognize and induce a nick or double-stranded break at a desired recognition site. Thus, an engineered nuclease agent can be derived from a natural, naturally occurring nuclease agent or can be artificially created or synthesized. The modification of a nuclease agent can be as little as one amino acid in a protein cleaving agent or one nucleotide in a nucleic acid cleaving agent. In some embodiments, the engineered nuclease induces a nick or double-stranded break at a recognition site that is not a sequence recognized by the native (unengineered or unmodified) nuclease agent. Creating a nick or double-stranded break in a recognition site or other DNA can be referred to herein as "cutting" or "cleaving" the recognition site or other DNA.

[0089] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein, e.g., a Cas9 protein, a CAR, or a TCR, or a polynucleotide, e.g., a gRNA. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon-optimized.

[0090] As used herein, "complementary" or "complementary" refers to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to the property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at every position are complementary.

[0091] The various aspects described herein are described in further detail in the following subsections.

[0092] II. Modified immune cells The success of cellular immunotherapy for solid tumors is limited due to the exhaustion of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. Continuous exposure to tumor antigens leads to T cell exhaustion, characterized by a progressive loss of cytotoxicity and cytokine production and increased expression of inhibitory markers, such as PD-1 (Wherry et al., Nat. Rev. Immunol. 15, 486-499 (2015)). Furthermore, exhausted TILs upregulate and substitute for transcription factors, specifically the NR4A family (Chen et al., Nature 567, 530-534 (2019)).

[0093] The present disclosure provides modified immune cells, i.e., cells that have been modified, e.g., by gene editing, to have reduced expression levels of the NR4A gene and / or NR4A protein and overexpress c-Jun protein, resulting in improved function, e.g., sustained effector function and / or reduced exhaustion (e.g., T cells that overexpress c-Jun protein and have reduced expression levels of the NR4A gene and / or protein, e.g., CAR or TCR T cells that overexpress c-Jun protein and have reduced expression levels of the NR4A gene and / or protein).

[0094] In some aspects, the present disclosure provides a population of modified immune cells that express (i) a reduced expression level of a nuclear receptor subfamily 4 group A gene and / or protein selected from the group consisting of an NR4A member 1 (NR4A1) gene and / or protein, an NR4A member 2 (NR4A2) gene and / or protein, and an NR4A member 3 (NR4A3) gene and / or protein, and (ii) an increased expression level of a c-Jun protein. In some aspects, the NR4A gene and / or protein comprises an NR4A1 gene and / or protein. In some aspects, the NR4A gene and / or protein comprises an NR4A2 gene and / or protein. In some aspects, the NR4A gene and / or protein comprises an NR4A3 gene and / or protein. In some aspects, the NR4A gene and / or protein comprises any combination of an NR4A1 gene and / or protein, an NR4A2 gene and / or protein, and an NR4A3 gene and / or protein.

[0095] II.A.NR4A3 Nuclear receptor subfamily 4 group A member 3, commonly abbreviated as "NR4A3" (also known as MINOR, CSMF, NOR1, CHN, mitogen-induced nuclear orphan receptor, neuron-derived orphan receptor, nuclear hormone receptor NOR-1, "chondrosarcoma, extraskeletal myxoid type, EWS fusion type," and TEC), is a protein encoded by the NR4A3 gene in humans. The NR4A family of orphan nuclear receptors includes NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1). They act as transcription factors in a ligand-independent manner. Their function is primarily regulated by the rapid and transient induction of their expression by various extracellular signals, and therefore they are considered immediate-early genes. NR4As are involved in various cellular functions, including apoptosis, survival, proliferation, angiogenesis, inflammation, DNA repair, and fatty acid metabolism.

[0096] The NR4A3 gene is located on chromosome 9 (bases 99,821,885 to 99,866,893, 45,039 bases, plus-strand orientation, NCBI Reference Sequence: NC_000009.12). NR4A3 is a transcriptional activator that binds to regulatory elements in promoter regions in a cell- and response element (target)-specific manner. NR4A3 induces gene expression (by affinity) by binding to the NR4A1 response element (NBRE) 5'-AAAAGGTCA-3' (SEQ ID NO: 100) site as a monomer and to the Nur response element (NurRE) as a homodimer within the promoters of their regulated target genes, and plays a role in regulating proliferation, survival, and differentiation of many different cell types.

[0097] The NR4A3 protein has three isoforms produced by alternative splicing, the sequences of which are shown in Table 1 below. [Table 1]

[0098] In some aspects, cell compositions useful in the present disclosure comprise a population of modified immune cells that (i) overexpress c-Jun, e.g., recombinantly produced c-Jun protein, (ii) have reduced levels of the NR4A3 gene and / or NR4A3 protein, and (iii) express a ligand-binding protein (e.g., a CAR or TCR) (e.g., that specifically binds ROR1), and further have endogenous expression of the NR4A1 and NR4A2 genes and proteins. In some aspects, such modified immune cells (e.g., that overexpress c-Jun and have reduced levels of the NR4A3 gene and / or NR4A3 protein) also have reduced levels of one of the following: (i) the NR4A1 gene and / or NR4A1 protein, (ii) the NR4A2 gene and / or NR4A2 protein, or both (i) and (ii). Therefore, unless otherwise specified, modified immune cells having reduced levels of the NR4A3 gene and / or NR4A3 protein may have endogenous expression of other members of the NR4A family, or may have reduced expression of such other members. As used herein, the term "NR4A3 gene" refers to any transcript, genomic DNA, pre-mRNA, or mRNA. As used herein, the term "NR4A3 protein" refers to the isoform alpha, isoform beta, or isoform 3 disclosed above, as well as variants and mutants thereof. As used herein, the term NR4A3 protein also encompasses any fragment or variant of any of the isoforms disclosed herein that has at least one function of the wild-type NR4A3 protein.

[0099] As used herein, the terms "reduced levels," "lower levels," "reduced expression levels," or "lower levels" (or variants thereof) refer to both physical levels (e.g., a reduction in gene sequence due to editing from the genome, or a reduction in protein due to reduced protein expression) and functional levels. For example, a reduction in the level of the NR4A3 gene can refer to a reduction in gene function due to, e.g., the introduction of a mutation that introduces a stop codon or frameshift, an epigenetic modification that alters transcription, or a mutation or other change in the promoter gene or another gene that regulates the expression of NR4A3. In some embodiments, a reduction in the level of the NR4A3 gene in a modified cell refers to a reduction in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA capable of encoding functional NR4A3 protein, e.g., wild-type NR4A3 protein, compared to a reference cell. Similarly, a decrease in NR4A3 protein can refer to changes that result in the expression of a functional NR4A3 protein, e.g., a wild-type NR4A3 protein, including, but not limited to, changes (e.g., mutations or post-translational modifications) that cause loss of function (partial or complete), or the activity of molecules that bind to functional sites of NR4A3, e.g., that alter interactions with other cell signaling partners.

[0100] The level of the NR4A3 gene (e.g., the presence / absence of the entire gene or a portion thereof, or gene function) can be measured by various methods known in the art. The level of the NR4A3 protein (e.g., the presence / absence, or quantification or protein function of the NR4A3 protein or a fragment thereof) can be measured by various methods known in the art.

[0101] In some embodiments, the expression level of the NR4A3 gene and / or the expression level of the NR4A3 protein in a population of immune cells (e.g., CAR- or TCR-expressing cells) is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding population of cells that have not been modified to reduce the expression level of the NR4A3 gene and / or NR4A3 protein. In some embodiments, expression of the NR4A3 gene and / or NR4A3 protein in proteins of the population of immune cells (e.g., a population of T cells, CAR-expressing cells, or TCR-expressing cells) is completely suppressed following the modification.

[0102] In some embodiments, the expression level of the NR4A3 gene in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding cell population that has not been modified to reduce the expression level of the NR4A3 gene.

[0103] In some embodiments, the expression level of NR4A3 protein in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding cell population that has not been modified to reduce the expression level of NR4A3 protein.

[0104] In some aspects, the expression levels of the NR4A3 gene and NR4A3 protein in a population of immune cells are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., corresponding cells that have not been modified to reduce the expression levels of the NR4A3 gene and NR4A3 protein.

[0105] In some embodiments, the modified immune cells disclosed herein (i.e., cells having reduced expression levels of the NR4A3 gene and / or NR4A3 protein) comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the modified immune cells disclosed herein (i.e., a population of cells having reduced expression levels of the NR4A3 gene and / or NR4A3 protein) comprise lymphocytes. In some embodiments, the lymphocytes are T cells, e.g., CD8+ T cells and / or CD4+ T cells. As used herein, "modified immune cells" includes progeny of the originally modified immune cells, which also have reduced expression levels of the NR4A3 gene and / or NR4A3 protein.

[0106] II.B.NR4A2 Nuclear receptor subfamily 4 group A member 2 (also known as NOT, RNR1, HZF-3, NURR1, and TINUR), commonly abbreviated as NR4A2, is a protein encoded by the NR4A2 gene in humans, which is located on chromosome 2 (bases 156,324,432 to 156,332,724; NCBI reference sequence: NC_000002.12).

[0107] The NR4A2 protein has two isoforms produced by alternative splicing, the sequences of which are shown in Table 2 below. [Table 2]

[0108] In some aspects, cell compositions useful in the present disclosure comprise a population of modified immune cells that (i) overexpress c-Jun, e.g., recombinantly produced c-Jun protein, (ii) have reduced levels of the NR4A2 gene and / or NR4A2 protein, and (iii) express a ligand-binding protein (e.g., a CAR or TCR) (e.g., that specifically binds ROR1), and further have endogenous expression of the NR4A1 and NR4A3 genes and proteins. In some aspects, such modified immune cells (e.g., that overexpress c-Jun and have reduced levels of the NR4A2 gene and / or NR4A2 protein) also have reduced levels of one of the following: (i) the NR4A1 gene and / or NR4A1 protein, (ii) the NR4A3 gene and / or NR4A3 protein, or both (i) and (ii). Thus, unless otherwise specified, modified immune cells having reduced levels of the NR4A2 gene and / or NR4A2 protein may have endogenous expression of other members of the NR4A family, or may have reduced expression of such other members. As used herein, the term "NR4A2 gene" refers to any transcript, genomic DNA, pre-mRNA, or mRNA. As used herein, the term "NR4A2 protein" refers to isoforms 1 or 2 disclosed above, as well as variants and mutants thereof. As used herein, the term NR4A2 protein also encompasses any fragment or variant of any of the isoforms disclosed herein that retains at least one function of the wild-type NR4A2 protein.

[0109] As used herein, the terms "reduced levels," "lower levels," "reduced expression levels," or "lower levels" (or variants thereof) refer to both physical levels (e.g., a reduction in gene sequence due to editing from the genome, or a reduction in protein due to reduced protein expression) and functional levels. For example, a reduction in the level of the NR4A2 gene can refer to a reduction in gene function due to, e.g., the introduction of a mutation that introduces a stop codon or frameshift, an epigenetic modification that alters transcription, or a mutation or other change in the promoter gene or another gene that regulates the expression of NR4A2. In some embodiments, a reduction in the level of the NR4A2 gene in a modified cell refers to a reduction in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA capable of encoding a functional NR4A2 protein, e.g., a wild-type NR4A2 protein, compared to a reference cell. Similarly, a decrease in NR4A2 protein can refer to changes that result in the expression of a functional NR4A2 protein, e.g., a wild-type NR4A2 protein, including, but not limited to, changes (e.g., mutations or post-translational modifications) that cause loss of function (partial or complete), or the activity of molecules that bind to functional sites of NR4A2, e.g., that alter interactions with other cell signaling partners.

[0110] The level of the NR4A2 gene (e.g., the presence / absence of the entire gene or a portion thereof, or gene function) can be measured by various methods known in the art. The level of the NR4A2 protein (e.g., the presence / absence, or quantification or protein function of the NR4A2 protein or a fragment thereof) can be measured by various methods known in the art.

[0111] In some embodiments, the expression level of the NR4A2 gene and / or the expression level of the NR4A2 protein in a population of immune cells (e.g., CAR- or TCR-expressing cells) is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding population of cells that have not been modified to reduce the expression level of the NR4A2 gene and / or NR4A2 protein. In some embodiments, expression of the NR4A2 gene and / or NR4A2 protein in the population of immune cells (e.g., a population of CAR- or TCR-expressing cells) is completely suppressed following the modification.

[0112] In some embodiments, the expression level of the NR4A2 gene in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding cell population that has not been modified to reduce the expression level of the NR4A2 gene.

[0113] In some embodiments, the expression level of NR4A2 protein in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding cell population that has not been modified to reduce the expression level of NR4A2 protein.

[0114] In some aspects, the expression levels of the NR4A2 gene and NR4A2 protein in a population of immune cells are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., corresponding cells that have not been modified to reduce the expression levels of the NR4A2 gene and NR4A2 protein.

[0115] In some embodiments, the modified immune cells disclosed herein (i.e., cells having reduced expression levels of the NR4A2 gene and / or NR4A2 protein) comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the modified immune cells disclosed herein (i.e., a population of cells having reduced expression levels of the NR4A2 gene and / or NR4A2 protein) comprise lymphocytes. In some embodiments, the lymphocytes are T cells, e.g., CD8+ T cells and / or CD4+ T cells. As used herein, "modified immune cells" includes progeny of the originally modified immune cells, which also have reduced expression levels of the NR4A2 gene and / or NR4A2 protein.

[0116] II.C.NR4A1 Nuclear receptor subfamily 4 group A member 1 (also known as HMR, N10, TR3, NP10, GFRP1, NAK-1, NGFIB, and NUR77), commonly abbreviated as NR4A1, is a protein encoded in humans by the NR4A1 gene, which is located on chromosome 12 (bases 52022832 to 52059507, NCBI reference sequence NC_000012.12).

[0117] The NR4A1 protein has three isoforms produced by alternative splicing, the sequences of which are shown in Table 3 below. [Table 3]

[0118] In some aspects, cell compositions useful in the present disclosure comprise a population of modified immune cells that (i) overexpress c-Jun, e.g., recombinantly produced c-Jun protein, (ii) have reduced levels of the NR4A1 gene and / or NR4A1 protein, and (iii) express a ligand-binding protein (e.g., a CAR or TCR) (e.g., that specifically binds ROR1), and further have endogenous expression of the NR4A2 and NR4A3 genes and proteins. In some aspects, such modified immune cells (e.g., that overexpress c-Jun and have reduced levels of the NR4A1 gene and / or NR4A1 protein) also have reduced levels of one of the following: (i) the NR4A2 gene and / or NR4A2 protein, (ii) the NR4A3 gene and / or NR4A3 protein, or both (i) and (ii). Thus, unless otherwise specified, modified immune cells having reduced levels of the NR4A1 gene and / or NR4A1 protein may have endogenous expression of other members of the NR4A family, or may have reduced expression of such other members. As used herein, the term "NR4A1 gene" refers to any transcript, genomic DNA, pre-mRNA, or mRNA. As used herein, the term "NR4A1 protein" refers to isoform 1, isoform 2, or isoform 3 disclosed above, as well as variants and mutants thereof. As used herein, the term NR4A1 protein also encompasses any fragment or variant of any of the isoforms disclosed herein that retains at least one function of the wild-type NR4A1 protein.

