Compositions and methods for inhibiting T cell exhaustion

Abstract

To provide compositions and methods for inhibiting T cell exhaustion.SOLUTION: The present invention provides compositions comprising isolated T cells, modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-references of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 62 / 738,687, filed September 28, 2018, and U.S. Provisional Patent Application No. 62 / 599,299, filed December 15, 2017, which are incorporated herein by reference in their entirety.

[0002] Field of Invention This invention relates to T cell compositions and methods of use in the context of therapy and treatment. In particular, the invention provides T cells that are modified (e.g., genetically and / or functionally) to maintain functionality under conditions in which unmodified T cells exhibit exhaustion. The compositions and methods disclosed herein find use in preventing exhaustion of modified T cells (e.g., chimeric antigen receptor (CAR) T cells) and unmodified T cells, thereby enhancing T cell function (e.g., activity against cancer or infectious diseases). The compositions and methods of the present invention find use in both clinical and research settings, for example, in the fields of biology, immunology, medicine, and oncology. [Background technology]

[0003] background T cells are immune cells that are activated via T cell receptor (TCR) signaling and co-stimulation after binding to an antigen. Physiological activation via the T cell receptor allows T cells to mediate potent antitumor and / or anti-infective effects. During the resolution of an acute inflammatory response, a subset of activated effector T cells differentiate into long-term survival memory cells. In contrast, in patients with chronic infections or cancer, T cells are not uncommonly subjected to pathological differentiation toward a state of dysfunction known as T cell exhaustion. T cell exhaustion is characterized by significant changes in metabolic function, transcriptional programming, effector function (e.g., cytokine secretion, killing ability), and co-expression of multiple surface inhibitory receptors. The underlying cause of T cell exhaustion is persistent antigen exposure that triggers continuous TCR signaling. Preventing or reversing T cell exhaustion has long been considered a means of enhancing T cell efficacy (e.g., in patients with cancer or chronic infections).

[0004] Chimeric antigen receptor (CAR) T cells exhibit impressive response rates in B-cell malignancies, but long-term disease control occurs in only about 50% of patients with B-ALL1 and large B-cell lymphoma (Reference 2; incorporated herein by reference in its entirety), and even less frequently in CLL (Reference 3; incorporated herein by reference in its entirety). Furthermore, despite numerous studies, CAR T cells have not mediated sustained antitumor effects in solid tumors (Reference 4; incorporated herein by reference in its entirety). The efficacy of CAR T cells is limited by many factors, including the need for heterogeneous antigen expression and high antigen density for optimal CAR function to enable rapid selection of antigen-loss variants (References 5-7; incorporated herein by reference in their entirety), the suppressive tumor microenvironment (Reference 8; incorporated herein by reference in its entirety), and intrinsic T-cell dysfunction as a result of T-cell exhaustion (References 3, 9, 10; incorporated herein by reference in their entirety). T cell exhaustion is increasingly considered a cause of T cell dysfunction in CAR T cells. Sustained antigen-independent signaling for scFv aggregation generally occurs in CAR-expressing T cells and can induce rapid exhaustion (Reference 9; incorporated herein by reference in its entirety). Incorporation of the CD28 endodomain into the second-generation CAR T cell receptor enhances expansion but also makes CAR T cells more susceptible to exhaustion, both in the setting of a persistently signaling receptor and in CD19-28z CAR T cells exposed to high tumor burden (Reference 9; incorporated herein by reference in its entirety). An increased frequency of exhausted T cells in CD19-BBz CAR grafts has recently been shown to differentiate unresponsive and responsive patients treated for CLL3. Broad-based foundational data from diverse studies suggest that intrinsic T cell dysfunction for T cell exhaustion is a major limiting factor in the effectiveness of CAR T cell therapies, and that substantially improving clinical outcomes could be achieved by reconstructing exhaustion-resistant CAR T cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Neelapu, SS et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B- Cell Lymphoma. N Engl J Med 377, 2531-2544, doi:10.1056 / NEJMoa1707447 (2017) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a composition and method for inhibiting T cell exhaustion. [Means for solving the problem]

[0007] overview The present invention relates to compositions and methods for use in preventing exhaustion of modified T cells (e.g., T cells modified to express synthetic receptors such as modified T cell receptors or chimeric antigen receptors (CARs)) and non-modified (e.g., native) T cells. Modified T cells according to the present invention (e.g., to prevent T cell exhaustion), compositions containing them, and methods of use enhance T cell function (e.g., activity against cancer or infectious diseases).

[0008] CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is a significant barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, during the development of the embodiments herein, we conducted experiments using model systems employing sustainedly signaling CARs that induce the exhaustion features described in other settings. The results show that exhaustion was associated with a severe defect in IL-2 production accompanied by increased chromatin accessibility of the AP-1 transcription factor motif, as well as overexpression of many bZIP and IRF transcription factors involved in inhibitory activity. Remodeling CAR T cells to overexpress AP-1 factor (e.g., c-Jun) enhanced growth potential, increased functional capacity, decreased terminal differentiation, and improved antitumor capacity in many in vivo tumor models.

[0009] Experiments conducted in the development of the embodiments described herein further demonstrate that functional deficiency in AP-1 factor (e.g., c-Jun) mediates dysfunction in exhausted human T cells, and that remodeling CAR T cells to overexpress AP-1 factor (e.g., c-Jun) makes T cells more resistant to exhaustion, thereby addressing a major barrier to progress in this new class of therapeutics.

[0010] Experiments conducted during the development of the embodiments described herein further demonstrate that knockdown of IRF4 dramatically increases the functional activity of exhausted HA-28z CAR T cells, that the enhancement of in vivo function of c-Jun-modified HA-28z CAR T cells cannot be replicated by exovivo provision of IL-2, that c-Jun enhances the cellular activity of Her2-BBz CAR T cells in the suppressive solid tumor microenvironment, that c-Jun overexpression increases the resistance of exhausted HA-28z CAR T cells to TGFβ-mediated repression, and that transcriptional changes in c-Jun-modified cells are consistent with reduced exhaustion and increased memory morphology.

[0011] Provided herein are modified T cells (e.g., T cells modified to express a modified T cell receptor or a synthetic receptor such as CAR) and non-modified (e.g., natural, native) T cells, which are modified to overexpress and / or include one or more activator protein 1 (AP-1) transcription factors (e.g., c-Fos, c-Jun, activating transcription factor (ATF), and Jun dimerized protein (JDP) families) at elevated levels (e.g., made to have physiologically elevated levels), and / or modified (e.g., genetically) to reduce the expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB, BATF3, and other BATF family members, IRF4, and ATF family members).

[0012] Accordingly, in one aspect, the present invention provides T cells that are modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors. For example, in one embodiment, c-Jun is expressed in T cells that are modified to express a synthetic receptor such as a modified T cell receptor or a chimeric antigen receptor (CAR). In another embodiment, c-Jun is expressed in T cells having a native, innate T cell receptor. The present invention is not limited to the means of expressing one or more AP-1 transcription factors. In one embodiment, when co-expressed with a modified TCR or CAR, c-Jun (and / or other AP-1 transcription factors) and the modified receptor are co-expressed from another viral vector. In another embodiment, they are expressed from a single vector construct using a bicistronic vector. c-Jun (and / or other AP-1 transcription factors) may be expressed constitutively or in a controlled manner (e.g., using a system that remotely controls expression via small molecules, or using an endogenously controlled system). In another embodiment, the c-Jun and / or other AP-1 transcription factor genes may be genetically incorporated into cellular DNA using retroviruses, lentiviruses, or other viral vectors, or via CRISPR / Cas9-based systems. In yet another embodiment, the c-Jun and / or other AP-1 transcription factors may be expressed via RNA or tumor-disintegrating viruses or other transient expression systems known in the art. The c-Jun and / or other AP-1 transcription factors may be delivered to T cells ex vivo for adoptive transfer or by in vivo gene transfer.

[0013] Similarly, the present invention is not limited to the type of T cell that is modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors, and / or to reduce the expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4 and ATF family members) (e.g., genetically). In some embodiments, the T cell is a CD3+ T cell (e.g., a combination of CD4+ and CD8+ T cells). In some embodiments, the T cell is a CD8+ T cell. In other embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a natural killer (NK) T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is a combination of CD4+ and CD8 T+ cells (e.g., CD3+). In some embodiments, the T cell is a memory T cell. In some embodiments, the memory T cell is a central memory T cell. In some aspects, memory T cells are effector memory T cells. In some aspects, T cells are tumor-infiltrating lymphocytes. In some aspects, T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and / or γδ T cells. In some aspects, T cells are cytokine-induced killer cells.

[0014] In some embodiments, T cells are antitumor T cells (T cells that are activated and grow in response to an antigen, and that have activity against tumors (e.g., autologous tumors)). In one embodiment, antitumor T cells (e.g., useful in adoptive T cell transplantation) include peripheral blood-derived T cells genetically modified with receptors that recognize and respond to tumor antigens. Such receptors generally consist of an extracellular domain containing a tumor antigen-specific single-chain antibody (scFv) linked to an intracellular T cell signaling motif (see, for example, Westwood, JA et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52):19051-19056). Other antitumor T cells include T cells obtained from excised tumors or tumor biopsies (e.g., tumor-infiltrating lymphocytes (TILs)). In another embodiment, the T cells are polyclonal or monoclonal tumor-reactive T cells (e.g., obtained by apheresis and ex vivo-magnified against tumor antigens presented by autologous or artificial antigen-presenting cells). In yet another embodiment, the T cells are remodeled to express human or mouse-derived T cell receptors that recognize tumor antigens. The present invention is not limited to the type of tumor antigen thus recognized. In fact, any T cells containing receptors that recognize tumor antigens will find use in the compositions and methods of the present invention.Examples include, but are not limited to, CD19, CD20, CD22, receptor tyrosine kinase-like orphan receptor 1 (ROR1), disiaroganglioside 2 (GD2), Epstein-Barr virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), CD33 / IL3Rα, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate-specific membrane antigen (PSMA), glycolipid F77, and epidermal growth factor receptor variol. Examples include T cells expressing receptors that recognize antigens selected from Ant III (EGFRvIII), NY-ESO-1, melanoma antigen gene (MAGE) family member A3 (MAGE-A3), melanoma antigen 1 recognized by T cells (MART-1), GP1000, p53, or other tumor antigens described herein (e.g., naturally occurring receptors, or receptors modified to express a modified T cell receptor or a synthetic receptor such as CAR).