[0119] As used herein, the terms "reduced levels," "lower levels," "reduced expression levels," or "lower levels" (or variants thereof) refer to both physical levels (e.g., a reduction in gene sequence due to editing from the genome, or a reduction in protein due to reduced protein expression) and functional levels. For example, a reduction in the level of the NR4A1 gene can refer to a reduction in gene function due to, e.g., the introduction of a mutation that introduces a stop codon or frameshift, an epigenetic modification that alters transcription, or a mutation or other change in the promoter gene or another gene that regulates the expression of NR4A1. In some embodiments, a reduction in the level of the NR4A1 gene in a modified cell refers to a reduction in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA capable of encoding a functional NR4A1 protein, e.g., a wild-type NR4A1 protein, compared to a reference cell. Similarly, a decrease in NR4A1 protein can refer to changes that result in the expression of a functional NR4A1 protein, e.g., a wild-type NR4A1 protein, including, but not limited to, changes (e.g., mutations or post-translational modifications) that cause loss of function (partial or complete), or the activity of molecules that bind to functional sites of NR4A1, e.g., that alter interactions with other cell signaling partners.

[0120] The level of the NR4A1 gene (e.g., the presence / absence of the entire gene or a portion thereof, or gene function) can be measured by various methods known in the art. The level of the NR4A1 protein (e.g., the presence / absence, or quantification or protein function of the NR4A1 protein or a fragment thereof) can be measured by various methods known in the art.

[0121] In some embodiments, the expression level of the NR4A1 gene and / or the expression level of the NR4A1 protein in a population of immune cells (e.g., CAR- or TCR-expressing cells) is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding population of cells that has not been modified to reduce the expression level of the NR4A1 gene and / or NR4A1 protein. In some embodiments, expression of the NR4A1 gene and / or NR4A1 protein in proteins of the population of immune cells (e.g., a population of CAR- or TCR-expressing cells) is completely suppressed following the modification.

[0122] In some embodiments, the expression level of the NR4A1 gene in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding cell population that has not been modified to reduce the expression level of the NR4A1 gene.

[0123] In some embodiments, the expression level of NR4A1 protein in a population of immune cells is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to a reference population of immune cells, e.g., a corresponding population of cells that have not been modified to reduce the expression level of NR4A1 protein.

[0124] In some aspects, the expression levels of the NR4A1 gene and NR4A1 protein in a population of immune cells are reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, compared to a population of reference immune cells, e.g., corresponding cells that have not been modified to reduce the expression levels of the NR4A1 gene and NR4A1 protein.

[0125] In some embodiments, the modified immune cells disclosed herein (i.e., cells having reduced expression levels of the NR4A1 gene and / or NR4A1 protein) comprise lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or a combination thereof. In some embodiments, the modified immune cells disclosed herein (i.e., a population of cells having reduced expression levels of the NR4A1 gene and / or NR4A1 protein) comprise lymphocytes. In some embodiments, the lymphocytes are T cells, e.g., CD8+ T cells and / or CD4+ T cells. As used herein, "modified immune cells" includes progeny of the originally modified immune cells, which also have reduced expression levels of the NR4A1 gene and / or NR4A1 protein.

[0126] II. Dc-Jun protein In addition to the reduced levels of the NR4A gene and / or protein described above, the modified immune cells (e.g., CAR or TCR expressing immune cells) of the present disclosure are also modified to increase the level of c-Jun protein. As described herein, in some embodiments, the immune cells are modified to include an exogenous nucleotide sequence encoding a c-Jun protein, such that the level of c-Jun protein is increased compared to a reference immune cell (e.g., a corresponding immune cell that has not been modified to include an exogenous nucleotide sequence encoding a c-Jun protein). In some embodiments, the c-Jun protein is encoded by a polycistronic polynucleotide, which encodes multiple proteins, including c-Jun and a ligand binding protein (e.g., a CAR or TCR), and, in some embodiments, one or more additional proteins (e.g., a safety switch protein such as EGFRt). In some embodiments, the modified immune cells having reduced levels of the NR4A gene and / or NR4A protein comprise a polynucleotide encoding a chimeric polypeptide comprising a c-Jun polypeptide and a ligand binding protein (e.g., a CAR or TCR). In some embodiments, such chimeric polypeptides can include a cleavable linker such that the c-Jun polypeptide and the ligand binding protein (e.g., CAR or TCR) are post-translationally cleaved into separate functional proteins. In some embodiments, the modified immune cells provided herein (i.e., having reduced levels of one or more members of the NR4A family genes and / or proteins) are capable of naturally expressing c-Jun protein (e.g., without modifying the cells with an exogenous nucleotide sequence encoding a c-Jun protein). In some embodiments, such immune cells are modified with a transcriptional activator (e.g., a CRISPR / Cas system-based transcriptional activator, e.g., CRISPRa), such that expression of endogenous c-Jun protein is increased compared to a reference cell (e.g., a corresponding cell not modified with the transcriptional activator).

[0127] As used herein, the term "transcriptional activator" refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to an enhancer or promoter-proximal element of a nucleic acid sequence, thereby inducing its transcription). Non-limiting examples of such transcriptional activators that can be used in the present disclosure include transcription activator-like effector (TALE)-based transcriptional activators, zinc finger protein (ZFP)-based transcriptional activators, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system-based transcriptional activators, or combinations thereof. See, for example, Kabadi et al., Methods 69(2):188-197 (Sep. 2014), which is incorporated herein by reference in its entirety.

[0128] In some embodiments, the cells described herein are modified with a CRISPR / Cas system-based transcription activator, e.g., CRISPR activator (CRISPRa). See, e.g., Nissim et al., Molecular Cell 54:1-13 (May 2014), incorporated herein by reference in its entirety. CRISPRa is a type of CRISPR tool that involves the use of a modified Cas protein that lacks endonuclease activity but retains its ability to bind to guide RNA and target DNA nucleic acid sequences. Non-limiting examples of such modified Cas proteins that can be used in the present disclosure are known in the art. See, e.g., Pandelakis et al., Cell Systems 10(1):1-14 (Jan. 2020), incorporated herein by reference in its entirety. In some embodiments, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as "dCas9"). In some embodiments, the modified Cas protein comprises a modified Cas12a protein. In some embodiments, modified Cas proteins useful in the present disclosure bind to a guide polynucleotide (e.g., a small guide RNA) (a "modified Cas guide complex"), where the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In some embodiments, the guide polynucleotide comprises a recognition sequence that is complementary to a promoter region of an endogenous nucleic acid sequence encoding the protein of interest. In some embodiments, one or more transcriptional activators are attached to the modified Cas guide complex (e.g., to the N-terminus and / or C-terminus of the modified Cas protein), such that when the modified Cas guide complex is introduced into a cell, the one or more transcriptional activators can bind to a regulatory element of the nucleic acid sequence (e.g., a promoter region), thereby inducing and / or increasing expression of the encoded protein (e.g., c-Jun).In some embodiments, the one or more transcriptional activators can bind to regulatory elements (e.g., promoter regions) of endogenous genes, thereby inducing and / or increasing expression of the encoded protein (e.g., c-Jun). Non-limiting examples of common activators that can be used include the omega subunit of RNAP, VP16, VP64, and p65. See, e.g., Kabadi and Gersbach, Methods 69:188-197 (2014), incorporated herein by reference in its entirety.

[0129] In some embodiments, one or more transcriptional repressors (e.g., Kruppel-associated box domains (KRAB)) can be attached to the modified Cas guide complex (e.g., at the N- and / or C-terminus of the modified Cas protein), and when introduced into a cell, the one or more transcriptional repressors can suppress or reduce transcription of genes, such as genes that can interfere with the expression of c-Jun (e.g., Bach2). See, e.g., US20200030379A1 and Yang et al., J Transl Med 19:459 (2021), each of which is incorporated herein by reference in its entirety. In some embodiments, modified Cas proteins useful in the present disclosure can bind both one or more transcriptional activators and one or more transcriptional repressors.

[0130] As will be apparent to one of skill in the art, in some embodiments, the cells described herein have been modified using a combination of approaches. For example, in some embodiments, the cells have been modified to reduce the level of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) and to contain (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., a ligand-binding protein, e.g., a CAR or TCR) and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases the expression of an endogenous protein (e.g., c-Jun). In some embodiments, the cells have been modified to reduce the level of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) and to contain (i) an exogenous nucleotide sequence encoding a first protein (e.g., a ligand-binding protein) and (ii) an exogenous nucleotide sequence encoding a second protein (e.g., a c-Jun protein). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first and second proteins may be part of a single polycistronic vector.

[0131] In some embodiments, due to such modifications (e.g., introduction of exogenously introduced c-Jun nucleotide sequences and / or transcriptional activators), the modified cells overexpress c-Jun protein, i.e., express higher levels (e.g., 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% more, or at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold) than corresponding cells lacking such modifications ("reference cells"). The phrases "increase the expression level [or amount] of," "overexpress," or "increase expression of" (and similar forms of phrases used herein) are used interchangeably.

[0132] c-Jun is an oncogenic transcription factor belonging to the activator protein-1 (AP-1) family. It interacts with various proteins (e.g., c-Fos) to form dimeric complexes that regulate various cell signaling pathways, including cell proliferation and tumor progression. Therefore, increased c-Jun expression has been observed in certain cancers, and there has been great interest in developing c-Jun antagonists to treat such cancers. See, for example, Brennan, A., et al., J Exp Clin Cancer Res 39(1):184 (Sep. 2020).

[0133] In humans, the c-Jun protein is encoded by the JUN gene, located on chromosome 1 (nucleotides 58,780,791 to 58,784,047, minus-strand orientation, of GenBank accession number NC_000001.11). Synonyms for the JUN gene and its encoded protein include "Jun proto-oncogene, AP-1 transcription factor subunit," "v-Jun avian sarcoma virus 17 oncogene homolog," "transcription factor AP-1," "Jun oncogene," "AP-1," "Jun activation domain-binding protein," "p39," and "enhancer-binding protein AP1." The wild-type human c-Jun protein sequence is 331 amino acids long. The amino acid and nucleic acid sequences of wild-type human c-Jun are shown in Tables 4 and 5, respectively. [Table 4] [Table 5]

[0134] II.D.1. Codon Optimization As described herein, modified immune cells having reduced levels of the NR4A gene and / or NR4A protein comprise a polynucleotide comprising a nucleotide sequence encoding a c-Jun protein, wherein the nucleotide sequence is codon-optimized. Thus, in some embodiments, the nucleotide sequence encoding the c-Jun protein described herein (also referred to herein as a "c-Jun nucleotide sequence") differs from that of a wild-type c-Jun nucleotide sequence (e.g., SEQ ID NO: 6).

[0135] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 7-16. In some embodiments, the nucleotide sequence encoding the c-Jun protein comprises any one of the nucleic acid sequences set forth in SEQ ID NOs: 7-16.

[0136] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

[0137] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 8.

[0138] In some embodiments, a nucleotide sequence encoding a c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, a nucleotide sequence encoding a c-Jun protein described herein has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 9.

[0139] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence has at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 10.

[0140] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 11.

[0141] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleotide sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 12.

[0142] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 13.

[0143] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 14.

[0144] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 15.

[0145] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 16.

[0146] In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence encoding the c-Jun protein has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 16. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0147] The c-Jun nucleotide sequences disclosed herein can be codon-optimized using any method known in the art. For example, in some embodiments, the codons of the c-Jun nucleotide sequences disclosed herein are optimized to alter (e.g., increase or decrease) one or more of the following parameters compared to a wild-type nucleotide sequence (e.g., SEQ ID NO: 6): (i) codon adaptation index (i.e., codon usage bias), (ii) guanine-cytosine (GC) nucleotide content, (iii) mRNA secondary structure and instability motifs, (iv) repetitive sequences (e.g., direct repeats, inverted repeats, diad repeats), (v) restriction enzyme recognition sites, or (vi) combinations thereof.

[0148] Without being bound by any theory, in some embodiments, such codon optimization can increase expression of the protein encoded by the nucleotide sequence. Thus, in some embodiments, the codon-optimized c-Jun nucleotide sequences of the present disclosure, when transfected, transduced, or otherwise introduced into a human cell, e.g., a human T cell, are capable of increasing expression of the encoded c-Jun transcription factor compared to the corresponding expression in a cell transfected with a wild-type nucleotide sequence (e.g., SEQ ID NO: 6). In some embodiments, the expression of the c-Jun transcription factor is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, An increase of 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0149] In some aspects, increased expression of the c-Jun transcription factor in modified immune cells having reduced levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) is associated with increased expression of the c-Jun transcription factor in the transfected cells (e.g., immune cells, e.g., CD4 + and / or CD8 +The therapeutic agent may improve and / or enhance one or more properties of T cells (e.g., T cells). Non-limiting examples of such properties include resistance to exhaustion (e.g., as indicated by decreased expression of exhaustion markers such as PD-1, CD39, TIM-3, and / or LAG-3, extended survival, and / or increased cytokine production), persistence / extended survival, increased expansion / proliferation, improved effector function (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or a combination thereof.

[0150] Assays useful for measuring exhaustion, cell phenotype, persistence, cytotoxicity and / or killing, proliferation, cytokine production / release, and gene expression profiles are known in the art and include, for example, flow cytometry, intracellular cytokine staining (ICS), INCUCYTE® immune cell killing assay, Meso Scale Discovery (MSD) or similar assays, sustained antigen stimulation assays, bulk and single cell RNAseq (see, e.g., Fron Genet. 2020;11:220, 2019 Bioinformatics 35:i436-445, 2019 Annual Review of Biomed. Data Sci. 2:139-173), cytotoxicity / killing assays, ELISA, Western blot, and other standard molecular and cell biology methods, such as those described herein or in, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021), or elsewhere.

[0151] In some embodiments, increased expression of the c-Jun transcription factor in modified immune cells with reduced levels of the NR4A gene and / or NR4A protein increases the cell's resistance to exhaustion. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.In some embodiments, the resistance to exhaustion is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least about 57 at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0152] In some embodiments, overexpression of the c-Jun transcription factor in modified immune cells having reduced levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) can reduce exhaustion in exhausted cells. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.In some aspects, exhaustion can be at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least about 57-fold, at least about 58-fold, at least about 59-fold, at least about 60-fold At least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold decrease.

[0153] In some embodiments, increased expression of the c-Jun transcription factor in modified immune cells having reduced levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) can extend the persistence / survival of the cells, e.g., when administered to a subject in vivo. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.In some embodiments, the persistence / survival of the cells is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0154] In some embodiments, increased expression of the c-Jun transcription factor in modified immune cells having reduced levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) can increase the expansion / proliferation of the cells, e.g., upon antigen stimulation. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpress c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.In some embodiments, the expansion / growth of the cells is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0155] In some embodiments, increased expression of the c-Jun transcription factor in modified immune cells having reduced levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) can increase the effector function of the cells, e.g., cytokine production, granzyme release, and / or cytotoxicity in response to sustained antigen stimulation. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the modified immune cells (i.e., overexpressing c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.In some embodiments, the effector function of the cells (e.g., in response to sustained antigenic stimulation) is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least about 57-fold, at least about 58- at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0156] Without being bound by any theory, overexpression of c-Jun in T cells engineered to reduce expression levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof) helps maintain the activation state of the cell, for example, by reducing or preventing T cell dysfunction (e.g., T cell exhaustion). The c-Jun nucleotide sequences provided herein (e.g., codon-optimized c-Jun described herein) can be used to engineer immune cells, e.g., T cells, engineered to reduce levels of the NR4A gene and / or NR4A protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof), which then exhibit sustained potent cytotoxicity against desired target cells (e.g., targets of endogenous TCRs or targets of chimeric binding proteins described herein). Compared to T cells that do not overexpress c-Jun, engineered T cells overexpressing the codon-optimized c-Jun disclosed herein show fewer signs of T cell exhaustion. In some embodiments, the modified T cells provided herein (i.e., overexpressing c-Jun) have reduced levels of the NR4A1 gene and / or protein. In some embodiments, the modified T cells provided herein (i.e., overexpressing c-Jun) have reduced levels of the NR4A2 gene and / or protein. In some embodiments, the modified T cells provided herein (i.e., overexpressing c-Jun) have reduced levels of the NR4A3 gene and / or protein. In some embodiments, the modified T cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the modified T cells (i.e., overexpressing c-Jun) have reduced levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein.In some embodiments, the engineered T cells (i.e., that overexpress c-Jun) have reduced levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein, hi some embodiments, the engineered T cells (i.e., that overexpress c-Jun) have reduced levels of each of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.

[0157] As described herein, in some aspects, the disclosure includes modified immune cells that overexpress c-Jun and have reduced expression levels of the NR4A3 gene and / or NR4A3 protein, while having endogenous levels of the NR4A1 and NR4A2 genes and proteins, and a ligand binding protein (e.g., a CAR or TCR) (e.g., that specifically binds to ROR1). In some aspects, the disclosure includes modified immune cells that overexpress c-Jun protein and have reduced expression levels of the NR4A2 gene and / or NR4A2 protein, while having endogenous levels of the NR4A1 and NR4A3 genes and proteins, and a ligand binding protein (e.g., a CAR or TCR) (e.g., that specifically binds to ROR1). In some aspects, the present disclosure includes engineered immune cells that overexpress c-Jun and have reduced expression levels of the NR4A1 gene and / or NR4A1 protein, while having endogenous levels of NR4A2 and NR4A2 genes and NR4A2 and NR4A3 proteins, and a ligand binding protein (e.g., CAR or TCR) (e.g., that specifically binds ROR1). As described herein, in some aspects, the engineered immune cells described herein have reduced levels of two members of the NR4A family. For example, in some aspects, the immune cells described herein have been engineered to (i) express a ligand binding protein (e.g., CAR or TCR) (e.g., that specifically binds ROR1), (ii) overexpress c-Jun protein, and (iii) have reduced levels of both the NR4A1 gene and / or NR4A1 protein and the NR4A2 gene and / or NR4A2 protein. In some embodiments, the immune cells are modified to (i) express a ligand-binding protein (e.g., a CAR or TCR) (e.g., that specifically binds to ROR1), (ii) overexpress c-Jun protein, and (iii) reduce the levels of both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein.In some embodiments, immune cells are modified to (i) express a ligand binding protein (e.g., a CAR or TCR) (e.g., that specifically binds to ROR1), (ii) overexpress a c-Jun protein, and (iii) reduce the levels of both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. As described herein, in some embodiments, the modified immune cells described herein have reduced levels of all members of the NR4A family. Thus, in some embodiments, immune cells provided herein are modified to (i) express a ligand binding protein (e.g., a CAR or TCR) (e.g., that specifically binds to ROR1), (ii) overexpress a c-Jun protein, and (iii) reduce the levels of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.

[0158] Alternatively, a c-Jun protein useful in the present disclosure can be a mutant human c-Jun protein, so long as the mutant c-Jun protein does not affect the ability of the mutant to rescue dysfunctional (exhausted) T cells. In some embodiments, the mutant c-Jun protein comprises at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to the C-terminal amino acid residues (e.g., the C-terminal 50, 75, 100, 150, 200, or 250 or more residues), C-terminal portion (e.g., one-quarter, one-third, or one-half) or C-terminal domain (e.g., epsilon, bZIP, and amino acids C-terminal thereto) of a wild-type c-Jun protein. In some embodiments, the N-terminal amino acid residues (e.g., the N-terminal 50, 75, 100, or 150 or more), N-terminal portion (e.g., one-quarter, one-third, or one-half), or N-terminal domain (e.g., the delta, transactivation domain, and amino acids N-terminal thereto) of the wild-type c-Jun protein are deleted, mutated, or otherwise inactivated.

[0159] In some embodiments, the c-Jun protein comprises an inactivating mutation (e.g., a substitution, deletion, or insertion) in its transactivation domain and / or its delta domain. In some embodiments, the c-Jun protein comprises one or both of an S63A and an S73A mutation. In some embodiments, the c-Jun protein has a deletion between residues 2 and 102 or between residues 30 and 50 compared to wild-type human c-Jun.

[0160] In some embodiments, c-Jun polypeptides useful in the present modified immune cells include the truncated c-Jun polypeptides disclosed in WO2019 / 118902, the entire contents of which are expressly incorporated herein by reference.

[0161] In some aspects, in conjunction with reduced expression of the NR4A gene and / or protein, the overexpressed c-Jun polypeptide, when overexpressed in a cell, can prevent and / or reduce exhaustion of the cell (e.g., an immune cell (e.g., an anti-ROR1 CAR T cell) that expresses a CAR or TCR and has reduced expression of the NR4A3 gene and / or NR4A3 protein and has endogenous expression of the NR4A1 and NR4A2 genes and / or proteins, an immune cell (e.g., an anti-ROR1 CAR T cell) that expresses a CAR or TCR and has reduced expression of the NR4A2 gene and / or NR4A2 protein and has endogenous expression of the NR4A1 and NR4A3 genes and / or proteins, or an immune cell (e.g., an anti-ROR1 CAR T cell) that expresses a CAR or TCR and has reduced expression of the NR4A1 gene and / or NR4A1 protein and has endogenous expression of the NR4A2 and NR4A3 genes and / or proteins). Without wishing to be bound by any theory, in some embodiments, cells that overexpress c-Jun protein with reduced expression of the NR4A gene and / or protein are exhaustion resistant, thereby addressing a major barrier to the advancement of adoptive cell therapy (e.g., CAR T cell therapy).In some embodiments, the resistance to exhaustion is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 50-fold, at least about 51-fold, at least about 52-fold, at least about 53-fold, at least about 54-fold, at least about 55-fold, at least about 56-fold, at least about 57-fold, at least about 5 at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more.

[0162] Overexpression of c-Jun protein in immune cells, e.g., T cells, in conjunction with reduced expression of NR4A genes (e.g., NR4A1, NR4A2, or NR4A3) and / or proteins, in some embodiments, serves to maintain the activation state of the cells, for example, by reducing or preventing T cell dysfunction (e.g., T cell exhaustion). The engineered immune cells, e.g., T cells, exhibit sustained and potent cytotoxicity against antigen-bearing cells (e.g., ROR1-bearing tumor cells). Compared to T cells that do not overexpress c-Jun protein and do not have reduced expression of NR4A (e.g., NR4A1, NR4A2, and / or NR4A3) genes and / or proteins, the engineered T cells show reduced signs of T cell exhaustion and increased signs of effector cells that can persist and function longer.

[0163] In some embodiments, the immune cells provided herein (e.g., expressing a CAR or TCR, e.g., anti-ROR1 CAR-engineered cells described herein, and overexpressing c-Jun protein and having reduced expression of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) have reduced expression of one or more exhaustion markers, including, but not limited to, TIGIT, PD-1, and CD39. Exhaustion marker expression can be measured in bulk populations by flow cytometry using bulk RNASeq transcriptome analysis, or in some embodiments, individual cell transcriptome analysis can be performed using single-cell RNASeq. ... The expression of one or more exhaustion markers in CAR-engineered T cells, and in CAR-engineered T cells that overexpress c-Jun protein and have reduced expression of NR4A (e.g., NR4A1, NR4A2, and / or NR4A3) genes and / or proteins, is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, or at least about 3.0-fold increased compared to reference cells (e.g., corresponding cells that have not been engineered to overexpress the c-Jun protein and that express endogenous levels of NR4A1, NR4A2, and NR4A3 genes and / or proteins). , at least about 3.5-fold, at least about 4-fold, at least 4.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold or more decreased.In some aspects, the expression of TIGIT in an immune cell described herein (e.g., an immune cell expressing a CAR or TCR, e.g., an anti-ROR1 CAR-engineered T cell, and overexpressing a c-Jun protein and having reduced expression of the NR4A1, NR4A2, and / or NR4A3 gene and / or protein) is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, or less than or equal to ... at least about 2 at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold or more decreased.In some aspects, the expression of PD-1 in an immune cell described herein (e.g., an immune cell expressing a CAR or TCR, e.g., an anti-ROR1 CAR-engineered T cell, and overexpressing a c-Jun protein and having reduced expression of the NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3.0-fold, at least about A decrease of 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 55 fold, at least about 60 fold, at least about 65 fold, at least about 70 fold, at least about 75 fold, at least about 80 fold, at least about 85 fold, at least about 90 fold, at least about 95 fold, or at least about 100 fold.In some aspects, the expression of CD39 in an immune cell described herein (e.g., an immune cell expressing a CAR or TCR, e.g., an anti-ROR1 CAR-engineered T cell, and overexpressing a c-Jun protein and having reduced expression of the NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 7.0-fold, at least about 8.0-fold, at least about 9.0-fold, at least about 10.0-fold, at least about 11.0-fold, at least about 12.0-fold, at least about 13.0-fold, at least about 14.0-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, at least about 30.0-fold, at least about 31.0-fold, at least about 32.0-fold, at least about 33.0-fold, at least about 34.0-fold, at least about 35.0-fold, at least about 36.0-fold, at least about 37.0 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 55 fold, at least about 60 fold, at least about 65 fold, at least about 70 fold, at least about 75 fold, at least about 80 fold, at least about 85 fold, at least about 90 fold, at least about 95 fold, or at least about 100 fold or more decreased.

[0164] In some embodiments, after antigen stimulation, immune cells described herein (e.g., expressing a CAR or TCR, e.g., an engineered anti-ROR1 CAR) are administered to the recipient cells. T cells, and populations of T cells that overexpress c-Jun protein and have reduced expression of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins, secrete at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold, at least about 125-fold, or at least about 150-fold or more IL-2, IFN-γ, and / or TNF-α compared to a corresponding control population of cells that do not overexpress c-Jun protein and do not have reduced levels of NR4A1, NR4A2, and NR4A3 genes and / or proteins. In some aspects, a population of immune cells described herein (e.g., expressing a CAR or TCR, e.g., engineered anti-ROR1 CAR T cells, and overexpressing a c-Jun protein and having reduced levels of the NR4A1, NR4A2, and / or NR4A3 gene and / or protein) is at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, At least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold or more IL-2, IFN-γ, and / or TNF-α are expressed on days 0 and / or 14 of sustained antigen stimulation at E:T ratios of 1:1, 1:5, 1:10, and / or 1:20.Cytokine secretion can be measured using methods known in the art, such as ELISA or MSD analysis.

[0165] In some aspects, a population of immune cells described herein (e.g., expressing a CAR or TCR, e.g., engineered anti-ROR1 CAR T cells, and overexpressing a c-Jun protein and having reduced levels of the NR4A1, NR4A2, and / or NR4A3 gene and / or protein) exhibits at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, or at least about 250-fold or more improved killing efficiency, e.g., as quantified by area under the curve (AUC), compared to a corresponding control population of cells that do not overexpress the c-Jun protein and do not have reduced levels of the NR4A1, NR4A2, and NR4A3 gene and / or protein.

[0166] In some aspects, a population of immune cells described herein (e.g., expressing a CAR or TCR, e.g., engineered anti-ROR1 CAR T cells, and overexpressing a c-Jun protein and having reduced levels of the NR4A1, NR4A2, and / or NR4A3 gene and / or protein) has at least the same, or at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3 ... In some embodiments, the cells exhibit 5-fold, at least about 4-fold, at least about 5-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 125-fold, at least about 150-fold, at least about 200-fold, at least about 225-fold, at least about 250-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold or more proliferation in response to the antigen. Antigen-induced proliferation can be determined using proliferation assays known in the art, such as those described herein.

[0167] Assays useful for measuring one or more characteristics of the cells described herein (e.g., anti-ROR1 CAR T cells), such as exhaustion, cell phenotype, persistence, cytotoxicity and / or killing, proliferation, cytokine release, and gene expression profile, are known in the art and include, for example, flow cytometry, intracellular cytokine staining (ICS), IncuCyte immune cell killing assay, Meso Scale Discovery (MSD) or similar assays, sustained antigen stimulation assays, continuous antigen stimulation assays (similar to sustained antigen stimulation assays but without resetting the E:T cell ratio with each restimulation), bulk and single cell RNAseq (see, e.g., Fron Genet. 2020;11:220, 2019 Bioinformatics 35:i436-445, 2019 Annual Review of Biomed. Data Sci. 2:139-173), cytotoxicity / killing assays, ELISA, Western blot, and other standard molecular and cell biology methods, such as those described herein or described in, for example, Current Protocols Examples include those described in *Current Protocols in Molecular Biology* or *Current Protocols in Immunology* (John Wiley & Sons, Inc., 1999-2021) or elsewhere.

[0168] In some embodiments, the population of immune cells is a pure population. In some embodiments, the pure population comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least 99% cells belonging to the same immune cell type (e.g., 99% of the immune cells are lymphocytes). In some embodiments, the population of immune cells comprises one, two, three, four, or five different cell types; for example, a population of immune cells comprising two cell types may comprise lymphocytes and dendritic cells.

[0169] In some aspects, the population of engineered immune cells disclosed herein comprises, consists of, or consists essentially of lymphocytes. In some aspects, the population of engineered immune cells disclosed herein comprises lymphocytes, wherein the lymphocytes are selected from the group consisting of T cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells, natural killer (NK) T cells, and any combination thereof. In some specific aspects, the lymphocytes are T cells. In some specific aspects, the lymphocytes are NK cells.