[0015] In some embodiments, T cells can be remodeled to express CARs. The present invention is not limited by the type of CAR. In fact, any CAR that specifically binds to a desired antigen (e.g., a tumor antigen) can be modified as disclosed and described herein to overexpress and / or include one or more AP-1 transcription factors (e.g., c-Jun) at elevated levels (e.g., made to have physiologically elevated levels). In some embodiments, the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising heavy-chain and light-chain variable regions that specifically bind to a desired antigen. In some embodiments, the CAR further comprises a transmembrane domain (e.g., a T cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain (e.g., a T cell receptor signaling domain (e.g., a TCRζ chain)) containing one or more immunoreceptor tyrosine system activating motifs (ITAMs). In some embodiments, the CAR comprises one or more co-stimulatory domains (e.g., domains that provide a second signal to stimulate T cell activation). The present invention is not limited by the type of co-stimulatory domain. In fact, any co-stimulatory domain known in the art may be used, but is not limited to CD28, OX40 / CD134, 4-1BB / CD137 / TNFRSF9, high affinity immunoglobulin E receptor γ subunit (FcERIγ, ICOS / CD278, interleukin 2 subunit β (ILRβ) or CD122, cytokine receptor common subunit γ (IL-2Rγ) or CD132 and CD40). In one embodiment, the co-stimulatory domain is 4-1BB.

[0016] In one aspect, the present invention provides a method for treating a disease or condition in a subject (e.g., a patient) having the disease or condition, comprising the step of administering an effective amount of T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and / or modified (e.g., genetically) for reduced expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4, and ATF family members). The present invention is not limited by the type of disease or condition being treated. In fact, any disease or condition treatable by T cell administration (e.g., alleviation of disease signs or symptoms upon treatment) can be treated in an improved and more effective manner using the compositions and methods of the present invention (including and / or using T cells modified to express and / or contain, for example, elevated levels of one or more AP-1 transcription factors). In one aspect, the disease or condition is cancer. In another aspect, the disease or condition is an infectious disease. The present invention is not limited by the type of cancer or the type of infectious disease. In fact, any cancer known in the art for which T-cell therapy is used for treatment may be treated using the compositions and methods of the present invention. In a similar manner, any infectious disease known in the art for which T-cell therapy is used for treatment may be treated using the compositions and methods of the present invention. In one embodiment, administration of an effective amount of T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and / or reduced expression and / or activity of one or more AP-1 inhibitory complex members to a subject (e.g., a patient) having a disease or condition inhibits T cell exhaustion (compared to a subject receiving the same amount of remodeled T cells (e.g., CAR T cells or T cells containing recombinant TCRs) that are not modified to express and / or contain elevated levels of one or more AP-1 transcription factors or to have reduced expression and / or activity of one or more AP-1 inhibitory complex members).

[0017] Thus, in one aspect, the present invention provides a method of inhibiting T cell exhaustion (e.g., maintaining the functionality of T cells exposed to excessive antigen (e.g., in the context of treating a disease or condition)) by modifying T cells to express and / or comprise a reduced expression and / or activity of one or more AP-1 transcription factors and / or one or more AP-1 inhibitory complex members at elevated levels, compared to, for example, control T cells not so modified. In one aspect, T cells modified to express and / or comprise one or more AP-1 transcription factors at elevated levels (e.g., c-Jun) exhibit increased functionality and / or activity (e.g., increased antigen-induced cytokine production, enhanced killing ability (e.g., increased recognition of tumor targets with low surface antigens), increased memory cell formation, and / or enhanced proliferation in response to antigen) and / or characteristics of reduced exhaustion (e.g., lower levels of markers indicative of exhaustion (e.g., PD-1, TIM-3, LAG-3) and / or lower levels of programmed cell death). In some aspects, the T cells described herein modified to express and / or comprise a decreased expression and / or activity of one or more AP-1 transcription factors and / or one or more AP-1 inhibitory complex members at elevated levels significantly enhance the clinical efficacy (e.g., of engineered T cells (e.g., CAR T cells) and / or unengineered natural T cells).

[0018] In some embodiments, the present invention demonstrates that treatment of a subject with cancer with a therapeutically effective dose of a composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors is superior to treatment of a subject with cancer with T cells expressing normal levels of one or more AP-1 transcription factors. In some embodiments, treatment of an animal suffering from cancer (e.g., human) with a therapeutically effective dose of an immunotherapy composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors inhibits the development or growth of cancer cells and / or makes the cancer cells a more sensitive population to other treatments (e.g., cancer drugs or the cell death-inducing activity of radiotherapy). Accordingly, the compositions and methods of the present invention can be used as monotherapies (for example, to kill cancer cells and / or reduce or inhibit cancer cell growth, or to induce apoptosis and / or cell cycle arrest in cancer cells), or when administered in combination with one or more further agents (e.g., cell death-inducing or cell cycle-disrupting cancer drugs or radiotherapy) such as other anticancer agents to make a larger proportion of cancer cells susceptible to killing, they inhibited cancer cell growth or induced apoptosis and / or cell cycle arrest compared to the corresponding proportion of cells in animals treated with cancer drugs or radiotherapy alone.

[0019] Thus, in one aspect, the present invention provides a method of treating cancer or delaying cancer progression in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising T cells that have been (e.g., genetically) modified to express and / or contain elevated levels of one or more AP-1 transcription factors (e.g., c-Jun) and / or that have been (e.g., genetically) modified with respect to reduced expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4, and ATF family members). In one aspect, the therapeutically effective amount of the modified T cell composition reduces the number of cancer cells in the patient after such treatment. In one aspect, the therapeutically effective amount of the modified T cell composition reduces and / or eliminates tumor burden in the patient after such treatment. In one aspect, the method further comprises administering radiation therapy to the patient. In one aspect, the radiation therapy is administered before, at the same time as, and / or after the patient receives a therapeutically effective amount of the modified T cell composition. In one aspect, the method further comprises administering to the patient one or more anti-cancer agents and / or one or more chemotherapeutic agents. In one aspect, the one or more anti-cancer agents and / or one or more chemotherapeutic agents are administered before, at the same time as, and / or after the patient receives a therapeutically effective amount of the modified T cell composition. In one aspect, the combination treatment of the patient using a therapeutically effective amount of the modified T cells and a series of anti-cancer agents results in a greater tumor response and clinical benefit in such patient compared to patients treated with the modified T cells or anti-cancer drug / radiation alone. Since all approved dosages for anti-cancer drugs and radiation therapy are known, the present invention contemplates various combinations of such dosages with the modified T cells.

[0020] In one embodiment, the present invention provides a therapeutically effective amount of a composition (e.g., an immunotherapy composition) comprising T cells modified in accordance with this disclosure (for use, for example, in the treatment of cancer or delaying the progression of cancer in a subject). As described herein, the composition may further comprise one or more anticancer agents, e.g., one or more chemotherapeutic agents. The present invention also provides the use of the composition for inducing cell cycle arrest and / or apoptosis. The present invention also relates to the use of the composition for sensitizing cells to further agents (one or more), such as agents that induce apoptosis and / or cell cycle arrest, and for chemoprotection of normal cells by inducing cell cycle arrest prior to treatment with chemotherapeutic agents. The compositions of the present invention are useful in treating, improving or preventing any type of cancer or infectious disease and any disorder such as any cell responsive to the induction of apoptotic cell death (disorders characterized by impaired apoptosis control, including hyperproliferative diseases such as cancer). In one embodiment, the composition may be used to treat, improve or prevent cancers further characterized by resistance to cancer therapy (e.g., these cancer cells are chemoresistant, radioresistant, hormone-resistant, etc.). The present invention also provides pharmaceutical compositions comprising a composition (e.g., an immunotherapy composition) containing the modified T cells of the present invention in a pharmaceutically acceptable carrier.

[0021] In another embodiment, the present invention provides a method for treating cancer or delaying cancer progression in a patient, comprising the step of administering to the patient a therapeutically effective amount of a composition comprising T cells that have been (e.g., genetically) modified to express and / or contain one or more AP-1 transcription factors (e.g., c-Jun) at elevated levels, in combination with a therapeutically effective amount of a TCR signaling inhibitor (e.g., to prevent T cell exhaustion). In one embodiment, the TCR signaling inhibitor is a tyrosine kinase inhibitor. In another embodiment, the tyrosine kinase inhibitor inhibits Lck kinase. The TCR signaling inhibitor may be administered by any suitable dosage form, but is typically administered orally. Multiple cycles of treatment may be administered to the subject. In one embodiment, the TCR signaling inhibitor is administered according to a standard dosing schedule (e.g., daily or intermittently). In another embodiment, the TCR signaling inhibitor is administered for a time sufficient to repair at least partial T cell function, and then discontinued.

[0022] These and other aspects of the present invention will be readily apparent to those skilled in the art in consideration of the disclosure herein.