[0170] In some embodiments, the modified immune cells disclosed herein are T cells. In some embodiments, the T cells comprise a CAR. In some embodiments, modified T cells (CAR T cells) that can be prepared to express the CAR include, for example, CD8 + T cells or CD4 + The CAR-expressing cells disclosed herein are T cells. In some embodiments, the CAR-expressing cells disclosed herein are CAR T cells, e.g., mono-CAR T cells, genome-edited CAR T cells, dual-CAR T cells, or tandem-CAR T cells. In some embodiments, the engineered cells disclosed herein are NK cells. In some embodiments, the NK cells comprise a CAR. In some embodiments, the CAR NK cells are mono-CAR NK cells, dual-CAR NK cells, or tandem-CAR NKT cells. In some embodiments, the engineered cells of the present disclosure comprise both T cells and NK cells. In some embodiments, the T cells and NK cells both comprise a CAR. Examples of such CAR T cells and CAR NK cells are provided in International Application No. PCT / US2019 / 044195. In some embodiments, the T cells, NK cells, or both comprise any of the other ligand-binding proteins described herein. For example, in some embodiments, the T cells comprise a TCR, e.g., an engineered TCR. In some embodiments, the NK cells comprise a TCR, eg, an engineered TCR.

[0171] In some embodiments, the modified immune cells can be any immune cell type. In some embodiments, the cells are modified immune cells for any adoptive cell transfer (ACT) therapy (also known as adoptive cell therapy). ACT therapy can be autologous therapy or allogeneic therapy. In some embodiments, the ACT therapy includes but is not limited to CAR T therapy, tumor-infiltrating lymphocyte (TIL) therapy, NK cell therapy, or any combination thereof.

[0172] In some embodiments, the modified immune cells are TILs for TIL therapy. The use of TILs as adoptive cell transfer therapy for cancer treatment 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 single-cell suspensions isolated from tumor fragments. Multiple individual cultures are established, grown separately, and assayed for specific tumor recognition. TILs are expanded over several weeks. The specific TIL line that shows the best tumor reactivity is then further expanded using a "rapid expansion protocol" (REP), typically using anti-CD3 activation for two weeks. The cultured TILs can be modified at any point during the ex vivo process to decrease expression of the NR4A1, NR4A2, or NR4A3 gene and / or NR4A1, NR4A2, or NR4A3 protein, including combinations thereof, and increase expression of c-Jun protein. The final TILs after REP are infused back into the patient. The process may also include a preliminary chemotherapy regimen to deplete endogenous lymphocytes, allowing the adoptively transferred TILs to approach the tumor site sufficiently to surround it.

[0173] As described herein, immune cells of the present disclosure (e.g., that overexpress c-Jun protein and have reduced levels of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) can comprise a ligand binding protein (also referred to herein as a "chimeric binding protein"). Non-limiting examples of ligand binding proteins (e.g., chimeric binding proteins) useful in the present disclosure include chimeric antigen receptors (CARs), T cell receptors (TCRs) (e.g., engineered TCRs), chimeric antibody-T cell receptors (caTCRs), chimeric signaling receptors (CSRs), T cell receptor mimetics (TCR mimetics), and combinations thereof.

[0174] In some aspects, the immune cells, e.g., T cells, disclosed herein may comprise a chimeric antigen receptor (CAR) that specifically binds to an antigen (e.g., a tumor antigen). Non-limiting examples of CARs that can be used in the present disclosure are known in the art. See, for example, US2020 / 0172879A1 and US2019 / 0183932A1, each of which is incorporated herein by reference in its entirety.

[0175] In some embodiments, the immune cells, e.g., T cells, disclosed herein comprise a T cell receptor (TCR), e.g., an engineered T cell receptor (also known as a "transgenic TCR"). T cell receptors are heterodimers composed of two distinct transmembrane polypeptide chains, an α chain and a β chain, each consisting 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 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. 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 population of T cells.

[0176] In some embodiments, the immune cells, e.g., T cells, disclosed herein comprise a chimeric antibody-T cell receptor (caTCR). As used herein, a "chimeric antibody-T cell receptor" or "caTCR" comprises (i) an antibody portion that specifically binds to an antigen of interest and (ii) a T cell receptor module capable of recruiting at least one TCR-associated signaling molecule. In some embodiments, the antibody portion and the T cell receptor module are fused together. In some embodiments, the chimeric binding protein comprises a chimeric signaling receptor (CSR). A "chimeric signaling receptor" or "CSR" comprises a ligand-binding domain that specifically binds to a target ligand and a costimulatory signaling domain that is capable of providing a stimulatory signal to an immune cell that expresses the CSR. Non-limiting examples of caTCRs and CSRs are further described in U.S. Pat. No. 10,822,413 B2, which is incorporated herein by reference in its entirety.

[0177] In some embodiments, the immune cells, e.g., T cells, disclosed herein comprise a T cell receptor mimic (TCR mimic). As used herein, the term "T cell receptor mimic" or "TCR mimic" refers to an antibody (or fragment thereof) engineered to recognize a tumor antigen, where the tumor antigen is presented in the context of an HLA molecule. As will be apparent to those skilled in the art, these antibodies can mimic the specificity of a TCR. Non-limiting examples of TCR mimics are provided, for example, in US2009 / 0226474A1 and US2019 / 0092876A1, each of which is incorporated herein by reference in its entirety.

[0178] In some embodiments, the ligand binding protein (e.g., a CAR or TCR) that can be expressed in the modified cells disclosed herein specifically binds to (i.e., targets) one or more antigens expressed on tumor cells, e.g., malignant B cells, malignant T cells, or malignant plasma cells.

[0179] In some embodiments, the ligand binding protein (e.g., CAR or TCR) is selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5 , HMWMAA, o-acetyl-GD2, folate receptor beta, 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-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant (e.g., including KRAS, HRAS, and NRAS mutant proteins), hTERT, sarcoma translocation breakpoints, 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, mutSpecifically binds to (i.e., targets) an antigen selected from the group consisting of hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of APRIL protein, and any combination thereof.

[0180] In some embodiments, the immune cells (e.g., modified CAR or TCR-engineered cells) described herein may target major types of antigens (e.g., tumor antigens), namely, 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 tumor virus antigens. Human papillomavirus (HPV) E6 and HPV E7 proteins belong to the category of tumor virus antigens.

[0181] In some aspects, the immune cells (e.g., engineered CAR or TCR engineered cells) described herein can target CT antigens, such as melanoma-associated antigens (MAGEs), including, but not limited to, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12. In some aspects, the immune cells (e.g., engineered CAR or TCR engineered cells) described herein can target a glycoprotein found primarily in melanoma and normal melanocytes (gp100), a melanoma antigen recognized by T cells (MART-1), and / or tyrosinase. In some embodiments, the immune cells (e.g., engineered CAR or TCR engineered cells) described herein can target Wilms' tumor 1 (WT1), an overexpressed antigen that is highly expressed in most acute myeloid leukemias (AMLs), acute lymphoblastic leukemias, almost all types of solid tumors, and several critical tissues, e.g., cardiac tissue. In some embodiments, the immune cells (e.g., engineered CAR or TCR engineered cells) described herein can target mesothelin, another overexpressed antigen that is highly expressed in mesotheliomas but is also present on mesothelial cells in several tissues, including the trachea.

[0182] In some embodiments, the modified immune cells of the present disclosure, e.g., CAR T cells or NK cells or TCR-engineered T cells, can target any one of the tumor antigens disclosed above or a combination thereof. As described herein, in some embodiments, the immune cells provided herein can specifically target the ROR1 antigen. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in approximately 57% of triple-negative breast cancer (TNBC) patients and 42% of non-small cell lung cancer (NSCLC) adenocarcinoma patients (Balakrishnan 2017), representing a highly attractive target for chimeric antigen receptor (CAR) T cells. Receptor tyrosine kinase-like orphan receptor 1 positive (ROR1) +Solid tumors can be safely targeted with anti-ROR1 CAR T cells (Specht 2020), but efficacy is limited in part because CAR T cells exhibit exhaustion or dysfunction after infusion in patients with solid tumor malignancies. Furthermore, solid tumors possess an immunosuppressive barrier that limits the antitumor activity of immunotherapies such as CAR T cells (Newick 2016, Srivastava 2018, Martinez 2019).

[0183] Without wishing to be bound by any theory, cells expressing the anti-ROR1 chimeric binding proteins described herein (e.g., anti-ROR1 CARs or anti-ROR1 TCRs) have been modified to overexpress c-Jun protein while simultaneously reducing the expression levels of the NR4A1, NR4A2, and / or NR4A3 genes and / or NR4A1, NR4A2, and / or NR4A3 proteins. As described herein, in some embodiments, cells expressing the anti-ROR1 chimeric binding proteins (e.g., anti-ROR1 CARs or anti-ROR1 TCRs) have been modified to overexpress c-Jun protein while simultaneously reducing the levels of multiple members of the NR4A family. These modified cells are more resistant to exhaustion and exhibit improved effector function compared to other anti-ROR1 cells available in the art (e.g., that express a ligand-binding protein but have not been modified to overexpress c-Jun protein and / or reduce the levels of multiple members of the NR4A family).

[0184] In some embodiments, the modified immune cells described herein (e.g., overexpressing c-Jun protein and having reduced levels of the NR4A1, NR4A2, and / or NR4A3 genes and / or NR4A1, NR4A2, and / or NR4A3 proteins) comprise a ROR1-binding chimeric antigen receptor ("anti-ROR1 CAR"). Exemplary anti-ROR1 CARs are described in Hudecek, et al. Clin. Cancer Res. 19.12(2013):3153-64, which is incorporated herein by reference in its entirety. In some embodiments, CAR T cells of the present disclosure comprising an anti-ROR1 CAR are generated as described in Hudecek et al. (e.g., as described in Hudecek et al., page 3155, first paragraph in its entirety, which is incorporated herein by reference in its entirety). In some aspects, the anti-ROR1 CARs of the present disclosure include antibodies or fragments thereof comprising the VH and / or VL sequences of the 2A2, R11, and R12 anti-ROR1 monoclonal antibodies described in Hudecek et al. (paragraphs spanning pages 3154-55), Baskar et al. MAbs 4(2012):349-61, and Yang et al. PLoS ONE 6(2011):e21018, which are incorporated herein by reference in their entireties.

[0185] In some embodiments, an anti-ROR1 chimeric binding protein (e.g., an anti-ROR1 CAR or anti-ROR1 TCR) useful in the present disclosure can cross-compete with an anti-ROR1 antibody, e.g., the R12 antibody. The R12 antibody sequence is shown in Table 7. In some embodiments, an anti-ROR1 chimeric binding protein (e.g., an anti-ROR1 CAR or anti-ROR1 TCR) useful in the present disclosure binds to the same epitope as the R12 antibody. As will be apparent to one of skill in the art, any anti-ROR1 antibody known in the art can be used in the present disclosure. Non-limiting examples of such antibodies include the 2A2 and R11 antibodies described in Hudecek, et al. Clin. Cancer Res. 19.12(2013):3153-64, Baskar et al. MAbs 4(2012):349-61, and Yang et al. PLoS ONE 6(2011):e21018, US9,316,646B2, and US9,758,586B2, each of which is incorporated herein by reference in its entirety. [Table 7]

[0186] In some aspects, the antigen-binding domain of an anti-ROR1 chimeric binding protein (e.g., an anti-ROR1 CAR) described herein that can be expressed in an engineered immune cell disclosed herein comprises the VH CDR3 of the R12 antibody. In some aspects, the antigen-binding domain of an anti-ROR1 chimeric binding protein (e.g., an anti-ROR1 CAR) of the present disclosure comprises the VH CDR1, VH CDR2, and VH CDR3 of the R12 antibody. In some aspects, the antigen-binding domain of an anti-ROR1 chimeric binding protein (e.g., an anti-ROR1 CAR) of the present disclosure comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the R12 antibody. In some aspects, the antigen-binding domain of an anti-ROR1 chimeric binding protein of the present disclosure, e.g., an R12 scFv, comprises the VH and VL of the R12 antibody.

[0187] In some embodiments, the intracellular domain of a chimeric binding protein (e.g., any of the CARs or TCRs provided herein, e.g., an anti-ROR1 CAR) that can be expressed in an engineered immune cell disclosed herein includes a signaling domain, e.g., one derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the chimeric binding protein (e.g., a CAR or a TCR) further comprises a costimulatory domain, such as those derived from 2B4, HVEM, ICOS, LAG3, DAP10, DAP12, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. In some embodiments, the chimeric binding protein (e.g., a CAR or a TCR) comprises a 4-1BB costimulatory domain.

[0188] In some embodiments, the transmembrane domain of a chimeric binding protein (e.g., a CAR or TCR provided herein) that can be expressed in an engineered immune cell disclosed herein can be any of a variety of binding domains, including, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD1 03, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, or CD19.

[0189] In some embodiments, a chimeric binding protein (e.g., a CAR or TCR) that can be expressed in an engineered immune cell disclosed herein further comprises a sequence encoding a costimulatory domain, e.g., a costimulatory domain described herein. In some embodiments, the costimulatory domain comprises a costimulatory domain of interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, Lck-binding deficient CD28 (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds to CD83, or any combination thereof.

[0190] As further described herein, in some aspects, the immune cells described herein are modified, e.g., by gene editing tools, to reduce expression of the NR4A1, NR4A2, or NR4A3 gene and / or protein (including combinations thereof). Reduced expression of the NR4A1, NR4A2, and / or NR4A3 gene can be achieved, for example, by editing the entire NR4A1, NR4A2, and / or NR4A3 gene, by editing a portion of the NR4A1, NR4A2, and / or NR4A3 gene, or by editing a regulatory region controlling expression of the NR4A1, NR4A2, and / or NR4A3 gene. Thus, methods known in the art for reducing gene and / or protein expression in cells can be used to reduce expression of the NR4A1, NR4A2, and / or NR4A3 gene and / or protein (including combinations thereof) in immune cells expressing a ligand-binding protein (e.g., a CAR-expressing cell or a TCR-expressing cell provided herein). For example, in some embodiments, the expression of NR4A1, NR4A2, and / or NR4A3 genes and / or their encoded proteins in an immune cell (e.g., a CAR-expressing cell or a TCR-expressing cell) provided herein can be reduced by contacting the cell with a gene editing tool capable of reducing the expression levels of the NR4A1, NR4A2, and / or NR4A3 genes and their encoded proteins. Non-limiting examples of gene editing tools are provided below. In some specific embodiments, the gene editing tool comprises, for example, an shRNA, an siRNA, an miRNA, an antisense oligonucleotide, a CRISPR, a zinc finger nuclease, a TALEN, a meganuclease, a restriction endonuclease, or any combination thereof. In some embodiments, the gene editing tool is a CRISPR. In some embodiments, the gene editing tool comprises a guide RNA (gRNA) that specifically targets the NR4A family member. Non-limiting examples of such gRNAs are provided in Tables A, C, and D.