[0023] In other words, the gist of this invention relates to the following: Item 1 A composition comprising isolated T cells modified to overexpress and / or contain one or more AP-1 transcription factors at elevated levels. Section 2 The composition according to item 1, wherein isolated T cells are further modified to express recombinant receptors. Section 3 The composition according to item 2, wherein the recombinant receptor is a T cell receptor (TCR). Section 4 The composition according to item 2, wherein the recombinant receptor is a chimeric antigen receptor (CAR). Section 5 The composition according to item 2, wherein the recombinant receptor is specific to the tumor antigen. Section 6 The composition according to item 5, wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000, and p53. Section 7 The composition according to item 1, wherein the isolated T cells are natural, naturally occurring T cells. Section 8 The composition according to item 7, wherein natural, naturally occurring T cells are obtained from an excised tumor. Section 9 Natural, naturally occurring T cells are obtained by leukocyte extraction from a blood sample. The composition described in item 7. Section 10 The composition according to item 9, wherein T cells are expanded exovivoically. Section 11 The composition according to item 1, wherein the T cells are selected from the group consisting of CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer (NK) T cells, γδ T cells, combinations of CD4+ cells and CD8T+ cells, memory T cells, cytokine-induced killer cells, and combinations thereof. Item 12 The composition according to item 1, wherein one or more AP-1 transcription factors are selected from the group consisting of c-Fos, c-Jun, activating transcription factor (ATF), and Jun dimerized protein (JDP). Section 13 The composition according to item 1, wherein the AP-1 transcription factor is c-Jun. Section 14 The composition according to item 1, wherein the AP-1 transcription factor is a mutated / cleaved AP-1 transcription factor. Item 15 The composition according to item 1, wherein the mutated / cleaved AP-1 transcription factor contains an N-terminal deletion. Section 16 The composition according to claim 1, wherein the mutated / cleaved AP-1 transcription factor (i) lacks a transactivation domain or (ii) contains an inactive transactivation domain. Item 17 The composition according to item 1, wherein isolated T cells co-express c-Jun and a remodeled receptor. Section 18 The composition according to item 17, wherein c-Jun and a modified receptor are expressed from another expression vector construct. Section 19 The composition according to item 17, wherein c-Jun and a modified receptor are co-expressed from a single expression vector construct. Item 20 The composition according to item 1, wherein the isolated T cells are T cells having specificity and activity against tumors. Section 21 The composition according to item 20, wherein the T cells are peripheral blood-derived T cells that have been genetically modified to express receptors that recognize tumors and respond to tumors. Section 22 The composition according to claim 1, further modified to reduce and / or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members in T cells. Section 23 The composition according to item 22, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, BATF family members, IRF4, ATF family members, or combinations thereof. Section 24 The composition according to item 22, wherein the AP-1 inhibitory complex member is JunB and / or BATF3. Section 25 A method for treating a disease or pathological condition in a subject, comprising the step of administering to a subject having a disease or pathological condition an effective amount of a composition comprising T cells modified to express and / or contain one or more AP-1 transcription factors at elevated levels. Section 26 The method described in section 25, wherein the AP-1 transcription factor is c-Jun. Section 27 The method described in section 26, wherein the AP-1 transcription factor is a mutated / cleaved transcription factor. Section 28 The method according to paragraph 26, wherein T cells are further modified to express recombinant receptors. Section 29 c-Jun and recombinant receptors are expressed from a different expression vector construct, or c-Jun and recombinant receptor are co-expressed from a single expression vector construct. The method described in item 28. Section 30 The method according to paragraph 25, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are less susceptible to T cell exhaustion. Section 31 The method according to paragraph 25, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors have increased responsiveness to low antigen densities on target cells of the T cells. Section 32 The method according to paragraph 25, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are less likely to experience a decrease in memory form. Section 33 The method according to paragraph 25, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are less likely to experience a decrease in proliferative capacity. Section 34 The method described in paragraph 25, wherein the disease or condition is a tumor or cancer. Section 35 The method according to paragraph 34, wherein T cells are modified to express recombinant receptors specific to tumors or cancer. Section 36 The method described in item 35, wherein the recombinant receptor is a chimeric antigen receptor (CAR). Section 37 The method according to item 36, wherein the CAR is specific to a tumor antigen selected from the group consisting of CD19, CD20, CD22, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000, and p53. Section 38 The method described in paragraph 25, wherein the disease or condition is a viral, bacterial and / or parasitic infection. Section 39 The method according to paragraph 25, wherein T cells are further modified to reduce and / or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members. Section 40 The method according to item 39, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, BATF family members, IRF4, ATF family members, or combinations thereof. Section 41 The method according to item 40, wherein the AP-1 inhibitory complex member is JunB and / or BATF3. Section 42 A method for treating cancer or slowing the progression of cancer in a patient, comprising the step of administering to the patient a therapeutically effective amount of a composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR). Section 43 The method according to paragraph 42, wherein the administration step reduces the number of cancer cells in the patient. Section 44 The method according to paragraph 42, wherein the administration step reduces and / or eliminates the tumor burden in the patient. Section 45 The method according to claim 42, further comprising the step of administering one or more anticancer agents and / or one or more chemotherapeutic agents to a patient. Section 46 The method according to paragraph 42, wherein the administration step occurs before, concurrently with, and / or after the patient receives radiotherapy. Section 47 The method according to paragraph 42, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less susceptible to T cell exhaustion. Section 48 The method according to paragraph 42, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) have increased responsiveness to low antigen densities on target cells of the T cells. Section 49 The method according to paragraph 42, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less likely to experience a decrease in memory form. Section 50 The method according to paragraph 42, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less likely to experience a decrease in proliferative capacity. Section 51 A therapeutically effective amount of a composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express tumor antigen-specific chimeric antigen receptors (CARs), for use in the treatment of cancer or delaying cancer progression in a subject. Section 52 A composition for use according to item 51, further comprising one or more anticancer agents and / or one or more chemotherapeutic agents. Section 53 A composition for use according to item 51, which reduces the number of cancerous cells in a patient and reduces or eliminates the tumor burden in a patient. Section 54 The method according to paragraph 51, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less susceptible to T cell exhaustion. Section 55 The method according to paragraph 51, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) have increased responsiveness to low antigen densities on target cells of the T cells. Section 56 The method according to paragraph 51, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less likely to experience a decrease in memory form. Section 57 The method according to paragraph 51, wherein T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors and to express a tumor antigen-specific chimeric antigen receptor (CAR) are less likely to experience a decrease in proliferative capacity. Section 58 A method for treating a disease or condition in a patient, comprising the step of administering to the patient a disease or condition an effective amount of a composition comprising T cells modified to reduce and / or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members. Section 59 The method described in paragraph 58, where the disease or condition is cancer. Item 60 The method according to item 59, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, BATF3, BATF family members, IRF4, IRF family members, ATF family members, or combinations thereof. Section 61 The method according to section 58, wherein the AP-1 inhibitory complex members are JunB, BATF3 and / or IRF4. Section 62 The method according to paragraph 58, wherein T cells are modified by CRISPR-Cas9, shRNA, siRNA, RNAi, microRNA, degron, a controllable promoter, or pharmacological inhibition. Section 63 A composition comprising isolated T cells modified to reduce and / or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members. Section 64 The composition according to claim 63, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, BATF3, BATF family members, IRF4, IRF family members, ATF family members, or combinations thereof. Section 65 The composition according to claim 63, wherein the AP-1 inhibitory complex members are JunB, BATF3 and / or IRF4. Section 66 The composition according to item 1, wherein isolated T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors are less susceptible to T cell exhaustion. Section 67 The composition according to claim 1, wherein isolated T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors have increased responsiveness to low antigen densities on target cells of the T cells. Section 68 The composition according to item 1, wherein isolated T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors are less likely to experience a decrease in memory form. Section 69 The composition according to item 1, wherein isolated T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors are less likely to experience a decrease in proliferative capacity. [Effects of the Invention]

[0024] The present invention may provide compositions and methods for inhibiting T cell exhaustion. [Brief explanation of the drawing]