[0191] In some aspects, a population of immune cells provided herein (e.g., CAR- or TCR-expressing cells produced by the methods disclosed herein, i.e., having reduced levels of expression of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins and increased levels of c-Jun protein) exhibit enhanced or improved one or more properties compared to reference immune cells (i.e., corresponding immune cells that have not been modified to have reduced levels of NR4A1, NR4A2, and NR4A3 genes and / or proteins and to express the c-Jun protein). In some aspects, improving one or more properties of the immune cells disclosed herein can be useful in treating tumors (e.g., reducing tumor volume and / or tumor weight). The one or more properties that can be improved by the present disclosure include any property of the immune cells disclosed herein that can be useful in treating cancer. For example, in some embodiments, a population of immune cells (e.g., CAR- or TCR-expressing cells produced by the methods disclosed herein, i.e., having reduced expression levels of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins and increased levels of c-Jun protein) may exhibit increased effector activity compared to reference cells (e.g., CAR- or TCR-expressing cells that have not been modified to express the c-Jun protein such that the expression levels of the NR4A1, NR4A2, and NR4A3 genes and proteins are reduced).

[0192] In some aspects, the improved property of the engineered immune cells relative to a reference cell is: (i) increasing the expansion and / or proliferation of said immune cells; (ii) an increase in the cytotoxicity of the immune cells; (iii) an increase in cytokine expression in the immune cells; or (iv) Any combination thereof.

[0193] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein that overexpress c-Jun and have reduced levels of one or more of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein) are more resistant to exhaustion and / or dysfunction compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein).

[0194] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein that overexpress c-Jun and have reduced levels of one or more of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein) are apoptosis-resistant, i.e., exhibit reduced or no apoptosis, compared to reference immune cells (i.e., corresponding immune cells that have not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein).

[0195] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein that overexpress c-Jun and have reduced levels of one or more of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein) are immune checkpoint resistant, i.e., exhibit reduced or no immune checkpoint activity, compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein).

[0196] In some aspects, modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein that overexpress c-Jun and have reduced levels of one or more of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein) exhibit improved T cell activation compared to reference immune cells (i.e., corresponding immune cells that have not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein). In some aspects, such improved T cell activation can be evidenced, for example, by modified immune cells that exhibit improved proliferation, improved cytotoxicity, improved cytokine expression, or any combination thereof, compared to reference immune cells (i.e., corresponding immune cells that have not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein).

[0197] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein that overexpress c-Jun and have reduced levels of one or more of the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein) maintain anti-tumor function in the tumor microenvironment (TME) compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to reduce expression of the NR4A1, NR4A2, and NR4A3 genes and / or proteins and to overexpress the c-Jun protein).

[0198] The present disclosure also provides pharmaceutical compositions comprising a population of modified immune cells disclosed herein and a pharmaceutically acceptable carrier, such pharmaceutical compositions being further described elsewhere in this disclosure.

[0199] III. Treatment method Provided herein are methods for treating a tumor (or cancer) in a subject in need thereof, the methods comprising administering to the subject a cell composition of the present disclosure, e.g., cells that overexpress c-Jun protein and have a reduced expression level of the NR4A (e.g., NR4A1, NR4A2, and / or NR4A3) gene and / or its encoded protein, i.e., the NR4A protein. As used herein, the term "cell composition" refers to the immune cells alone or in combination with one or more additional agents (e.g., excipients). In some embodiments, the cells comprise a ligand-binding protein (e.g., CAR or TCR) that specifically binds to a tumor antigen described herein. In some embodiments, the tumor antigen comprises ROR1. Thus, in some embodiments, methods of treating a tumor provided herein comprise administering to the subject a cell composition described herein, wherein the cells (i) overexpress a c-Jun protein, (ii) have reduced levels of an NR4A gene and / or protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof), and (iii) express a CAR that specifically targets a tumor antigen. In some embodiments, methods of treating a tumor provided herein comprise administering to the subject a cell composition described herein, wherein the cells (i) overexpress a c-Jun protein, (ii) have reduced levels of an NR4A gene and / or protein (e.g., NR4A1, NR4A2, NR4A3, or a combination thereof), and (iii) express a TCR (e.g., an engineered TCR) that specifically targets a tumor antigen.

[0200] In some embodiments, the expression level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene is reduced by at least about 5%, at least about 10%, at least about 15%, 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 reduce the expression level of the NR4A (NR4A1, NR4A2, or NR4A3) gene). In some embodiments, the expression level of the NR4A (NR4A1, NR4A2, and / or NR4A3) protein is reduced by at least about 5%, at least about 10%, at least about 15%, 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 reduce the expression level of the NR4A (NR4A1, NR4A2, or NR4A3) protein). In some embodiments, the expression levels of both the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and protein are reduced by at least about 5%, at least about 10%, at least about 15%, 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 reduce the expression level of the NR4A (NR4A1, NR4A2, or NR4A3) gene and / or protein). Methods for reducing the expression levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein are provided elsewhere in this disclosure.

[0201] In some embodiments, administration of the cell composition of the present disclosure 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 some embodiments, the reference tumor volume is the tumor volume in a non-treated subject. Unless otherwise specified, a "non-treated subject" (or variant thereof) includes any of the following: (1) a non-treated subject (e.g., afflicted with the same tumor) receiving corresponding cells that express endogenous levels of c-Jun protein and all members of the NR4A family; (2) a non-treated subject receiving corresponding cells that overexpress c-Jun but have endogenous levels of all members of the NR4A family; (3) a non-treated subject receiving corresponding cells that have reduced levels of one or more members of the NR4A family but do not overexpress c-Jun; and (4) any combination of (1)-(3). In some embodiments, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, 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 the reference tumor volume after administration.

[0202] In some embodiments, treating a tumor comprises reducing tumor weight in the subject. In some 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 15%, 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 a reference tumor weight. In some embodiments, the reference tumor weight is the tumor weight in the subject before administration of the modified cells. In some embodiments, the reference tumor weight is the tumor weight in a corresponding subject that did not receive the administration.

[0203] In some embodiments, for example, administration of a cell composition of the present disclosure to a subject suffering from a tumor increases TILs (e.g., CD4 + or CD8 + In some embodiments, the number and / or percentage of TILs in the tumor and / or TME can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% 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). %, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more. In some embodiments, for example, administration of a cell composition of the present disclosure to a subject afflicted with a tumor increases TIL (e.g., CD4 + or CD8 +) can be increased by at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 50-fold, at least about 75-fold, or at least about 100-fold as compared to a reference (e.g., a subject that did not receive the modified cells or the corresponding value in the same subject prior to administration of the modified cells).

[0204] In some embodiments, administration of the cell compositions of the present disclosure may reduce the number and / or percentage of regulatory T cells in a subject's tumor and / or TME, hi some embodiments, the number and / or percentage of regulatory T cells in the tumor and / or TME is reduced by at least about 5%, at least about 10%, at least about 15%, 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 percentage in a subject who did not receive the modified cells).

[0205] In some embodiments, administration of the cell compositions of the present disclosure may reduce the number and / or percentage of myeloid-derived suppressor cells (MDSCs) in a subject's tumor and / or TME. In some embodiments, the MDSCs are monocytic MDSCs (M-MDSCs). In some embodiments, the MDSCs are polymorphonuclear MDSCs (PMN-MDSCs). In some embodiments, the MDSCs include both M-MDSCs and PMN-MDSCs. In some embodiments, the number and / or percentage of MDSCs in the tumor and / or TME is reduced by at least about 5%, at least about 10%, at least about 15%, 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 administered the modified cells).

[0206] In addition to the above, administration of the cell compositions of the present disclosure can have other effects that contribute to the treatment of tumors, which are further described below.

[0207] As described herein, the cell compositions of the present disclosure (i.e., overexpressing c-Jun protein and having reduced expression levels of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins, and expressing a binding molecule that specifically binds to a tumor antigen, e.g., ROR1) can be used to treat tumors derived from various cancer types, including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. A comprehensive, non-limiting list of cancer indications is provided in the indications section of this application.

[0208] In some embodiments, the cell compositions of the present disclosure can be used in combination with other therapeutic agents (e.g., anti-cancer agents and / or immunomodulatory agents). Thus, in some embodiments, the methods of treating tumors disclosed herein comprise administering the cell compositions of the present disclosure in combination with one or more additional therapeutic agents. In some embodiments, the cell compositions of the present disclosure can be used in combination with one or more anti-cancer agents, such that multiple components of the immune pathway can be targeted. In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods 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. A comprehensive and non-limiting list of combination therapies is disclosed in detail in the Combination Therapies section of this application.

[0209] In some aspects, the cell composition of the present disclosure is administered to the subject before or after administration of the additional therapeutic agent. In some aspects, the cell composition of the present disclosure is administered to the subject simultaneously with the additional therapeutic agent. In some aspects, the cell composition of the present disclosure and the additional therapeutic agent may be administered simultaneously as a single composition contained in a pharmaceutically acceptable carrier. In some aspects, the cell composition of the present disclosure and the additional therapeutic agent are administered simultaneously as separate compositions.

[0210] In some aspects, subjects that can be treated according to the present disclosure are non-human animals, e.g., rats or mice. In some aspects, subjects that can be treated are humans.

[0211] In some embodiments, treating a tumor, e.g., with the methods disclosed herein, includes enhancing the activation of T cells (e.g., tumor-specific T cells). As used herein, the phrase "enhancing T cell activation" refers to altering the signaling of T cells during activation to promote retention of their memory.

[0212] Thus, in some aspects, the present disclosure relates to methods of increasing activation of T cells by overexpressing c-Jun protein and reducing the expression level of NR4A (NR4A1, NR4A2, or NR4A3) genes and / or proteins in the cells. The activation state of a cell can be determined by any method known in the art, for example, by analyzing one or more functional properties of the cell (e.g., proliferation, cytotoxicity, cytokine production) or by analyzing the phenotypic expression of the cell. In some aspects, increasing the activation of T cells (e.g., tumor-specific T cells) can result in one or more of the following improved properties in the cell: (i) increased proliferation, (ii) increased cytotoxicity, (iii) increased cytokine expression, or (iv) any combination thereof.

[0213] In some embodiments, increasing activation of T cells (e.g., tumor-specific T cells) results in increased proliferation of the cells. In some embodiments, the proliferation of the T cells is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 90% relative to the proliferation of a reference cell (e.g., a corresponding cell that has not been modified to reduce the expression level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and to overexpress the c-Jun protein). 0%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more. In some embodiments, the improved proliferation can result in, for example, an increased number of the engineered T cells (i.e., that overexpress c-Jun protein and have reduced expression levels of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) in the subject.In some embodiments, the number of the modified T cells is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or less compared to the number of reference cells (e.g., corresponding cells that have not been modified to reduce the expression level of the NR4A gene and / or protein and to overexpress the c-Jun protein). an increase of at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more.

[0214] In some embodiments, increasing the activation of T cells (e.g., tumor-specific T cells) improves the cytotoxicity of the cells. As used herein, the term "cytotoxicity" refers to the ability of a cellular composition (e.g., tumor-specific T cells) of the present disclosure to attack tumor cells and induce tumor cell damage. A cellular composition (e.g., tumor-specific T cells) of the present disclosure can attack tumor cells and induce tumor cell damage by any method known in the art, for example, by inducing apoptosis of tumor cells via the release of cytotoxic molecules (e.g., perforin, granzymes, and granulysin) or via Fas-Fas ligand interaction. In some embodiments, the cytotoxicity of the T cells is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, at least about 550%, at least about 550%, at least about 600%, at least about 650%, at least about 0%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more improved (i.e., increased).

[0215] In some embodiments, increasing activation of T cells (e.g., tumor-specific T cells) results in increased cytokine expression in the cells. In some embodiments, the cytokine expression is increased (i.e., increased) by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more, compared to cytokine expression in a reference cell (e.g., a corresponding cell that has not been modified to overexpress the c-Jun protein and reduce the expression level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). As used herein, the term "cytokine" refers to any cytokine that may be useful in the treatment of cancer. Non-limiting examples of such cytokines include IFN-γ, TNF-α, IL-2, and any combination thereof.

[0216] In some embodiments, the expansion and / or proliferation of the immune cells, the cytotoxicity of the immune cells, or the cytokine expression of the immune cells is increased by about 2-fold to about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, or more. In some embodiments, the expansion and / or proliferation of the immune cells, the cytotoxicity of the immune cells, or the cytokine expression of the immune cells is increased by about 10-fold to about 500-fold, about 20-fold to about 400-fold, about 25-fold to about 250-fold, or about 10-fold to about 50-fold. In some aspects, the expansion and / or proliferation of the immune cells, the cytotoxicity of the immune cells, or the cytokine expression of the immune cells is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold or more compared to that of a reference cell (e.g., a corresponding immune cell that has not been modified to overexpress the c-Jun protein and to reduce the expression level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein).

[0217] In some embodiments, modified immune cells according to the present disclosure exhibit increased cytokine expression relative to a reference cell, hi some embodiments, the cytokine is interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or any combination thereof.

[0218] In some embodiments, the expression level of IL-2 in the modified immune cell is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, or at least about 100 fold or more compared to the expression level of IL-2 in a reference immune cell.

[0219] In some embodiments, the expression level of IFN-γ in the modified immune cell is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold or more compared to the expression level of IFN-γ in a reference immune cell.

[0220] In some embodiments, the expression level of TNF-α in the modified immune cell is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, or at least about 100 fold or more compared to the expression level of TNF-α in a reference immune cell.

[0221] In some embodiments, immune cells (e.g., CAR- or TCR-expressing cells) that express a ligand binding protein disclosed herein (i.e., that overexpress a c-Jun protein and have reduced expression levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or NR4A protein) produce increased amounts of IL-2 when stimulated with an antigen, e.g., an alloantigen (e.g., a tumor antigen), e.g., by continuous and / or chronic stimulation. In some embodiments, the amount of IL-2 produced by the immune cells (e.g., CAR- or TCR-expressing cells) is at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, or less than that of a reference cell (e.g., a corresponding cell that has not been modified to overexpress the c-Jun protein and have reduced expression levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold or more increase.

[0222] In some aspects, immune cells (e.g., CAR- or TCR-expressing cells) that express a ligand binding protein disclosed herein (i.e., that overexpress c-Jun protein and have reduced expression levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein) produce increased amounts of IFN-γ when stimulated with an antigen, e.g., an alloantigen (e.g., a tumor antigen), e.g., by continuous and / or chronic stimulation. In some aspects, the amount of IFN-γ produced by the immune cell (e.g., a CAR- or TCR-expressing cell) is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold or more compared to a reference cell (e.g., a corresponding cell that has not been modified to overexpress the c-Jun protein and to reduce the expression level of an NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein).

[0223] In some embodiments, immune cells (e.g., CAR- or TCR-expressing cells) that express a ligand binding protein disclosed herein (i.e., that overexpress a c-Jun protein and have reduced expression levels of NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins) produce increased amounts of TNF-α when stimulated with an antigen, e.g., an alloantigen (e.g., a tumor antigen), e.g., by continuous and / or chronic stimulation. In some embodiments, the amount of TNF-α produced by the immune cells (e.g., CAR- or TCR-expressing cells) is at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, or less than that of a reference cell (e.g., a corresponding cell that has not been modified to overexpress the c-Jun protein and have reduced expression levels of NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins). at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, or at least about 100-fold or more increase.

[0224] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein) exhibit increased cell expansion and / or cell proliferation compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to overexpress the c-Jun protein and to reduce levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). In some embodiments, cell expansion and / or cell proliferation in the modified immune cells is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, or at least about 100 fold or more compared to that of the reference immune cells.

[0225] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein) exhibit extended persistence and / or survival compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to overexpress the c-Jun protein and to reduce levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). In some embodiments, persistence and / or survival in the modified immune cells is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, or at least about 100 fold or more compared to that of the reference immune cells.

[0226] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein) exhibit increased anti-tumor activity compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to overexpress the c-Jun protein and to reduce levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). In some embodiments, the anti-tumor activity in the engineered immune cells is increased by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, or at least about 100 fold or more relative to that of the reference immune cells.

[0227] In some aspects, the modified immune cells disclosed herein (e.g., CAR- or TCR-expressing cells described herein) exhibit reduced exhaustion or dysfunction compared to a reference immune cell (i.e., a corresponding immune cell that has not been modified to overexpress the c-Jun protein and to reduce levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein). In some aspects, the exhaustion or dysfunction in the modified immune cell is reduced by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the reference immune cell.

[0228] 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 some embodiments, the methods of treating tumors disclosed herein comprise administering to a subject the modified cells of the present disclosure in combination with one or more additional therapeutic agents. Such agents can include, for example, chemotherapeutic agents, targeted anti-cancer therapies, oncolytic agents, cytotoxic agents, immune-based therapies, cytokines, surgery, radiation therapy, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some embodiments, the modified cells disclosed herein (i.e., overexpressing c-Jun protein and having reduced expression levels of NR4A1, NR4A2, and / or NR4A3 genes and / or proteins) can be used in combination with standard therapies (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.

[0229] In some embodiments, the modified cells of the present disclosure can be used in combination with one or more anti-cancer agents so that multiple components of the immune pathway can be targeted. 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), negative immune modulation, e.g., by inhibiting the CTLA-4 and / or PD1 / PD-L1 / PD-L2 pathways and / or T reg or other immunosuppressive cells (e.g., myeloid-derived suppressor cells) by depleting or blocking them; stimulating positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathways and / or stimulate T cell effector function; systemically increasing the frequency of anti-tumor T cells; reg , e.g., tumor T regThese include therapies that deplete or inhibit CD25, for example, using CD25 agonists (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion, therapies that affect the function of suppressor myeloid cells contained in the tumor, therapies that increase the immunogenicity of tumor cells (e.g., anthracyclines), adoptive T cell or NK cell transfer, including genetically engineered cells, for example, cells modified with chimeric antigen receptors (CAR-T therapy), therapies that inhibit metabolic enzymes, for example, 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 stimulatory cytokines, blockade of immune suppressive cytokines, or any combination thereof.

[0230] In some embodiments, the anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through a specific immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods 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).

[0231] In some embodiments, the anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through a specific immune checkpoint pathway). In some 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), a TIM3 agonist (e.g., an anti-TIM3 antibody), or a combination thereof.

[0232] In some aspects, the modified cells disclosed herein are administered to the subject before or after administration of the additional therapeutic agent. In some aspects, the modified cells are administered to the subject simultaneously with the additional therapeutic agent. In some aspects, the modified cells and the additional therapeutic agent may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier. In some aspects, the modified cells and the additional therapeutic agent are administered simultaneously as separate compositions. In some aspects, the additional therapeutic agent and the modified immune cells are administered sequentially.

[0233] IV. Methods for Producing Modified Immune Cells The present disclosure provides methods for generating or preparing cells that overexpress c-Jun protein and have reduced levels of NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins, the methods comprising, for example, (i) modifying the cells with a gene editing tool, wherein the gene editing tool reduces expression of the NR4A gene and / or protein, and (ii) modifying the cells to overexpress c-Jun protein. In some embodiments, the cells can be modified by transducing the cells with a polynucleotide comprising a nucleotide sequence that expresses c-Jun protein. As described herein, in some embodiments, the cells can be modified with a transcriptional activator capable of increasing expression of endogenous c-Jun protein. In some embodiments, the NR4A gene and / or protein comprises NR4A1 and / or NR4A1 protein. In some embodiments, the NR4A gene and / or protein comprises NR4A2 and / or NR4A2 protein. In some embodiments, the NR4A genes and / or proteins comprise NR4A3 and / or NR4A3 proteins. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein. In some aspects, increased expression of c-Jun protein in combination with decreased expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins synergistically reduces or inhibits the exhaustion of the cells.

[0234] Accordingly, the present disclosure also provides a method of reducing or inhibiting exhaustion of a cell expressing a ligand-binding protein (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)), the method comprising modifying the cell to have a reduced expression level of an NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and to overexpress a c-Jun protein. In some aspects, the cell is an immune cell. The present disclosure also provides a method of promoting the persistence of effector function in an immune cell, the method comprising modifying the cell to have a reduced expression level of an NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and to overexpress a c-Jun protein. In some aspects, the NR4A gene and / or protein comprises NR4A1 and / or NR4A1 protein. In some aspects, the NR4A gene and / or protein comprises NR4A2 and / or NR4A2 protein. In some embodiments, the NR4A genes and / or proteins comprise NR4A3 and / or NR4A3 proteins. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A1 gene and / or protein and the NR4A2 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A1 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise both the NR4A2 gene and / or protein and the NR4A3 gene and / or protein. In some embodiments, the NR4A genes and / or proteins comprise the NR4A1 gene and / or protein, the NR4A2 gene and / or protein, and the NR4A3 gene and / or protein.

[0235] Gene editing, e.g., base editing, can be performed using any editing tool known in the art. For example, in some embodiments, modified cells (e.g., immune cells) can be modified using techniques such as CRISPR / Cas, TALEN, zinc finger nucleases (ZFNs), meganucleases, restriction endonucleases, interfering RNA (RNAi), or antisense oligonucleotides. In some embodiments, NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or expression can also be modified using shRNA, siRNA, or miRNA. All of these techniques are described in more detail below. In some embodiments, the method used to reduce the expression of NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins includes using one or more gene editing tools (e.g., two, three, or more tools). In some embodiments, methods used to reduce expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein include at least one method acting on NR4A DNA (e.g., CRISPR) or RNA (e.g., antisense oligonucleotides) and at least one method acting on the NR4A protein (e.g., inhibition of binding to a cell signaling partner or post-translational modification).

[0236] In some aspects, cells (e.g., immune cells) modified to reduce or disable NR4A (NR4A1, NR4A2, and / or NR4A3) gene levels, e.g., using gene editing tools as disclosed herein, can be further modified to express a ligand binding protein (e.g., a CAR or TCR). Thus, in some aspects, methods of preparing immune cells described herein include modifying immune cells with (i) a gene editing tool (e.g., capable of specifically targeting one or more members of the NR4A family), (ii) a nucleotide sequence encoding a c-Jun protein, and (iii) a nucleotide sequence encoding a ligand binding protein (e.g., a CAR or TCR). In some aspects, methods of preparing immune cells described herein include modifying immune cells with (i) a gene editing tool (e.g., capable of specifically targeting one or more members of the NR4A family), (ii) a transcriptional activator capable of increasing endogenous expression of c-Jun, and (iii) a nucleotide sequence encoding a ligand binding protein (e.g., a CAR or TCR). As described herein, in some embodiments, the gene editing tool comprises a guide RNA that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:94, and SEQ ID NO:96 (see, e.g., Tables A, C, and D). Non-limiting examples of other gene editing tools that can be used are further described elsewhere in this disclosure.

[0237] In some embodiments, a nucleotide sequence encoding a c-Jun protein that can be used with the above-described methods includes any of the c-Jun nucleotide sequences provided herein. For example, in some embodiments, the nucleotide sequence encoding the c-Jun protein is (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8. (c) a nucleic acid sequence having 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 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10; (d) a nucleic acid sequence having at least 79%, at least about 99%, at least about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11. (e) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12; %, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13; (f) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14;(h) a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15; or (i) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. ,

[0238] In some embodiments, immune cells modified according to the gene editing methods disclosed herein to express a CAR or TCR may have improved anti-cancer properties, non-limiting examples of which are described elsewhere in this disclosure.

[0239] While the methods for reducing expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein, e.g., gene editing, are provided in the context of CAR- or TCR-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 NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein. For example, in some embodiments, the methods for reducing expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein disclosed herein can be applied to immune cells. In some 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 some embodiments, the lymphocytes are T cells, e.g., CD4 + T cells or CD8 + In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes (TILs). In some embodiments, the TILs are CD8 + In some embodiments, the TILs are CD4 +TILs. Thus, the present disclosure provides cell compositions comprising modified cells (e.g., modified immune cells, the parent cells of which are, for example, any of the cells disclosed above) prepared according to the methods disclosed herein for reducing expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and overexpressing c-Jun protein, wherein the modified cells exhibit reduced expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and overexpression of c-Jun protein relative to reference cells (e.g., corresponding cells that have not been modified to reduce the expression levels of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein and to overexpress the c-Jun protein). In some aspects, these modified cells can be used to prepare pharmaceutical compositions.

[0240] In some aspects, modifying a cell described herein comprises (i) contacting the cell with a gene editing tool capable of reducing the expression level of an NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein in the cell, and (ii) contacting the cell with a polynucleotide comprising a nucleotide sequence encoding a c-Jun protein. In some aspects, modifying a cell described herein comprises (i) contacting the cell with a gene editing tool capable of reducing the expression level of an NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein in the cell, and (ii) contacting the cell with a transcriptional activator capable of increasing endogenous expression of c-Jun. In some embodiments, contacting the gene editing tool (or any other tool capable of reducing expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein) with the cell to be modified can occur in vivo, in vitro, ex vivo, or a combination thereof. In some embodiments, the contacting occurs in vivo (e.g., gene therapy). In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs ex vivo. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is a heterologous cell.In some embodiments, contacting the cell with the genetic tool (or any other tool capable of reducing expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein) reduces the expression level of the NR4A gene and / or protein in the cell by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, 20-fold, 30-fold, 40-fold, or at least 50-fold compared to the level of the NR4A gene and / or protein in a reference cell (e.g., a corresponding cell that has not been modified to reduce the expression level of the NR4A gene and / or NR4A protein).

[0241] In some embodiments, contacting a cell with a gene editing tool comprises various delivery routes. Generally, for the gene editing tools disclosed herein to reduce the expression of NR4A (NR4A1, NR4A2, and / or NR4A3) genes and / or proteins in a 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. For example, 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.

[0242] In some embodiments, a gene editing tool can mutate a gene encoding an NR4A (NR4A1, NR4A2, and / or NR4A3) protein to abolish expression of the functional protein. In some embodiments, a gene editing tool can remove the entire gene encoding an NR4A1, NR4A2, and / or NR4A3 protein, thereby abolishing expression of the protein. In some embodiments, a gene editing tool removes a portion (e.g., one or more exons) of a gene encoding an NR4A (NR4A1, NR4A2, and / or NR4A3) protein. In some embodiments, a gene editing tool, e.g., a base editor, modifies a specific nucleotide base without generating an indel. As used herein, the term "indel" refers to the insertion or deletion of a nucleotide base in a nucleic acid, which can result in a frameshift mutation within the coding region of a gene. Non-limiting examples of base editors are disclosed in U.S. Publication No. 2017 / 0121693, published May 4, 2017, which is incorporated herein by reference in its entirety.

[0243] In some embodiments, the method of preparing a modified immune cell described herein further comprises modifying the cell to express a ligand binding protein (e.g., a CAR or TCR). In some embodiments, the cell is further modified to express a CAR. In some embodiments, the cell is further modified to express a TCR (e.g., an engineered TCR). In some embodiments, modifying the cell to express a ligand binding protein (e.g., a CAR or TCR) comprises contacting the cell with a nucleic acid sequence encoding the ligand binding protein (e.g., a CAR or TCR). In some embodiments, the nucleic acid sequence encoding the ligand binding protein (e.g., a CAR or TCR) is expressed from a vector (e.g., an expression vector). In some embodiments, the vector may further comprise a nucleotide sequence encoding an additional protein of interest (e.g., a c-Jun protein).

[0244] In some aspects, the gene editing tool disclosed herein is expressed from a vector comprising a nucleic acid sequence encoding the gene editing tool. In some aspects, the nucleic acid sequence encoding the gene editing tool, the nucleic acid sequence encoding a c-Jun protein, and the nucleic acid sequence encoding the ligand binding protein (e.g., a CAR or TCR) are on separate vectors. In some aspects, the nucleic acid sequence encoding the gene editing tool and the nucleic acid sequence encoding the ligand binding protein (e.g., a CAR or TCR) are on the same vector. In some aspects, the nucleic acid sequence encoding the gene editing tool and the nucleic acid sequence encoding the c-Jun protein are on the same vector. In some aspects, the nucleic acid sequence encoding the c-Jun protein and the nucleic acid sequence encoding the ligand binding protein (e.g., a CAR or TCR) are on the same vector. In some aspects, the nucleic acid sequence encoding the gene editing tool, the nucleic acid sequence encoding the c-Jun protein, and the nucleic acid sequence encoding the ligand binding protein are all on the same vector.

[0245] IV.A. Gene Editing Tools One or more gene editing tools can be used to modify the cells of the present disclosure. Non-limiting examples of gene editing tools are disclosed below.

[0246] IV.A.1. CRISPR / Cas Systems In some embodiments, gene editing tools that can be used in the present disclosure include CRISPR / Cas systems. Such systems can, for example, use nucleic acid molecules encoding Cas9 nuclease, which are optionally codon-optimized for the desired cell type in which they are expressed (e.g., T cells, e.g., CAR-expressing or engineered TCR-expressing T cells). As further described herein, in some embodiments, such systems can include Cas9 nuclease proteins.

[0247] CRISPR / Cas system uses Cas nuclease, for example, Cas9 nuclease, which targets genome site by forming a complex with guide RNA (for example, synthetic guide RNA) (gRNA), which hybridizes with the target DNA sequence immediately before the NGG motif recognized by the Cas nuclease, for example, Cas9.This causes a double-strand break 3 nucleotide upstream of the NGG motif.The unique ability of CRISPR / Cas9 system is that it can simultaneously target multiple separate genome loci by co-expressing a single Cas9 protein with two or more gRNAs (for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 gRNAs).This system can also use guide RNA that comprises two separate molecules. In some 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.

[0248] A crRNA contains both the DNA-targeting segment (single strand) of the gRNA and a stretch of nucleotides that form 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 form 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 can be said to have a corresponding tracrRNA. The crRNA further provides a single-stranded DNA-targeting segment. Thus, the gRNA contains a sequence that hybridizes to the target sequence (e.g., NR4A1, NR4A2, and / or NR4A3 mRNA) and the tracrRNA. Thus, the crRNA and tracrRNA hybridize (as a corresponding 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.