[0025] Description of the drawing [Figure 1] Figures 1A and 1B show that the AP-1 transcription factors c-Fos and c-Jun are downregulated in GD2-28Z-exhausted CAR T cells. [Figure 2-1] Figures 2A-C show that enhanced AP-1 expression reduces the characteristics of CAR T cell exhaustion. [Figure 2-2] Figures 2D-E show that enhanced AP-1 expression reduces the characteristics of CAR T cell exhaustion. [Figure 3-1] Figures 3A-C show that the functional benefits of AP-1 are primarily due to c-Jun expression. [Figure 3-2] Figures 3D-E show that the functional benefits of AP-1 are primarily due to c-Jun expression. [Figure 3-3] Figure 3F shows that the functional benefits of AP-1 are primarily due to c-Jun expression. [Figure 4-1] Figures 4A-B show that bicistronic expression of c-Jun by CAR enhances CAR T functional activity. [Figure 4-2] Figure 4C shows that bicistronic expression of c-Jun by CAR enhances CAR T functional activity. [Figure 4-3] Figure 4D shows that bicistronic expression of c-Jun by CAR enhances CAR T functional activity. [Figure 4-4] Figure 4E shows that CAR-mediated bicistronic expression of c-Jun enhances CAR T functional activity. [Figure 5-1] Figure 5A shows that bicistronic expression of c-Jun by CAR enhances CAR T functional activity and the central memory phenotype. [Figure 5-2] Figure 5B shows that bicistronic expression of c-Jun by CAR enhances CAR T functional activity and the central memory phenotype. [Figure 5-3] Figure 5C shows that bicistronic expression of c-Jun by CAR enhances CAR T functional activity and the central memory phenotype. [Figure 6-1] Figures 6A-B show that bicistronic expression of c-Jun by CAR enhances CAR T cell pro-inflammatory cytokine production and reduces IL-10. [Figure 6-2]Figures 6C-F show that bicistronic expression of c-Jun by CAR enhances CAR T cell pro-inflammatory cytokine production and reduces IL-10. [Figure 7-1] Figures 7A-B show that bicistronic expression of c-Jun enhances CD19 and CD22 CAR T cell activity in response to tumor cells with low levels of antigen. [Figure 7-2] Figure 7C shows that bicistronic expression of c-Jun enhances CD19 and CD22 CAR T cell activity in response to tumor cells with low levels of antigen. [Figure 7-3] Figures 7D-E show that bicistronic expression of c-Jun enhances CD19 and CD22 CAR T cell activity in response to tumor cells with low levels of antigen. [Figure 8-1] Figures 8A-B show that knockdown of inhibitory AP-1 family members JunB and BATF3 increases IL2 production in exhausted CAR T cells. (A) CRISPR gene knockout (KO) of JunB in HA-28Z exhausted CAR T cells dramatically increases IL2 (top) and IFNg (bottom) production after exposure to the GD2+ cell line Nalm6-GD2 (left), 143B osteosarcoma (middle), and Kelly neuroblastoma (right). This increase was considerably greater than that for c-Jun overexpression (OE) alone. Dual JUNB-KO and cJUN-OE T cells showed no benefit compared to JUNB-KO alone. (B) CRISPR gene knockout (KO) of JunB in GD2-BBZ CAR T cells significantly increased IL2 (top) and IFNg (bottom) production after exposure to the GD2+ cell line Nalm6-GD2 (left), 143B osteosarcoma (center), and Kelly neuroblastoma (right), but c-Jun overexpression (OE) GD2-BBZ CAR T cells showed the greatest functional benefit. Dual JUNB-KO and cJUN-OE T cells showed no benefit compared to cJUN-OE alone. [Figure 8-2]Figures 8C-D show that knockdown of inhibitory AP-1 family members JunB and BATF3 increases IL2 production in exhausted CAR T cells. (C) CRISPR gene knockout (KO) of JunB in CD19-28Z (left) or CD19-BBZ (right) CAR T cells did not affect IL2 (top) production after exposure to Nalm6-GD2 leukemia cells, suggesting that JunB is a potent inhibitor only in persistently signaled / exhausted GD2 CAR T cells. (D) CRISPR gene knockout (KO) of BATF3 in HA-28Z exhausted CAR T cells increased IL2 (top) production after exposure to Nalm6-GD2 (left) and Kelly neuroblastoma (right), but IFNγ production remained unchanged. HA-28Z-exhausted CAR T cells edited using three independent gRNAs targeting BATF3 all showed increased IL-2 production compared to controls or ZB2-edited controls. [Figure 9] Figures 9A-B show that c-Jun-expressing HA-GD2 CAR T cells exhibit superior therapeutic in vivo activity compared to unmodified HA-GD2 CAR T cells. Growth of Nalm6-GD2 leukemia cells stably expressing firefly luciferase was tracked in vivo using bioluminescence imaging after adoptive transfer of 2(10⁶ CAR+ or mock (non-transduced) T cells. (A) Bioluminescence quantified over time. (B) Images showing individual mice. n=5 mice per group. (All scales are 1(10⁴-1(10⁶) except for adjusting the (mock d32) scale. [Figure 10] Figure 10 shows that c-Jun-modified GD2-BBZ CAR T cells exhibit superior in vivo activity in an aggressive 143B osteosarcoma solid tumor model. Growth of intramuscularly implanted 143B osteosarcoma tumor cells was tracked in vivo using calipathometry after adoptive transfer of 1(107CAR+ or mock (non-transduction) T cells. (A) Tumor growth quantified over time for n=5 mice per group. [Figure 11]Figures 11A-D show that c-Jun modified CD19 CAR T cells exhibit enhanced in vivo activity against CD19-low Nalm6 leukemia. 3(106) CAR+ T cells were IV-delivered to mice with CD19-low clone F Nalm6 leukemia tumors. (A-B) Tumor growth (A) and survival (B) of mice treated with CD19-BBZ CAR T cells + / - c-Jun. c-Jun modified CD19-BBZ CAR T cells show reduced tumor growth and significantly enhanced survival. (C-D) Tumor growth (C) and survival (D) of mice treated with CD19-28Z CAR T cells + / - c-Jun. c-Jun modified CD19-28Z CAR T cells show reduced tumor growth initially, but the CD19-negative disease eventually grew again in both groups, with no survival benefit (p>0.05). [Figure 12-1] Figures 12A-D show that HA-28z CAR T cells exhibit phenotypic, functional, transcriptional, and epigenetic T cell exhaustion characteristics. a) Decreased expansion of HA-28z vs. CD19-28z CAR T cells during the first expansion culture. D0 = bead activation, D2 = transduction. Error bars show the mean ± SEM from n=10 donors. b) Surface expression of exhaustion-related markers (D10). c) CD19-28z mainly contains T stem cell memory (CD45RA+CD62L+) and central memory (CD45RA-CD62L+), while HA-28z mainly contains CD45RA-CD62L- effector memory cells (D10). d) IL-2 (left) and IFNg (right) are released 24 hours after co-culture with CD19+GD2+Nalm6-GD2 leukemia cells. Error bars represent the mean ± SD derived from triple wells. One representative donor is shown for each assay. N-naive, CM-central memory. *p<.05, **p<.01, ***p<.001. ns p>.05. [Figure 12-2]Figures 12E-H show that HA-28z CAR T cells exhibit phenotypic, functional, transcriptional, and epigenetic T cell exhaustion characteristics. e) Principal component analysis (PCA) of the overall transcriptional profiles of naive and CM-derived CD19 or HA CAR T cells at days 7, 10, and 14 of culture. PC1 (39.3% variation) separates CD19 and HA CAR T cells. f) Gene expression of the top 200 genes driving PC1. The genes of interest in each cluster are listed above. g) Differentially accessible chromatin regions (D10) within CD8+ CD19 and HA-28z CAR T cells. Both N and CM subsets are incorporated into their respective CARs. h) PCA (D10) of ATAC-seq chromatin accessibility in CD19 or HA-28z CAR T cells. PC1 (76.9% variation) separates CD19 and HA CAR samples. N-Naive, CM-Central Memory. *p<.05, **p<.01, ***p<.001. ns p>.05. [Figure 12-3] Figures 12I-J show that HA-28z CAR T cells exhibit phenotypic, functional, transcriptional, and epigenetic T cell exhaustion characteristics. i) Overall chromatin accessibility profile (D10) of subset-derived CD19 and HA-28z CAR T cells. Top 5000 differentially accessible regions (peaks). j) Differentially accessible enhancer regions within CD19 and HA CAR T cells at the CTLA4 (upper) or IL7R (lower) locus. N-naive, CM-central memory. *p<.05, **p<.01, ***p<.001. ns p>.05. [Figure 13-1]Figures 13A-C show the signatures of the AP-1 family in exhausted CAR T cells. a) Deviation scores of the top 25 transcription factor motifs in 10-day HA vs. CD19-CAR expressing T cells by chromVAR analysis reveal numerous AP-1 (bZIP) family members in CD4+ and CD8+ T cells from N or CM subsets (D10). b) TF motif enrichment analysis in N CD8+ HA-28z CAR T cells shows that the AP-1 (bZIP) family motif is the most significantly enriched. c) Bulk RNA-seq expression (FPKM) of AP-1 (bZIP) and IRF family members shown in CD19 and HA-28z CAR T cells. Error bars show the mean ± SEM from n=6 samples across three donors showing paired CD19 vs. HA expression for each gene. The p-values ​​were calculated using the Wilcoxon matched-pair signed-rank test. *p<.05, **p<.01, ***p<.001. ns p>.05. [Figure 13-2]Figures 13D-E show the signatures of the AP-1 family in exhausted CAR T cells. d) CD19-28z and HA-28z CAR T cells were lysed, and the expression of the shown AP-1 family proteins was evaluated by Western blotting. Increased protein expression of JunB, BATF3, and IRF4 in HA-28z CAR T cells compared to CD19 CAR T cells was confirmed at days 7, 10, and 14 of culture. e) Correlation network of exhaustion-related transcription factors in N-derived CD8+ (left) and CD4+ (right) GD2-28z CAR T cells using single-cell RNA-seq analysis. Transcription factor genes identified as differentially expressed (p<0.05) by DESeq2 form nodes in the network. Color indicates log2 scaling change (FC) (GD2 vs. CD19 CAR). Border thickness indicates the magnitude of the correlation of expression between related pairs of genes across cells. The network was constructed using correlation scores >0.1. *p<.05, **p<.01, ***p<.001. ns p>.05. [Figure 14-1] Figures 14A-E show that c-Jun overexpression enhances the function of exhausted CAR T cells. a) Schematic diagram of the JUN-P2A-CAR expression vector. b) Intracellular flow cytometry (D10) showing total c-Jun expression in control and JUN-modified CAR T cells. c) Western blot (D10) for total c-Jun and phospho-c-JunSer73 in control and JUN-modified CD19 and HA CAR T cells. (d) IL-2 and (e) IFNg production after 24-hour co-culture of control (blue) or JUN-modified (red) CD19 and HA CAR T cells in response to antigen + tumor cells. Error bars represent the mean ± SD of the triple wells. Data from one representative donor are shown. Fold increases in IL-2 or IFNg production in JUN vs. control CAR T cells across multiple donors may be seen in Figure 24. *p<.05, **p<.01, ***p<.001. ns p >.05. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 14-2]Figures 14F-G show that c-Jun overexpression enhances the function of exhausted CAR T cells. f) Left: Flow cytometry plot (D10) showing representative CD45RA / CD62L expression in control vs. JUN-CAR T cells. Right: Relative frequencies of effector (E, RA+62L-), stem cell memory (SCM, RA+62L+), central memory (RA-62L+), and effector memory (RA-62L-) in CD4+ (top) or CD8+ (bottom) control or JUN-HA-28z CAR T cells. n=6 donors from independent experiments. Lines indicate paired samples from the same donor. Paired two-sided t-tests were performed. g) On day 39 of culture, 1x10⁶ viable T cells from Figure 24c were again cultured on plates and cultured for 7 days with or without IL-2. *p<.05, **p<.01, ***p<.001. ns p > 0.05. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 14-3] Figures 14H-K show that c-Jun overexpression enhances the function of exhausted CAR T cells. h-j) Cell surface phenotype of control or JUN-CD19-28z CAR T cells derived from (g) at day 46. h) CD4 vs. CD8 expression. i) Later expanding CD8+ JUN-modified CD19-28z CAR T cells have a stem cell memory phenotype (CD45RA+CD62L+). j) Later expanding CD8+ JUN-modified CD19-28z CAR T cells showed reduced exhaustion marker expression compared to controls. k) T cells derived from g were cryopreserved at D10, thawed, and left to stand overnight in IL-2. 5 x 10⁶ control, JUN-modified CD19-28z, or CD19-BBz CAR T cells were injected IV into healthy NSG mice. Peripheral blood T cell counts were quantified by flow cytometry 25 days after injection. Error bars represent the mean ± SEM of n=5 mice per group. *p<.05, **p<.01, ***p<.001. ns p >.05. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 15-1]Figures 15A-D demonstrate that functional rescue of exhausted HA-28z CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation and is JNP-independent. a) Proposed mechanism of c-Jun-mediated rescue of T cell exhaustion. AP-1-i represents the inhibitory AP-1 complex. b) Schematic diagram of a DD-regulated JUN expression vector. c) Schematic diagram of drug-induced stabilization of JUN-DD expression. Yellow diamonds represent TMP-stabilizing molecules. d) Total c-Jun expression (D10) in control, JUN-WT, and JUN-DD HA-28z CAR T cells by intracellular flow cytometry (left) and Western blot (right). e) IL-2 (left) and IFNg (right) production (D10) in control, JUN-WT, or JUN-DD (off, on) modified HA-28z CAR T cells 24 hours after stimulation with Nalm6-GD2 or 143B target cells or medium alone (baseline). In d-e), off indicates the absence of TMP, and on indicates T cells cultured in the presence of 10 μM TMP between D4-derived and co-culture. *p<.05, **p<.01, ***p<.001. ns p>.05. HTM - hinge / transmembrane, ICD - intracellular domain, DD - destabilization domain derived from E. coli DHFR, TMP - trimethoprim, WT - wild type. [Figure 15-2]Figures 15E–G show that functional rescue of exhausted HA-28z CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation and is JNP-independent. In d–e), "off" shows the absence of TMP, and "on" shows T cells cultured in the presence of 10 μM TMP during D4-derived and co-culture. In f–g), TMP was added either during T cell expansion (initiated at D4) or only during co-culture with tumor cells, as shown in f. For on-off and off-on conditions, TMP was removed / added 18 hours prior to co-culture and before antigen exposure to ensure complete c-Jun degradation / stabilization, respectively. g) IL-2 expression and IL-2 multiplier increase in one representative donor (left, SD across triple wells) (SEM of n=6 independent experiments showing three different donors against off-off conditions). Error bars represent the mean ± SD of the triple wells. Representative of three independent experiments. *p<.05, **p<.01, ***p<.001. ns p>.05. HTM-hinge / transmembrane domain, ICD-intracellular domain, DD-destabilizing domain derived from E. coli DHFR, TMP-trimethoprim, WT-wild type. [Figure 15-3]Figures 15H-J show that functional rescue of exhausted HA-28z CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation and is independent of JNPs. h-j) Increased functional activity of JUN-CAR T cells is independent of Jun N-terminal phosphorylation (JNP). h) Schematic diagram of the c-Jun protein showing the N-terminal transactivation domain (TAD). Asterisks represent JNP sites at Ser63 and Ser73, which are mutated to alanine in JUN-AA mutants. i) Western blot of total c-Jun and c-Jun-PSer73 in control, JUN-WT, and JUN-AA HA-28z CAR T cells. j) IL-2 (left) and IFNg (right) release in control, JUN-WT, and JUN-AA HA-28z CAR T cells 24 hours after stimulation with Nalm6-GD2 or 143B target cells or medium alone (baseline). Error bars represent the mean ± SD of the triple wells. Representative of three independent experiments. *p<.05, **p<.01, ***p<.001. ns p>.05. HTM - hinge / transmembrane, ICD - intracellular domain, DD - E. coli DHFR-derived destabilizing domain, TMP - trimethoprim, WT - wild type. [Figure 16-1]Figures 16A–E show that JUN-modified CAR T cells increase in vivo activity against leukemia and solid tumors. In a–c, NSG mice were inoculated with 1x Nalm6-GD2 leukemia cells via IV injection. 3x10⁶ mock, HA-28z, or JUN-HA-28z CAR+ T cells were administered IV at d3. Tumor progression was monitored using bioluminescence imaging (a–b). Scales are standardized for all time points. c) JUN-HA-28z CAR T cells induced long-term tumor-free survival. Error bars represent the mean ± SEM for n=5 mice / group. This finding was reproducible in >3 independent experiments, although in some experiments, long-term survival was reduced due to the growth of GD2(-)Nalm6 clones. d) Schematic diagram of the JUN-Her2-BBz retroviral vector construct. e) Her2-BBz CAR T cell lysis of GFP+ Nalm6-Her2 target cells at an E:T ratio of 1:8. Error bars represent the mean ± SD of triple wells. Representative of two independent experiments. *p<.05, **p<.01, ***p<.001. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 16-2] Figures 16F-H show that JUN-modified CAR T cells increase in vivo activity against leukemia and solid tumors. In f-h, NSG mice were inoculated with 1x10⁶ 143b-19 osteosarcoma cells by intramuscular injection. 1x10⁷ mock, Her2-BBz, or JUN-Her2-BBz CAR T cells were administered IV on d7. f) Tumor growth was monitored by calipathometry. g) Mice treated with JUN-Her2-BBz CAR T cells maintained long-term tumor-free survival. h) Peripheral blood T cells were quantified on d20 after tumor cell implantation in mice treated similarly to f. Error bars represent the mean ± SEM of n=5 mice / group. Representative of two independent experiments with similar results. *p<.05, **p<.01, ***p<.001. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 17-1]Figures 17A-E show that JUN-CAR T cells enhance T cell function under suboptimal stimulation. a) IL-2 and b) IFNg production 24 hours after stimulation of control or JUN-modified HA-28z CAR T cells with fixed 1A7 anti-CAR idiotype antibody. Each curve was fitted to a nonlinear dose-response kinetics to determine EC50. The smaller graph on the right visualizes the curves for antibody concentrations of 0-1 ug / mL. c) Vector schematic diagram of the JUN-CD22-BBz retroviral vector construct. d) CD22 surface expression on parental Nalm6, Nalm6-22KO, and Nalm6-22KO+CD22low. e) IL-2 (left) and IFNg (right) release after co-culture of control or JUN CD22-BBz CAR T cells exposed to Nalm6 and Nalm6-22low. Error bars show the mean ± SD of the triple wells. Representative of three independent experiments. *p<.05, **p<.01, ***p<.001. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 17-2] Figures 17F-I show that JUN-CAR T cells enhance T cell function under suboptimal stimulation. In f-i), NSG mice were inoculated with 1 x 10⁶ Nalm6-22low leukemia cells on day 0. On day 4, 3 x 10⁶ control or JUN-CD22-BBz CAR+ T cells or 3 x 10⁶ mock transduction T cells were transplanted IV. Tumor growth was monitored by bioluminescence imaging (f) using image (i). g) Mice receiving JUN-22BBz CAR T cells showed an increase in peripheral blood T cells on day 23. h) JUN expression significantly improved long-term survival in CAR-treated mice. In f-g, error bars represent the mean ± SEM of n=5 mice per group. Representative of two independent experiments with similar results. *p<.05, **p<.01, ***p<.001. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 18-1]Figures 18A-B show that high-affinity (HA) 14g2a-GD2E101K CAR T cells exhibit a more severe exhaustion signature compared to the original 14g2a-GD2 CAR. a) Surface inhibitory receptor expression in CD19, GD2, and HA-GD2E101K CAR T cells at day 10 of culture. The high-affinity E101K mutation results in increased inhibitory receptor expression in CD4+ and CD8+ CAR T cells compared to the parental GD2 CAR. b) IL-2 secretion after 24 hours of co-culture of HA-GD2E101K or original GD2-28z CAR T cells with GD2+ target cells. The increased exhaustion profile of HA-GD2E101K CAR T cells corresponds to decreased functional activity, as measured by their ability to produce IL-2 upon stimulation. Error bars represent the mean ± SD of the triple wells. Representatives from at least four independent experiments with similar results. *p<.05, **p<.01, ***p<.001. PCA - Principal Component Analysis, NES - Standardized Enrichment Score. [Figure 18-2] Figures 18C-F show that high-affinity (HA) 14g2a-GD2E101K CAR T cells exhibit a worsened exhaustion signature compared to the original 14g2a-GD2 CAR. c) Bulk RNA-seq PCA shows greater variation between HA-GD2E101K and CD19 CAR T cells, while GD2-28z(sh) CAR T cells are intermediate. Left - CD4+ T cell. Right - CD8+ CAR T cell, naive origin. d-e) HA-GD2E101K CAR expression induces enhanced inhibitory receptor expression (d) and decreased memory formation (e) in CD4+ CAR T cells (CD8+ data in Figure 12). f) RNA-seqPCA from Figure 12e showing PC2 isolation is driven by CM vs. N, and PC3 isolation is driven by CD4 vs. CD8. *p<.05, **p<.01, ***p<.001. PCA - Principal Component Analysis, NES - Standardized Enrichment Score. [Figure 18-3]Figure 18G shows that high-affinity (HA) 14g2a-GD2E101K CAR T cells exhibit a worsened exhaustion signature compared to the original 14g2a-GD2 CARs. g) GSEA: The set of genes upregulated in HA-28z CAR T cells versus CD19-28z CAR T cells at day 10 showed significant overlap with the genes upregulated in exhausted vs. memory CD8+ (left), exhausted vs. effector CD8+ (middle), and exhausted vs. naive CD8+ (right) in a mouse model of chronic viral infection (Wherry et al. Immunity, 2007). *p<.05, **p<.01, ***p<.001. PCA - principal component analysis, NES - standardized enrichment score. [Figure 19-1] Figures 19A-B show that GD2-28z CAR T cells exhibit exhaustion signatures at the single-cell level. a) Venn diagram showing single-cell data (red) from bulk RNA-seq and duplicated genes in differential expression analysis of the top 200 genes driving the isolation of CD19 and HA-28z CAR T cells. Of the top 200 genes derived from bulk RNA-seq, 79 are differentially expressed by DESeq2 analysis in GD2-28z vs. CD19-28z single cells. Cross-emphasized genes include inhibitory receptors (CTLA4, LAG3, GITR), effector molecules CD25, IFNG, GZMB, cytokines IL13 and IL1A, and AP-1 / bZIP family transcription factors BATF3 and IRF4. b) Heatmap clustering of the top 50 differentially expressed genes in GD2-28z vs. CD19-28z single-cell transcriptome analysis. Each row represents one cell. [Figure 19-2]Figure 19C shows that GD2-28z CAR T cells exhibit an exhaustion signature at the single-cell level. c) Violin plots showing individual gene expression in CD8+ GD2-28z and CD19-28z single CAR T cells. Upregulated genes in GD2 CAR T cells include inhibitory receptors, effector molecules, and AP-1 family transcription factors, while CD19 CAR T cells showed increased expression of memory-related genes. The P-values ​​shown for each gene above the individual plots were calculated using the unpaired two-tailed Wilcoxon-Mann-Whitney U test. [Figure 20-1] Figures 20A-B show ATAC-seq data quality control. a) insertion length and b) insertion distance from transcription start site (TSS) for combined samples (top) and individual samples (bottom). [Figure 20-2] Figure 20C shows ATAC-seq data quality control. c) Correlation between replicated samples. [Figure 20-3] Figure 20D shows ATAC-seq data quality control. d) Mapped peak locations in each sample by total number of peaks (top) and total frequency (bottom). [Figure 21-1] Figures 21A-B show that the AP-1 family contains the transcription factor motifs most significantly enriched in HA-28z-exhausted CAR T cells. a) Differentially accessible chromatin region (D10) in CD4+ CD19 and HA CAR T cells. Both N and CM subsets are incorporated for each CAR. b) PCA derived from Figure 1h showing PC2 separation is driven by CM vs. N, and PC3 separation is driven by CD4 vs. CD8. (N-naive, CM-central memory). [Figure 21-2]Figure 21C shows that the AP-1 family contains the most significantly enriched transcription factor motifs in HA-28z-exhausted CAR T cells. c) The top transcription factor motifs enriched within differentially accessible chromatin regions in HA-28z CAR T cells include AP-1 / bZIP family factors in all initiation T cell subsets. The CD8+ naive subset is shown in Figure 2. (N-naive, CM-central memory). [Figure 21-3] Figure 21D shows that the AP-1 family contains the transcription factor motifs that were most significantly enriched in HA-28z-exhausted CAR T cells. d) Peak clustering by shared regulatory motifs (left) and enrichment heatmaps of transcription factor motifs in each cluster (right). Ten different clusters including clusters associated with exhausted (EX1-EX4) or healthy (HLT1-HLT2) CAR T cells, CM (CM) or N (naive) initiation subsets, and CD4 or CD8 T cell subsets. The gene of interest in each cluster is highlighted to the right. (N-naive, CM-central memory). [Figure 22]Figures 22A-C show that AP-1 / bZIP family transcription factors are upregulated in HA-28z CAR T cells and form an immune regulatory complex. a) Scale change in gene expression (HA / CD19) for the shown AP-1 / bZIP and IRF family genes from RNA sequencing data derived from Figure 2. Error bars represent the mean ± SEM of n=6 samples from three independent donors. b) Scale change in protein expression (HA / CD19) for the shown AP-1 / bZIP and IRF family proteins was determined by concentration meter analysis of Western blots. Error bars represent the mean ± SEM of n=4 experiments from three independent donors. *p<.05, **p<.01, ***p<.001. c) Western blot analysis of the shown AP-1 / bZIP and IRF family member proteins after immunoprecipitation for input (left column) or c-Jun (middle column) or JunB (right column) in CD19 and HA-28z CAR T cells. The numbers below represent the multiplicative increase in protein expression for HA versus CD19 under each condition, and the colored shapes indicate identified complexes of a size drawn in constant proportion. IP-Western blots show increased presence of c-Jun / JunB, c-Jun / IRF4, c-Jun / BATF, and c-Jun / BATF3 complexes in HA-28z CAR T cells. IRF4 also binds to JunB in a similar ratio, while BATF and BATF3 show preferential complexation with JunB. [Figure 23]Figures 23A-E show that the enhancement of AP1-modified CAR T cell activity depends on c-Jun rather than c-Fos. a-c) CAR T cells were co-transduced with or without lentiviral vectors encoding both AP1 transcription factors Fos and Jun (AP1) and with cleaved NGFR (tNGFR) surface-selective markers. a) Schematic diagram of the lentiviral construct. b) Representative transduction efficiency of AP1-modified CAR T cells as measured by NGFR surface expression in CD4+ and CD8+ CAR T cells shown. c) IL-2 production in control or AP1-modified CAR T cells 24 hours after stimulation with 143B-19 target cells. AP1-modified HA-28z CAR T cells show increased IL-2 production compared to control CAR T cells. Representative experiments from two independent experiments with similar results. d-e) CAR T cells were co-transduced with lentiviral vectors encoding either AP1 transcription factor Fos or Jun and with cleaved NGFR (tNGFR) surface-selective markers. d) Schematic diagrams of Fos and Jun lentiviral constructs. e) IL-2 production in control, Fos, or Jun-modified CAR T cells 24 hours after stimulation with Nalm6-GD2 target cells. Error bars represent the mean ± SD of the triple wells. Representative experiments from two independent experiments with similar results. In a and d, * indicates a stop codon. *p<.05, **p<.01, ***p<.001, ns p>0.05. [Figure 24]Figures 24A-C show an extended functional evaluation of JUN-modified CAR T cells. a-b) Multiplier increase in IL-2(a) and IFNg(b) release after 24 hours of co-culture with indicated target cells in JUN vs. control CD19 and HA-28z CAR T cells. Each dot represents one independent experiment from a different donor. c) Extended expansion of control or JUN-modified CAR T cells in vitro in three independent experiments using three different healthy donors. At the indicated time points, T cells were again cultured in fresh T cell medium + 100 IU / mL IL2. T cells were counted and nourished every 2-3 days to maintain cells at 0.5 x 10⁶ / mL. For donor-1, 5 x 10⁶ viable T cells were again cultured on days 14 and 28. For donor-2, 5 x 10⁶ viable T cells were again cultured on days 14, 28, 42 and 56. For donor-3, 5x10⁶ viable T cells were again cultured on plates on days 10, 17, 24, and 31. [Figure 25]Figures 25A-E show that c-Jun overexpression reduces the prevalence and complexation of inhibitory AP-1 family members JunB, BATF, and BATF3. a) Kinetics of drug-induced c-Jun stability in JUN-DD CAR T cells as assessed by Western blotting. At time 0, 10 μM TMP was added to untreated cells (on) or washed from pre-treated cells (off). Cells were removed from each condition at times 1, 2, 4, 8, 24, and 48 hours and prepared for Western blotting analysis of c-Jun expression. Observed bands correspond to the size of JUN-DD cells. b) Concentration metric analysis was performed on the blots derived from (a) and standardized against a loading control. Expression was plotted against time, and first-order kinetic curves were fitted to the data to determine t1 / 2 for off and on kinetics. c) Western blot analysis (D10) for AP-1 / bZIP and IRF family member proteins shown in control and JUN-CAR T cells. The numbers below represent the fold change in protein expression compared to CD19. d) Corresponding decrease in mRNA expression of BATF, BATF3, and JUNB in ​​JUN-HA-28z CAR T cells compared to HA-28z. n=3 donors, standardized against CD19 mRNA. e) IP-Western blot analysis shows that c-Jun overexpression reduces inhibitory JunB / BATF and JunB / BATF3 complexes. Input (left column), immunoprecipitation for c-Jun (middle column), or JunB (right column) in control or JUN-HA-28z CAR T cells. IRF4 protein, mRNA, and complexation with c-Jun remain unchanged. [Figure 26]Figures 26A-E show that JUN-CAR T cells enhance GD2-BBz CAR T cell function in solid tumors. a) Vector schematic of the JUN-GD2-BBz retroviral vector construct. b) IL-2 (left) and IFNg (right) production in JUN-modified (red) or control (blue) GD2-BBz CAR T cells 24 hours after stimulation with Nalm6-GD2 or 143B target cells. c) Lysis of GD2-BBz CAR T cells in GFP+ Nalm6-GD2 target cells at E:T ratios of 1:1 (left) or 1:4 (right). In a-c, error bars represent the mean ± SD of triple wells. Representative of at least three independent experiments. In d-e, NSG mice were inoculated with 0.5 x 10⁶ 143B-19 osteosarcoma cells by intramuscular injection. 1x10⁷ mock, GD2-BBz, or JUN-GD2-BBz CAR T cells were administered IV on day 3. d) Tumor growth was monitored by calyx measurement. e) Peripheral blood CD4+ (top) or CD8+ (bottom) T cell count 14 days after tumor implantation. Error bars represent the mean ± SEM of n=5 mice per group. Representative of two independent experiments, but early death (unrelated to tumor size) was excluded from the survival curve in both models. *p<.05, **p<.01, ***p<.001. HTM - hinge / transmembrane. ICD - intracellular domain. [Figure 27-1] Figures 27A-C demonstrate that the N-terminal mutation of c-Jun can rescue exhausted HA-28z CAR T cells. a) Different c-Jun mutations were cloned into HA-28z CAR T cell vectors. b) IL-2 (top) and IFNg (bottom) secretion 24 hours after co-culture with GD2+ 143B osteosarcoma target cells. c) In vitro lysis of GFP+ Nalm6-GD2 or 143B target cells was measured over 5 days at effector:target (E:T) ratios of 1:1, 1:2, or 1:4. At low E:T ratios and later time points, JUN-WT, JUN-AA, JUN-Dd, and JUN-DTAD showed improved control of tumor growth compared to JUN-De, JUN-Dbasic, JUN-DLeu, and JUN-DbZIP CAR T cells. [Figure 27-2]Figures 27D-E show that the N-terminal mutation in c-Jun can rescue exhausted HA-28z CAR T cells. d) Increased cytokine production was confirmed in both CD4+ and CD8+ HA-28z CAR T cells by intracellular cytokine staining and flow cytometry 5 hours after stimulation with Nalm6-GD2. e) Quantification of peripheral T cell counts 12 days after T cell injection into NSG mice with Nalm6-GD2 leukemia. [Figure 28] Figure 28 shows that knockdown of IRF4 dramatically increases the functional activity of exhausted HA-28z CAR T cells. [Figure 29-1] Figures 29A-B show that the transcription mutant (JUN-AA) also rescues functional activity and proliferative capacity in CD19 CAR T cells. [Figure 29-2] Figure 29C shows that the transcription mutant (JUN-AA) also rescues functional activity and proliferative capacity in CD19 CAR T cells. [Figure 30] Figure 30 shows that the enhanced in vivo function of c-Jun-modified HA-28z CAR T cells cannot be replicated by ex vivo supply of IL-2. [Figure 31-1] Figures 31A-D show that c-Jun enhanced Her2-BBz CAR T cell activity within the suppressive solid tumor microenvironment. [Figure 31-2] Figure 31E shows that c-Jun enhanced Her2-BBz CAR T cell activity within the suppressive solid tumor microenvironment. [Figure 32] Figure 32 shows that c-Jun overexpression increases resistance to TGFβ-mediated repression in exhausted HA-28z CAR T cells. [Figure 33] Figures 33A-D show that transcriptional changes in c-Jun modified cells are consistent with reduced fatigue and increased memory formation. [Modes for carrying out the invention]