[0249] Naturally occurring genes encoding the three elements (Cas9, tracrRNA, and crRNA) are typically organized into operons. 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 with it, and then bind to the Cas9 protein.

[0250] Alternatively, the CRISPR system used herein may further utilize a fusion crRNA-tracrRNA construct (i.e., a single transcript) that functions with codon-optimized Cas9. This single RNA is often referred to as a 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), a form of tracrRNA sequence (scaffold), and an appropriate promoter active in the cell and elements necessary for proper processing in eukaryotic cells. Many of these systems rely on custom-made complementary oligos that anneal to form double-stranded DNA, which is then cloned into the gRNA expression plasmid.

[0251] The gRNA expression cassette and the Cas9 expression cassette are then 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 herein by reference in its entirety.

[0252] In some embodiments, the Cas9 nuclease may be provided in the form of a protein. For example, in some embodiments, cells useful in the present disclosure (e.g., CAR- or TCR-expressing immune cells) may be modified (e.g., to reduce the level of the NR4A gene and / or NR4A protein) by introducing a Cas9 nuclease protein and a nucleic acid molecule comprising a gRNA. In some embodiments, the Cas9 nuclease protein and the nucleic acid molecule comprising the gRNA may be introduced into the cell sequentially. In some embodiments, the Cas9 nuclease protein and the nucleic acid molecule comprising the gRNA may be introduced into the cell simultaneously. For example, in some embodiments, the simultaneous administration comprises introducing the Cas9 nuclease protein and the nucleic acid molecule comprising the gRNA simultaneously as separate compositions. In some embodiments, the Cas9 protein may be provided in the form of a complex with the nucleic acid molecule comprising the gRNA (i.e., as a single composition).

[0253] In some embodiments, the Cas9 nuclease can be provided in the form of a nucleic acid encoding the protein. Thus, in some embodiments, cells useful in the present disclosure (e.g., CAR- or TCR-expressing immune cells) can be modified (e.g., to reduce levels of the NR4A gene and / or protein) by introducing a first nucleic acid molecule encoding a Cas9 nuclease protein and a second nucleic acid molecule comprising a gRNA. In some embodiments, the first and second nucleic acid molecules can be introduced into the cell sequentially. In some embodiments, the first and second nucleic acid molecules can be introduced into the cell simultaneously. For example, in some embodiments, the first and second nucleic acid molecules can be introduced into the cell simultaneously as separate compositions. In some embodiments, the first and second nucleic acid molecules can be part of a single polynucleotide, and the cell is modified to contain the single polynucleotide.

[0254] The nucleic acid encoding the Cas9 nuclease can be RNA (e.g., messenger RNA (mRNA)) or DNA. In some embodiments, the gRNA can be provided in the form of RNA. In some embodiments, the gRNA can be provided in the form of DNA encoding the RNA. In some embodiments, the gRNA can be provided in the form of separate crRNA and tracrRNA molecules, or separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0255] 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 some embodiments, the Cas protein is a type I Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the type II Cas protein is Cas9. In some embodiments, the type II Cas, e.g., Cas9, is a human codon-optimized Cas.

[0256] In some embodiments, the Cas protein is a "nickase" that can create a single-strand break (i.e., a "nick") in a target nucleic acid sequence without cleaving both strands of double-stranded DNA (dsDNA). Cas9, for example, contains two nuclease domains, a RuvC-like nuclease domain and an HNH-like nuclease domain, which are involved in cleaving opposing DNA strands. Mutations in either of these domains can create a nickase. Examples of mutations that create nickases can be found, for example, in WO / 2013 / 176772A1 and WO / 2013 / 142578A1, each of which is incorporated herein by reference.

[0257] In some embodiments, two separate 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 separate 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, a first nickase can create a single-stranded break in the first strand of dsDNA, and a second nickase can create a single-stranded break in the second strand of dsDNA, resulting in the creation of an overhanging sequence. The target sites of each nickase that create the single-stranded break can be selected so that the sequence of the created overhanging end is complementary to the sequence of the overhanging end of 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 first strand nickase is different from the target site for the second strand nickase.

[0258] In some embodiments, the expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) genes and their encoded NR4A proteins is reduced by contacting the cell with, for example, a CRISPR (e.g., a CRISPR-Cas9 system) specific for the NR4A (NR4A1, NR4A2, and / or NR4A3) genes. In some embodiments, the CRISPR is specific for the NR4A1 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) (i) has reduced levels of the NR4A1 gene and / or protein, (ii) has endogenous levels of the NR4A2 gene and / or protein, and (iii) has endogenous levels of the NR4A3 gene and / or protein. In some embodiments, the CRISPR is specific for the NR4A2 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) (i) has endogenous levels of the NR4A1 gene and / or protein, (ii) has reduced levels of the NR4A2 gene and / or protein, and (iii) has endogenous levels of the NR4A3 gene and / or protein. In some embodiments, the CRISPR is specific for the NR4A3 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) (i) has endogenous levels of the NR4A1 gene and / or protein, (ii) has endogenous levels of the NR4A2 gene and / or protein, and (iii) has reduced levels of the NR4A3 gene and / or protein.

[0259] As described herein, in some embodiments, the CRISPR targets multiple NR4A genes.For example, in some embodiments, the CRISPR can target both the NR4A1 gene and the NR4A2 gene.Therefore, in some embodiments, after contacting with the CRISPR, the cell (for example, CAR or TCR expressing immune cell) (i) reduces the level of the NR4A1 gene and / or protein, (ii) reduces the level of the NR4A2 gene and / or protein, and (iii) has endogenous level of the NR4A3 gene and / or protein.In some embodiments, the CRISPR can target both the NR4A1 gene and the NR4A3 gene. Thus, in some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) (i) has a reduced level of the NR4A1 gene and / or protein, (ii) has endogenous levels of the NR4A2 gene and / or protein, and (iii) has a reduced level of the NR4A3 gene and / or protein. In some embodiments, the CRISPR can target both the NR4A2 gene and / or the NR4A3 gene. In some embodiments, after contacting with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) (i) has endogenous levels of the NR4A1 gene and / or protein, (ii) has a reduced level of the NR4A2 gene and / or protein, and (iii) has a reduced level of the NR4A3 gene and / or protein. In some embodiments, the CRISPR can target the NR4A1 gene, the NR4A2 gene, and the NR4A3 gene. Thus, in some embodiments, after contact with the CRISPR, the cell (e.g., a CAR- or TCR-expressing immune cell) exhibits (i) a reduced level of the NR4A1 gene and / or protein, (ii) a reduced level of the NR4A2 gene and / or protein, and (iii) a reduced level of the NR4A3 gene and / or protein.

[0260] In some embodiments, gene editing using CRISPR reduces the level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% relative to the NR4A (NR4A1, NR4A2, and / or NR4A3) gene level observed in a reference cell (e.g., a corresponding cell that has not been subjected to gene editing using CRISPR). In some embodiments, the CRISPR completely disables expression of NR4A (NR4A1, NR4A2, and / or NR4A3) in the immune cell. In some embodiments, the level of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene can be measured using any technique known in the art, for example, digital droplet PCR.

[0261] In some embodiments, the nucleic acids encoding the gRNAs and / or Cas9s disclosed herein are RNA or DNA. In some embodiments, the RNAs or DNAs encoding the gRNAs and / or Cas9s disclosed herein are synthetic RNAs or DNAs, respectively. In some embodiments, the synthetic RNAs or DNAs comprise at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with unnatural nucleobases, such as 5-methoxyuridine or pseudouridine). In some embodiments, the polynucleotides (e.g., synthetic RNAs or DNAs) comprise only natural nucleobases, i.e., A, C, T, and U in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA or DNA. Generally, the CRISPR gene editing methods disclosed herein include contacting a cell, e.g., an immune cell, in vivo, in vitro, or ex vivo with (i) Cas9 or a nucleic acid encoding the Cas9, and (ii) a guide RNA (gRNA) for at least one NR4A (NR4A1, NR4A2, or NR4A3) gene or a nucleic acid encoding the gRNA, wherein the gRNA targets a sequence (e.g., an intron and / or exon sequence) contained in the NR4A gene, and contacting the cell with the Cas9 and at least one gRNA reduces expression of the NR4A (NR4A1, NR4A2, or NR4A3) gene and / or protein.

[0262] In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of any one or more of the sequences set forth in SEQ ID NOs: 30, 52-57, 58, 61, 65, 67, 68, 70, 71, 75, 76, 82, 83, 86, 94, and 96. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 30. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 30. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 52. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 52. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 52. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 53. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 53. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 53. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO:54.In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 54. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 54. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 55. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 55. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 56. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 56. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 56. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 57. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 57. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 57.In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 58. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 58. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 61. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 61. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 61. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 65. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 65. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 65. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 67. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 67. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 67.In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 67. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 68. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 68. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 68. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 70. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 70. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 70. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 70. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 71. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 71. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 71. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 75. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 75.In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 75. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 75. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 76. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 76. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 76. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 82. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 82. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 82. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 82. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 83. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 83. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 83. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 86.In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 86. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 86. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 86. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 94. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 94. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 94. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 94. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 96. In some embodiments, a gRNA that can be used to target the NR4A3 gene comprises the sequence set forth in SEQ ID NO: 96. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists of the sequence set forth in SEQ ID NO: 96. In some embodiments, a gRNA that can be used to target the NR4A3 gene consists essentially of the sequence set forth in SEQ ID NO: 96.

[0263] As described herein, in some embodiments, the gene editing method may further comprise reducing the levels of (i) the NR4A1 gene and / or NR4A1 protein, (ii) the NR4A2 gene and / or NR4A2 protein, or (iii) both (i) and (ii). In some embodiments, a gRNA that can be used to target the NR4A1 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 25. In some embodiments, a gRNA that can be used to target the NR4A1 gene comprises the sequence set forth in SEQ ID NO: 25. In some embodiments, a gRNA that can be used to target the NR4A1 gene consists essentially of the sequence set forth in SEQ ID NO: 25. In some embodiments, a gRNA that can be used to target the NR4A1 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA that can be used to target the NR4A1 gene comprises the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA that can be used to target the NR4A1 gene consists of the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA that can be used to target the NR4A1 gene consists essentially of the sequence set forth in SEQ ID NO: 26. In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 27. In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, a gRNA that can be used to target the NR4A2 gene consists essentially of the sequence set forth in SEQ ID NO: 27. In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 28.In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, a gRNA that can be used to target the NR4A2 gene consists of the sequence set forth in SEQ ID NO: 28. In some embodiments, a gRNA that can be used to target the NR4A2 gene consists essentially of the sequence set forth in SEQ ID NO: 28. In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises, consists of, or consists essentially of the sequence set forth in SEQ ID NO: 29. In some embodiments, a gRNA that can be used to target the NR4A2 gene comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, a gRNA that can be used to target the NR4A2 gene consists of the sequence set forth in SEQ ID NO: 29. In some embodiments, a gRNA that can be used to target the NR4A2 gene consists essentially of the sequence set forth in SEQ ID NO: 29.

[0264] As used herein, the term "contacting" (e.g., contacting a cell, e.g., an immune cell, with at least one gRNA and at least one Cas9) is intended to include incubating at least one gRNA and at least one Cas protein, e.g., Cas9, together in a cell in vitro (e.g., adding the gRNA and / or Cas protein, or nucleic acid(s) encoding the gRNA(s) and / or Cas9 protein(s), to a cell in culture), or contacting a cell in vivo or ex vivo.

[0265] The step of contacting the target sequence of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene with at least one gRNA and at least one Cas protein, e.g., Cas9 (or at least one nucleic acid sequence encoding them), as disclosed herein, can be carried out in any suitable manner. For example, the cells, e.g., immune cells, can be treated in cell culture conditions. Cells contacted with at least one gRNA and at least one Cas protein, e.g., Cas9 protein (or at least one nucleic acid sequence encoding them), as disclosed herein, can also be contacted simultaneously or sequentially with another agent, e.g., a vector comprising at least one nucleic acid sequence encoding a CAR or TCR. In some embodiments, after contacting the cells in vitro or ex vivo, the method further comprises introducing the cells into the subject, thereby treating or ameliorating symptoms of a disease or condition, e.g., cancer.

[0266] For ex vivo methods, the cells may comprise autologous cells, i.e., immune cell(s) harvested from a subject in whom the target polynucleotide sequence (e.g., the NR4A (NR4A1, NR4A2, and / or NR4A3) gene) contained in the immune cell(s) needs to be modified (i.e., the donor and recipient are the same individual). Autologous cells have the advantage of avoiding any immunologically based rejection of the cells. Alternatively, the cells may be xenogeneic, e.g., harvested from a donor. Typically, when the cells are derived from a donor, they are derived from a donor that is sufficiently immunologically compatible with the recipient, i.e., they are not subject to graft rejection and the need for immunosuppression is reduced or eliminated. In some embodiments, the cells are harvested from a xenogeneic source, i.e., from the recipient or a non-human mammal that has been genetically engineered to be sufficiently immunologically compatible with the recipient's species. Methods for determining immunological compatibility are known in the art and include tissue typing to assess donor-recipient compatibility with respect to HLA and ABO determinants. See, e.g., Transplantation Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

[0267] In some aspects, the present disclosure provides methods for generating modified immune cells, comprising modifying the NR4A (NR4A1, NR4A2, and / or NR4A3) gene sequence in a cell, e.g., an immune cell (e.g., a T cell), by ex vivo contacting the NR4A gene sequence in the cell with a Cas9 protein (or a nucleic acid encoding such a Cas9 protein) that targets a motif in the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and a gRNA (e.g., a motif located in exons 3 and 4 of NR4A3), where the gRNA directs the Cas9 protein to the target gene, hybridizes to the target motif, and cleaves the NR4A gene partially or completely, with an efficiency of about 10% to about 100%. Non-limiting examples of such gRNAs are provided herein (see, e.g., Tables A, C, and D). As described herein, in some embodiments, methods of generating modified immune cells described herein include altering the NR4A gene sequence by contacting the cell with a first nucleic acid molecule encoding the Cas9 protein and a second nucleic acid molecule comprising a gRNA that targets one or more members of the NR4A gene family. In some embodiments, the first and second nucleic acid molecules are contacted with the cell sequentially. In some embodiments, the first and second nucleic acid molecules are contacted with the cell simultaneously. For example, in some embodiments, the cell is contacted with a single polynucleotide comprising a first nucleic acid molecule encoding the Cas9 protein and a second nucleic acid molecule comprising the gRNA.

[0268] In some embodiments, the cells have been modified (e.g., transfected) with a nucleic acid (e.g., a vector) encoding a ligand-binding protein (e.g., a CAR or TCR) before, after, or simultaneously with the above-described modifying step. Additionally, as further described elsewhere in this disclosure, in some embodiments, the cells have been modified to increase levels of c-Jun protein before, after, or simultaneously with the above-described modifying step.