[0026] definition For the purpose of interpreting this specification, the following definitions apply, and wherever appropriate, a term used in the singular also includes the plural and vice versa. In the event of any conflict between any definition set forth below and any document incorporated herein by reference, the definition set forth below shall prevail.

[0027] As used herein, the terms “disease” and “pathological condition” are used interchangeably to describe a deviation from the normal or mean state for a member of a species or group (e.g., human), unless otherwise indicated herein, such deviation is harmful to the affected individual under conditions that are not detrimental to the majority of individuals of such species or group. Such deviation may manifest as a condition, sign and / or symptom (e.g., diarrhea, nausea, fever, pain, blisters, swelling, rash, immunosuppression, inflammation, etc.) associated with any damage to the normal state of the subject or any of its organs or tissues that interferes with or alters the performance of normal function. A disease or pathological condition may be caused by or arise from contact with microorganisms (e.g., pathogens or other infectious agents (e.g., viruses or bacteria)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards and / or the environment), or may be responsive to an inherent or strong defect in the organism (e.g., genetic abnormality) or a combination of these and other factors.

[0028] The terms “host,” “subject,” or “patient” are interchangeable herein to refer to an individual being treated (e.g., administered) by the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., humans, mice, rats, monkeys, horses, cattle, pigs, dogs, cats, etc.). In the context of the present invention, the term “subject” generally means an individual that has been administered or has been administered one or more compositions of the present invention (e.g., genetically modified or reconstructed T cells as described herein).

[0029] "T-cell exhaustion" refers to the loss of T-cell function that can result from infection (e.g., chronic infection) or disease. T-cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, increased apoptosis, and decreased cytokine secretion. Therefore, terms such as "improvement of T-cell exhaustion," "inhibition of T-cell exhaustion," and "reduction of T-cell exhaustion" refer to a state of restored T-cell function characterized by one or more of the following: decreased expression and / or levels of one or more of PD-1, TIM-3, and LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased production and / or secretion of antigen-inducing cytokines (e.g., IL-2); enhanced killing capacity; increased recognition of tumor targets with low surface antigens; and enhanced antigen-responsive proliferation.

[0030] The term "buffer" or "buffering agent" refers to a material that, when added to a solution, makes the solution resistant to changes in pH.

[0031] As used herein, the terms “cancer” and “tumor” mean a tissue or development containing cells that have lost the ability to control their growth and proliferation. Cancer and tumor cells are generally characterized by the loss of contact inhibition, and may be invasive and metastatic (for example, cancer and tumor cells have lost the ability to adhere to other cells / tissues). The present invention is not limited by the type of cancer or the type of treatment (for example, treated prophylactically and / or therapeutically). In fact, various cancers, including but not limited to brain cancer or other cancers of the central nervous system, melanoma, lymphoma, bone cancer, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, kidney cancer, melanoma, renal cancer, prostate cancer, sarcoma, carcinoma and / or combinations thereof, may be treated with the compositions and methods described herein.

[0032] "Metastasis," as used herein, refers to the process by which cancer spreads or migrates from its site of origin to other areas of the body, resulting in the development of similar cancerous lesions in new locations. "Metastatic" or "metastatic" cells are cells that have lost their adhesive contact with neighboring cells and have migrated from the primary site of disease through the bloodstream or lymphatic system to invade tissues in other parts of the body.

[0033] As used herein, the term “anti-cancer agent” means any therapeutic agent (e.g., chemotherapeutic compounds and / or molecular therapeutic compounds), antisense therapy, or radiotherapy used to treat hyperproliferative diseases such as cancer (e.g., in mammals, e.g., in humans).

[0034] "Effective dose" means the amount of a pharmaceutical composition, anticancer agent, or other drug that is effective in the dose and time interval required to achieve the desired therapeutic or preventive outcome (e.g., relief of some or all symptoms of the disease being treated).

[0035] The term “therapeutic effective dose,” as used herein, means an amount of therapeutic agent sufficient to produce improvement in one or more symptoms of a disorder, prevent the progression of a disorder, or cause a regression of a disorder. For example, in relation to the treatment of cancer, in one embodiment, a therapeutic effective dose means an amount of therapeutic agent that reduces the rate of tumor growth (e.g., reduces and / or eliminates the tumor burden in a patient), reduces tumor mass, reduces the number of metastases, reduces tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0036] The terms “sensitize” and “to sensitize,” as used herein, mean making an animal or cells within an animal more sensitive or responsive to the biological effects of the second drug (e.g., acceleration or delay of aspects of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, vascularization, necrosis, or apoptosis) by administration of the first drug. The sensitizing effect of the first drug on target cells may be measured as the difference in the intended biological effects (e.g., acceleration or delay of aspects of cellular function including, but not limited to, cell growth, proliferation, invasion, vascularization, or apoptosis) observed when the second drug is administered with and without the first drug. The response of sensitized cells may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% compared to the response in the absence of the first drug.

[0037] As used herein, the terms “purified” or “to purify” mean the removal of impurities or undesirable compounds from a sample or composition. As used herein, the terms “substantially purified” mean the removal of approximately 70–90% to 100% of impurities or undesirable compounds from a sample or composition.

[0038] As used herein, the terms “administer” and “to administer” refer to the act of providing a composition to a subject. Examples of routes of administration to the human body include, but are not limited to, the eyes (ocular), mouth (oral), skin (transdermal), nose (transnasal), lungs (inhalation), oral mucosa (buccal), ear, rectum, injection (e.g., intravenous, subcutaneous, intraperitoneal, intratumor, etc.), and topical administration. In one embodiment, the administration of T cells of the present invention is by intravenous infusion.

[0039] As used herein, the terms “co-administration” and “co-administration” mean the administration of at least two drugs (or more) (e.g., genetically modified immune cells and one or more other drugs—e.g., anticancer drugs) or therapeutic agents to a subject. In some embodiments, the co-administration of two or more drugs or therapeutic agents is simultaneous. In other embodiments, the first drug / therapeutic agent is administered before the second drug / therapeutic agent. In some embodiments, co-administration may be via the same or different routes of administration. Those skilled in the art will understand that the preparation and / or routes of administration of the various drugs or therapeutic agents used may differ. Appropriate doses for co-administration can be readily determined by those skilled in the art. In some embodiments, when drugs or therapeutic agents are co-administered, each drug or therapeutic agent is administered at a lower dose than that appropriate for their single administration. Therefore, co-administration is particularly desirable in embodiments where co-administration of drugs or therapeutic agents reduces the required dose of one or more drugs that are potently harmful (e.g., toxic), and / or where co-administration of two or more drugs causes subject sensitization to one of the beneficial effects of the drugs due to co-administration of other drugs.

[0040] The term "pharmaceutically acceptable" as used herein means a composition that, when administered to a subject, substantially does not produce any adverse reactions (e.g., toxicity, allergies, or other immunological reactions).

[0041] As used herein, the term “pharmaceutically acceptable carrier” means any standard pharmaceutical carrier comprising, but not limited to, phosphate-buffered saline, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coating agents, sodium lauryl sulfate, isotonic and absorption retardants, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, etc. The composition may also include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants are described and known in the art (see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), as incorporated herein by reference).

[0042] As used herein, the term “pharmaceutically acceptable salt” means any salt of the compositions of the present invention (e.g., obtained by reaction with an acid or base) that is physiologically acceptable in a target subject. “Salts” of the compositions of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, sulfonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids, such as oxalic acid, are not inherently pharmaceutically acceptable but may be used to prepare salts that are useful as intermediates in obtaining the compositions of the present invention and their pharmaceutically acceptable acid addition salts. Examples of bases, though not limited to them, include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of the formula NW4+ (where W is a C1-4 alkyl group).

[0043] Examples of salts, though not limited to them, include acetate, adipine, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Other examples of salts include anions of the compounds of the present invention that are combined with suitable cations such as Na+, NH4+, and NW4+ (wherein W is a C1-4 alkyl group). For therapeutic use, salts of the compounds of the present invention are intended to be pharmaceutically acceptable. However, salts of pharmaceutically unacceptable acids and bases may also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0044] For therapeutic use, salts of the compositions of the present invention are intended to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of pharmaceutically acceptable compositions.

[0045] As used herein, the term “at risk of disease” means a subject who is predisposed to experiencing a particular disease (e.g., an infectious disease). This predisposition may be genetic (e.g., a specific genetic tendency to experience a disease, such as a hereditary disorder) or due to other factors (e.g., environmental conditions, exposure to harmful compounds in the environment, etc.). Therefore, the present invention is not intended to be limited to any particular risk (e.g., a subject may be “at risk of disease” simply by being exposed to and interacting with other people), nor is the present invention intended to be limited to any particular disease (e.g., cancer).

[0046] As used herein, the term “kit” means any delivery system for delivering materials. In the context of immunotherapeutic agents, such a delivery system may include a system that enables the storage, transport, or delivery of immunogenic factors and / or supporting materials (e.g., written instructions for using the materials) from one location to another. For example, a kit includes one or more encapsulations (e.g., boxes) containing the relevant immunotherapeutic agent (e.g., modified T cells and / or supporting materials). As used herein, the term “fragmented kit” means a delivery system comprising two or more separate containers, each containing a sub-part of the components of the entire kit. The containers may be delivered together or separately to intended recipients. For example, the first container may contain a composition comprising an immunotherapeutic composition for a particular use, while the second container contains a second drug (e.g., a chemotherapeutic agent). In practice, any delivery system comprising two or more separate containers, each containing a sub-part of the components of the entire kit, falls under the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system that contains all the components necessary for a particular application in a single container (for example, in a box containing each of the desired components). The term “kit” includes both fragmented kits and combined kits.

[0047] As used herein, the terms “immunoglobulin” or “antibody” refer to proteins that bind to one or more epitopes on a particular antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies, as well as the following classes: IgG, IgA, IgM, IgD, IgE, and the Fab and F(ab')2 fragments of secretory immunoglobulins (sIg). Immunoglobulins generally consist of two identical heavy chains and two light chains. However, the terms “antibody” and “immunoglobulin” also include single-chain and double-chain antibodies.

[0048] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The variable domain of the heavy chain may be referred to as "VH," and the variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of the antibody and contain the antigen-binding site.

[0049] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, where these domains are present on a single polypeptide chain. Generally, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure for antigen binding. For a review of scFv, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269–315.

[0050] As used herein, the term “antigen-binding protein” refers to a protein that binds to a specific antigen. Examples of “antigen-binding proteins” include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, and humanized antibodies; Fab fragments, F(ab')2 fragments, and Fab expression libraries; and single-chain antibodies.

[0051] As used herein, the term "epitope" refers to the portion of an antigen that creates contact with a particular immunoglobulin.

[0052] The term “specific binding” or “specific binding” is used in reference to the interaction between an antibody (or a part thereof, e.g., scFv) and a protein or peptide, where the interaction depends on the presence of a specific sequence or structure on the protein (e.g., an antigenic determinant or epitope); that is, an antibody (or a part thereof, e.g., scFv) generally recognizes and binds to a specific protein sequence or structure, rather than to the protein itself. For example, if an antibody is specific to epitope “A”, the presence of a protein containing epitope A (or free, unlabeled A) in a reaction involving labeled “A” and the antibody reduces the amount of labeled A that binds to the antibody.

[0053] As used herein, the terms “non-specific binding” and “background binding” refer to interactions that do not depend on the presence of a specific structure when used in relation to interactions between antibodies and proteins or peptides (i.e., antibodies bind to proteins in general, rather than to specific structures such as epitopes).

[0054] As used herein, the term “subject suspected of having cancer” means a subject who exhibits one or more symptoms indicative of cancer (e.g., a prominent lump or mass) or who has been screened for cancer (e.g., during a routine physical examination). A subject suspected of having cancer may also have one or more risk factors for developing cancer. Subjects suspected of having cancer are generally not tested for cancer. However, “subjects suspected of having cancer” include individuals who have undergone preliminary diagnosis (e.g., a CT scan showing a mass) but have not undergone confirmatory testing (e.g., biopsy and / or histology) or whose type and / or stage of cancer is unknown. The term further includes individuals who have previously had cancer (e.g., individuals in remission). A “subject suspected of having cancer” is sometimes diagnosed with cancer and sometimes found not to have cancer.

[0055] As used herein, the term “subject diagnosed with cancer” means a subject that has been tested and found to have cancerous cells. Cancer may be diagnosed using any appropriate method, including, but not limited to, biopsy, X-ray, blood tests, etc.

[0056] As used herein, the term “postoperative tumor tissue” means cancerous tissue (e.g., organ tissue) removed from the subject (e.g., during surgery).

[0057] As used herein, the term “subject at risk of cancer” means a subject who has one or more risk factors for developing a particular cancer. Risk factors include, but are not limited to, sex, age, genetic predisposition, environmental exposure, and previous cases of cancer, previous non-cancerous diseases, and lifestyle.

[0058] As used herein, the term “characterizing cancer in a subject” means, but is not limited to, the identification of one or more characteristics of a cancerous specimen in a subject, including the presence of benign, precancerous, or cancerous tissue and the stage of cancer.

[0059] As used herein, the term “characterizing tissue in a subject” means identifying one or more properties of a tissue sample, including, for example, the presence of cancerous tissue, precancerous tissue, and cancerous tissue that may metastasize.

[0060] As used herein, the term “stage of cancer” refers to a qualitative or quantitative assessment of the level of cancer progression. Criteria used to determine the stage of cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body, and where the cancer has spread (e.g., within the same organ or region of the body or to another organ).

[0061] As used herein, the term “primary tumor cells” means cancer cells isolated from tumors in mammals and not extensively cultured in vitro.

[0062] As used herein, the terms “treatment,” “therapeutic use,” or “medical use” mean any and all uses of the compositions and methods of the present invention to treat a disease state or symptom, or to prevent, inhibit, delay, or reverse the progression of any disease or other undesirable symptom in any other way. For example, the terms “treatment of cancer” or “treatment of tumor” or their grammatical equivalents herein mean suppression, regression, or partial or complete disappearance of a previously existing cancer or tumor. The definition includes any reduction in the size, aggressiveness, or growth rate of a previously existing cancer or tumor.