[0269] In some embodiments, the efficiency of the cleavage is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0270] The CRISPR / Cas system of the present disclosure can use gRNA spacer sequences of various lengths depending on the Cas used, e.g., Cas9. Cas9s from different species must pair with their corresponding gRNAs to form functional ribonucleoprotein (RNP) complexes; in other words, chimeric gRNA frames engineered from different bacterial species may have different lengths due to differences in spacer and chimeric frame sequences.

[0271] In some embodiments, the gRNA spacer sequence can be at least about 18 nucleotides (e.g., about 18, about 19, about 20, about 21, or about 22 nucleotides) in length. For example, the S. pyogenes gRNA spacer sequence contained in a gRNA that binds to S. pyogenes Cas9 is 20 nucleotides in length, while the S. aureus gRNA spacer sequence contained in a gRNA that binds to S. aureus Cas9 is 21 nucleotides in length. In some embodiments, the gRNA spacer sequence can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0272] Although a perfect match between the gRNA spacer sequence and the DNA strand to which it binds on the NR4A (NR4A1, NR4A2, and / or NR4A3) gene is preferred, mismatches between the gRNA spacer sequence and the NR4A target sequence are also acceptable as long as they result in reduced NR4A gene levels or reduced NR4A gene function. A "seed" sequence of about 8 to about 12 contiguous nucleotides on the gRNA that is perfectly complementary to the target NR4A sequence is preferred for proper recognition of the target sequence of the NR4A gene. The remainder of the gRNA spacer sequence can contain one or more mismatches.

[0273] Generally, gRNA activity is inversely correlated with the number of mismatches. Preferably, the spacer sequence of the gRNA of the present disclosure contains less than about 7 mismatches. In some embodiments, the spacer sequence of the gRNA contains 7, 6, 5, 4, 3, more preferably 2 or fewer, and even more preferably no mismatches with the corresponding target sequence of the NR4A gene. The fewer the number of nucleotides in the gRNA, the fewer the number of mismatches that can be tolerated. It is believed that binding affinity depends on the total number of matching gRNA-DNA combinations.

[0274] The spacer sequence of the gRNA of the present disclosure can be selected to minimize off-target effects of the CRISPR / Cas editing system. Thus, in some embodiments, the spacer sequence of the gRNA is selected to contain at least two mismatches when compared with all other genomic nucleotide sequences contained in the cell. In some embodiments, the spacer sequence of the gRNA is selected to contain at least one mismatch when compared with all other genomic nucleotide sequences contained in the cell. Those skilled in the art will understand that various techniques (e.g., bioinformatics analysis) can be used to select an appropriate spacer sequence of the gRNA to minimize off-target effects.

[0275] In some embodiments, the spacer sequence of the gRNA comprises, consists of, or consists essentially of the spacer sequence of SEQ ID NOs: 31-42.

[0276] In some embodiments, the spacer sequence of the gRNA comprises, consists of, or consists essentially of a spacer sequence that contains a mismatch of at least 1, 2, 3, 4, or 5 nucleotides compared to the DNA sequence of any one of SEQ ID NOs: 31-42.

[0277] In some embodiments, the effect of editing can be increased by targeting multiple locations.

[0278] In some embodiments, the two gRNAs are complementary to and / or hybridize with sequences on the same strand of the NR4A gene. In some embodiments, the two gRNAs are complementary to and / or hybridize with sequences on opposite strands of the NR4A gene. In some embodiments, the two gRNAs are not complementary to and / or hybridize with sequences on opposite strands of the NR4A gene. In some embodiments, the two gRNAs are complementary to and / or hybridize with overlapping target motifs of the NR4A gene. In some embodiments, the two gRNAs are complementary to and / or hybridize with offset target motifs of the NR4A gene.

[0279] In general, a gRNA of the present disclosure can include any variant or chemical modification of its sequence, so long as it allows the binding of a corresponding Cas protein, e.g., a Cas9 protein, to the target sequence and subsequent removal (in whole or in part) of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene.

[0280] The Cas proteins used in the methods disclosed herein, such as Cas9, are nucleic acid-cleaving endonucleases encoded by CRISPR loci in the genomes of many bacteria and involved in type II CRISPR systems. Cas9 proteins are produced by many species of bacteria, including Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Neisseria meningitidis, and others. Thus, Cas9 proteins useful in the present disclosure can be derived from any suitable bacterium known in the art. Non-limiting examples of such bacteria include Streptococcus pyogenes, Streptococcus mutans, Streptococcus pneumonia, Streptococcus aureus, Streptococcus thermophilus, Campylobacter jejuni, Neisseria meningitidis, Pasteurella multocida, Listeria innocua, and Francisella novicida. The methods disclosed herein can be performed with any Cas9 known in the art. In some embodiments, the Cas9 is a wild-type Cas9. In some embodiments, the Cas9 is a mutant Cas9 with improved enzymatic activity or a fusion protein comprising a Cas9 portion. In some embodiments, the Cas9 nuclease protein is a Streptococcus pyogenes Cas9 protein.

[0281] Because Cas9 nuclease proteins are typically expressed in bacteria, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing Cas9 recombinant proteins. Thus, in some embodiments, nucleic acids encoding Cas9 used in the methods disclosed herein are codon-optimized for expression in eukaryotic cells, e.g., in the cells of a human subject in need thereof.

[0282] In some embodiments, the Cas9 protein used in the methods disclosed herein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitutions and / or modifications can prevent or reduce proteolysis and / or extend the half-life of the polypeptide in cells. In some embodiments, the Cas9 protein can comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas9 protein can comprise naturally occurring amino acids. In some embodiments, the Cas9 protein can comprise alternative amino acids (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas9 protein can comprise modifications to include heterologous moieties (e.g., pegylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0283] While the methods disclosed herein are generally performed using a Cas9 protein, it is contemplated that in some embodiments, the Cas protein may be Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, or Cas8. In some embodiments, the Cas protein is a Cas9 protein or functional portion thereof from any bacterial species. In some specific embodiments, the Cas9 protein used in the methods disclosed herein is a Streptococcus pyogenes or Staphylococcus aureus Cas9 protein or functional portion thereof, or a nucleic acid encoding such a Cas9 or functional portion thereof. Non-limiting examples of other Cas nucleases that can be used are known in the art and are described, for example, in US9,970,001B2, US10,221,398B2, and US2020 / 0190487A1, each of which is incorporated herein by reference in its entirety. In some embodiments, Cas nucleases useful in the present disclosure comprise type I Cas proteins. Non-limiting examples of type I Cas proteins include Cas3, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and variants thereof. In some embodiments, Cas nucleases useful in the present disclosure comprise type II Cas proteins. Non-limiting examples of type II Cas proteins include Cas9, Csn2, Cas4, and variants thereof. In some embodiments, Cas nucleases useful in the present disclosure comprise type III Cas proteins. Non-limiting examples include Cas10, Csm2, Cmr5, Csx10, Csx11, and variants thereof. In some embodiments, Cas nucleases useful in the present disclosure comprise type IV Cas proteins. Non-limiting examples of such Cas proteins include Csf1. In some embodiments, Cas nucleases useful in the present disclosure comprise V-type Cas proteins.Non-limiting examples include Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14, C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), C2c4, C2c8, C2c9, and variants thereof. In some embodiments, Cas nucleases useful in the present disclosure comprise Type VI Cas proteins. Non-limiting examples of Type VI Cas proteins include Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, and variants thereof.

[0284] In some cases, Cas proteins useful in the present disclosure include orthologs or homologs of the above-described Cas proteins. The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. By way of further guidance, as used herein, a "homolog" of a protein is a protein of the same species that performs the same or similar function as its homologous protein. Homologous proteins may be, but are not required to be, structurally related, or are only partially structurally related. As used herein, an "ortholog" of a protein is a protein of a different species that performs the same or similar function as its orthologous protein. Orthologous proteins may be, but are not required to be, structurally related, or are only partially structurally related.

[0285] As used herein, "functional portion" refers to a peptide, e.g., a portion of Cas9, that retains the ability to form a complex with at least one gRNA, cleave a target sequence, and result in reduced expression of the NR4A (NR4A1, NR4A2, and / or NR4A3) gene and / or protein. In some embodiments, the functional portion comprises a combination of operably linked functional domains of the Cas9 protein selected from the group consisting of a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a non-covalent complex. In some embodiments, the functional domains form a fusion complex (e.g., a fusion protein). In some embodiments, the functional domains are chemically linked (e.g., via one or more spacers or linkers). In some embodiments, the functional domains are conjugated.

[0286] It should be understood that the present disclosure contemplates various methods of contacting the NR4A (NR4A1, NR4A2, and / or NR4A3) gene with at least one gRNA and at least one Cas protein, e.g., Cas9. In some embodiments, the exogenous Cas protein, e.g., Cas9, can be introduced into a cell in the form of a polypeptide. In some embodiments, the Cas protein, e.g., Cas9, can be conjugated or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates the uptake of a molecule into a cell. The cell-penetrating polypeptide can include a detectable label.

[0287] In some embodiments, a Cas protein, e.g., Cas9, can be conjugated or fused to a charged protein, e.g., a protein carrying a positive, negative, or overall neutral charge. Such attachment can be covalent. In some embodiments, the Cas protein, e.g., Cas9, can be fused to a peptide with a superpositive charge, significantly enhancing the ability of the Cas protein, e.g., Cas9, to penetrate cells. See Cronican et al. ACS Chem. Biol. 5(8):747-52 (2010). In some embodiments, the Cas protein, e.g., Cas9, can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include, but are not limited to, Tat, oligoarginine, and penetratin. Thus, in some specific embodiments, the methods disclosed herein may be practiced using a Cas protein, e.g., a Cas9 protein, including a Cas protein fused to a cell-penetrating peptide, a Cas protein fused to a PTD, a Cas protein fused to a tat domain, a Cas protein fused to an oligoarginine domain, a Cas protein fused to a penetratin domain, or combinations thereof.

[0288] In some embodiments, the Cas protein, e.g., Cas9, can be introduced into cells, e.g., immune cells, e.g., immune cells that express a CAR or TCR and have increased levels of c-Jun protein, and that contain a target polynucleotide sequence, e.g., the NR4A (NR4A1, NR4A2, and / or NR4A3) gene, in the form of a nucleic acid encoding the Cas protein, e.g., Cas9. The process of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection u...

Claims

1. A cell composition comprising a population of modified immune cells, showing (i) a decrease in the expression level of a nuclear receptor subfamily 4 group A gene and / or protein selected from the group consisting of NR4A member 1 (NR4A1) gene and / or NR4A1 protein, NR4A member 2 (NR4A2) gene and / or NR4A2 protein, and NR4A member 3 (NR4A3) gene and / or NR4A3 protein, and (ii) an increase in the expression level of c-Jun protein.

2. The cell composition according to claim 1, wherein the population of immune cells shows a decrease in the expression of NR4A3 gene and / or NR4A3 protein.

3. The cell composition according to claim 1, wherein the population of modified immune cells comprises lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, or any combination thereof.

4. The cell composition according to claim 3, wherein the lymphocytes include T cells, and the T cells include a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), for example, an engineered TCR.

5. The population of modified immune cells is modified with (1) a gene editing tool that reduces the expression level of the NR4A gene and / or protein, and (2) a nucleotide sequence encoding the c-Jun protein such that the population of modified immune cells shows an increase in the expression of the c-Jun protein, where optionally, (a) the gene editing tool comprises, consists of, or consists essentially of a guide RNA comprising a sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 96; (b) the nucleotide sequence encoding the c-Jun protein is (i) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7, (ii) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8, (iii) a nucleic acid sequence having 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 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10, (iv) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11, (v) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12, (vi) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13, (vii) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14, (viii)a nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15, or (ix)comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16; or (c) The cell composition according to claim 1, which is (a) and (b).

6. A pharmaceutical composition comprising the cell composition according to claim 1 and a pharmaceutically acceptable carrier.

7. The cell composition according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 for use in its treatment in a subject in need of treatment for a tumor.

8. The cell composition or pharmaceutical composition according to claim 7, wherein the tumor is derived from a cancer including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

9. A method for producing cells showing an increase in the expression level of c-Jun protein and a decrease in the level of NR4A gene and / or NR4A protein, comprising modifying said cells in vitro with (i) a nucleotide sequence encoding c-Jun protein and (ii) a gene editing tool, said gene editing tool comprising a guide RNA (gRNA) capable of decreasing the expression of said NR4A gene and / or NR4A protein, said gRNA comprising, consisting essentially of, or consisting of the sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 96, said method. **Claim 10** A method for reducing or inhibiting exhaustion of T cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR), comprising modifying said T cells in vitro such that the expression level of the NR4A gene and / or protein is decreased, and modifying said T cells in vitro such that c-Jun protein is overexpressed, said method. **Claim 11** Modifying the T cells such that the expression level of the NR4A gene and / or protein is decreased includes contacting the T cells with a gene editing tool capable of decreasing the expression level of the NR4A gene and / or protein in the T cells, wherein optionally, the gene editing tool includes a guide RNA (gRNA), and the gRNA includes, consists essentially of, or consists of the sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 96, the method according to claim 10.

12. A method of increasing cytokine production by T cells that express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) in response to antigen stimulation, the method comprising, in vitro, (i) modifying the T cells with a nucleotide sequence encoding the c-Jun protein such that the T cells overexpress the c-Jun protein after modification, and (ii) modifying the T cells with a gene editing tool, the gene editing tool including a guide RNA (gRNA) capable of decreasing the expression of the NR4A gene and / or NR4A protein, and the gRNA including, consisting essentially of, or consisting of the sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 96, the method.

13. A method of increasing the effector function of T cells expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR) in response to continuous antigen stimulation, the method comprising in vitro modifying the T cells with (i) a nucleotide sequence encoding the c-Jun protein such that the T cells overexpress the c-Jun protein after modification, and (ii) a gene editing tool, the gene editing tool comprising a guide RNA (gRNA) capable of reducing the expression of the NR4A gene and / or the NR4A protein, the gRNA comprising, consisting essentially of, or consisting of the sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO:

96.

14. The nucleotide sequence encoding the c-Jun protein is (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7, (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 8, (c) a nucleic acid sequence having 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 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 10, (d) A nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 11, (e) A nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12, (f) A nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 13, (g) A nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 14, (h) A nucleic acid sequence having at least 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15, or (i) The method according to any one of claims 9 to 13, comprising a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

16.

15. A cell composition comprising T cells that (a) express a ligand-binding protein (e.g., CAR or TCR), (b) have an increased level of c-Jun protein, and (b)(i) the expression level of the NR4A1 gene and / or NR4A1 protein, (ii) the NR4A2 gene and / or NR4A2 protein, (iii) the NR4A3 gene and / or NR4A3 protein, or (iv) any combination of (i) to (iii) is decreased, wherein the T cells are modified with a gRNA comprising, consisting of, or consisting essentially of the sequence set forth in any one of SEQ ID NO: 94, SEQ ID NO: 30, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 96.