[0063] As used herein, the terms “improved therapeutic outcome” and “enhanced therapeutic efficacy” in reference to cancer mean a delay or reduction in the growth of cancer cells or solid tumors, or a reduction in the total number of cancer cells or total tumor burden. “Improved therapeutic outcome” or “enhanced therapeutic efficacy” means an improvement in the individual’s condition according to any clinically acceptable criteria, including established tumor reversal, increased life expectancy, or improved quality of life.

[0064] As used herein, the term “gene transfer system” means any means of delivering a composition containing a nucleic acid sequence to a cell or tissue. Examples of gene transfer systems include, but are not limited to, vectors (e.g., retrovirus, adenovirus, lentivirus, adeno-associated virus, and other nucleic acid-based delivery systems), microinjection of naked nucleic acids, polymer-based delivery systems (e.g., liposome-based and metal particle-based systems), biolistic injection, transduction using transposase-based systems for gene integration, Crispr / Cas9-mediated gene integration, and non-integrated vectors such as RNA or adeno-associated viruses.

[0065] As used herein, the term “viral gene transfer system” refers to a gene transfer system comprising viral elements (e.g., intact viruses, modified viruses, and viral components such as nucleic acids or proteins) to facilitate the delivery of a sample to a desired cell or tissue. Non-limiting examples of viral gene transfer systems useful in the compositions and methods of the present invention are lentiviral and retroviral gene transfer systems.

[0066] As used herein, the term “site-directed recombination target sequence” refers to a nucleic acid sequence that provides a recognition sequence for a recombinant and a site where recombination occurs.

[0067] As used herein, the term “nucleic acid molecule” means any nucleic acid-containing molecule, including, but not limited to, DNA or RNA. The term includes, but is not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine The sequence includes any known base analog of DNA and RNA, including nin, 5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, β-D-mannosyl eosin (queosine), 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, and 2,6-diaminopurine.

[0068] As used herein, the term “heterogene” refers to a gene that is not found in its natural environment. For example, a heterogene is a gene of one species that has been introduced into another species. Heterogenes also include genes that are natural to an organism that has been modified in several ways (e.g., by mutation, appending to multiple copies, or ligating to a non-natural regulatory sequence). Heterogenes are distinguished from endogenous genes in that heterogene sequences typically bind to DNA sequences that are not naturally associated with gene sequences within a chromosome, or that are not naturally found in parts of a chromosome (e.g., genes expressed at loci where the gene is not normally expressed). Cells containing heterogenes are described herein as “modified” or “reconstructed” cells. For example, T cells containing a heterologous AP-1 transcription factor gene (e.g., a heterologous AP-1 transcription factor gene expression construct (used to overexpress AP-1 transcription factor in T cells)) and / or a heterologous receptor gene (e.g., a heterologous T cell receptor gene expression construct or a heterologous chimeric antigen receptor gene expression construct) are described herein as modified and / or reconstructed T cells.

[0069] As used herein, the term “gene expression” refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by “transcription” of the gene (i.e., by the enzymatic action of RNA polymerase), and, in the case of protein-coding genes, into protein by “translation” of mRNA. Gene expression can be controlled at many stages in this process. “Upregulation” or “activation” refers to control that increases the production of gene expression products (i.e., RNA or protein), while “downregulation” or “repression” refers to control that decreases production. Molecules involved in upregulation or downregulation (e.g., transcription factors) are sometimes referred to as “activators” and “repressors,” respectively.

[0070] In addition to containing introns, the genomic morphology of a gene may also include sequences located at both the 5' and 3' ends of sequences present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located at 5' or 3' relative to untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters or enhancers that control or influence gene transcription. The 3' flanking region may contain sequences that induce transcription termination, post-transcriptional cleavage, and polyadenylation.

[0071] As used herein, the terms “coding nucleic acid molecule,” “coding DNA sequence,” and “coding DNA” refer to the order or sequence of deoxyribonucleotides along a chain of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Therefore, a DNA sequence codes for an amino acid sequence.

[0072] As used herein, the terms “oligonucleotide having a gene-coding nucleotide sequence” and “polynucleotide having a gene-coding nucleotide sequence” mean a nucleic acid sequence containing a gene coding region, i.e., a nucleic acid sequence that codes for a gene product. The coding region may exist in cDNA, genomic DNA, or RNA form. If present in DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., sense strand) or double-stranded. Appropriate regulatory elements, such as enhancers / promoters, splicing sites, or polyadenylation signals, may be located very proximal to the gene coding region when it is necessary to enable proper initiation of transcription and / or accurate processing of the main RNA transcript. Alternatively, the coding region used in the expression vector of the present invention may include endogenous enhancers / promoters, splicing sites, intervening sequences, polyadenylation signals, or a combination of both endogenous and exogenous regulatory elements.

[0073] The terms “in a manipulative combination,” “in a manipulative order,” and “manipulatively linked,” as used herein, refer to the linking of nucleic acid sequences in a manner that produces a nucleic acid molecule capable of inducing the transcription of a given gene and / or the synthesis of a given protein molecule. The terms also refer to the linking of amino acid sequences in a manner that produces a functional protein.

[0074] The term “isolated,” when used in relation to nucleic acids, such as “isolated oligonucleotide” or “isolated polynucleotide,” refers to a nucleic acid sequence that has been identified and separated from at least one component or contaminant to which the nucleic acid sequence is normally bound in its natural supply. Therefore, isolated nucleic acids exist in a form or configuration different from that which is found in nature. In contrast, non-isolated nucleic acids are similar to nucleic acids such as DNA and RNA, which are found in their natural state. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome proximal to an adjacent gene; an RNA sequence, such as a specific mRNA sequence encoding a particular protein, is found in a cell as a mixture with many other mRNAs encoding many proteins. However, examples of isolated nucleic acids encoding a particular protein include, for instance, when the nucleic acid is located at a chromosomal location different from its natural cell location, or when it is adjacent to a nucleic acid sequence otherwise different from that which is found in nature, and such nucleic acids are typically found in cells that express a particular protein. Isolated nucleic acids, oligonucleotides, or polynucleotides can exist in single-stranded or double-stranded form. When isolated nucleic acids, oligonucleotides, or polynucleotides are used to express proteins, the oligonucleotide or polynucleotide may contain at least a sense strand or a coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both a sense strand and an antisense strand (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0075] The term "natural protein," as used herein, indicates that a protein does not contain the amino acid residues encoded by the vector sequence; that is, a natural protein contains only those amino acids that would be present in a protein if it were naturally occurring. Natural proteins may be produced by recombinant means or isolated from naturally occurring sources.

[0076] As used herein, the term "protein" means, when relating to a protein (as in "a portion of a given protein"), a fragment of that protein. Fragments can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

[0077] As used herein, the term “vector” is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which a vector is bound. One type of vector is a “plasmid,” which is a circular double-stranded DNA to which further DNA fragments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, where further DNA fragments can be ligated into a viral genome. Some vectors (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors) can self-replicate in the host cell to which they are introduced. Lentiviral vectors or retroviral vectors may be used (e.g., to introduce DNA encoding one or more AP-1 transcription factors and / or CAR constructs into cells (e.g., T cells)). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, some vectors can induce the expression of a gene to which the vector is operationally linked. Such vectors are referred herein as “recombinant expression vectors” or simply “expression vectors.” Generally, expression vectors useful in recombinant DNA technology are sometimes in the form of plasmids. Since plasmids are the most commonly used vector form, the terms "plasmid" and "vector" may be used interchangeably in this specification.

[0078] The term “expression vector,” as used herein, refers to a recombinant DNA molecule containing the appropriate nucleic acid sequences necessary for the expression of a desired coding sequence and a operably linked coding sequence within a particular host organism. Nucleic acid sequences required for expression in prokaryotes typically include promoters, operators (optional), and ribosome-binding sites, sometimes in addition to other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0079] As used herein, the term "transfection" refers to the introduction of foreign DNA into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybren-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and gene guns.

[0080] The term "stable transfection" or "stable transfection" refers to the introduction and integration of foreign DNA into the genome of a transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into its genomic DNA. The term "transient transfection" or "transient transfection" refers to the introduction of foreign DNA into a cell in which the foreign DNA is not integrated into the genome of the transfected cell. The foreign DNA remains in the nucleus of the transfected cell (for example, for several days). During this time, the foreign DNA is used for regulatory control that governs the expression of endogenous genes within the chromosome. The term "transient transfectant" refers to a cell that has taken up foreign DNA but failed to integrate this DNA.

[0081] As used herein, the term “selectable marker” refers to the use of a gene encoding enzyme activity that confers the ability to grow in a medium lacking otherwise essential nutrients; further, selectable markers may confer resistance to antibiotics or drugs to cells expressing the selectable marker. Selectable markers can be “dominant”; dominant selectable markers encode enzyme activity that can be detected in any eukaryotic cell line. Examples of dominant selectable markers include the bacterial aminoglycoside-3'-phosphotransferase gene (also known as the neo gene) that confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase (hyg) gene that confers resistance to the antibiotic hygromycin, and the bacterial xanthine-guanine phosphoribosyltransferase gene (also known as the gpt gene) that confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are not dominant in that their use must be associated with cell lines lacking the relevant enzyme activity. An example of a non-dominant selectable marker is TK-negative (TK - ) Thymidine kinase (TK) gene used with cell lines, CAD gene used with CAD-deficient cells and HPRT-negative (HPRT - An example of a marker used in conjunction with a cell line is the mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene. A review of the use of selectable markers in mammalian cell lines is presented in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) pp.16.9-16.15.

[0082] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within such an environment. An in vitro environment may consist of, but is not limited to, test tubes and cell cultures. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and any process or reaction occurring within such a environment.

[0083] As used herein, the term “cell culture” means any in vitro culture of cells. This term includes serial cell lines (e.g., those with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., untransformed cells), and any other cell populations maintained in vitro.

[0084] As used herein, the term “sample” is used in its broadest sense. In one sense, it means a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and may include fluids, solids, tissues, and gases. Examples of biological samples include blood products such as plasma and serum. However, such examples are not to be construed as limiting the types of samples to which the present invention can be applied.

[0085] Detailed description of the form The present invention is based on the finding that T cells modified (e.g., genetically) to overexpress and / or contain elevated (e.g., hyperphysiological) levels of one or more AP-1 transcription factors (e.g., c-Jun) exhibit reduced levels of T cell exhaustion (compared to unmodified T cells expressing normal levels of AP-1 transcription factors). As described in detail herein, the expression of AP-1 transcription factors fos and c-Jun is lower / downregulated in exhausted T cells, and T cells modified to overexpress and / or have elevated levels of c-Jun or other AP-1 transcription factors exhibit reduced levels of T cell exhaustion. For example, overexpression of AP-1 transcription factors (particularly c-Jun) prevented the onset of T cell exhaustion and maintained T cell function (even after exposure to high levels of antigen) (see Examples 1-6). GD2 CAR T cells expressing increased levels of AP-1 transcription factors (particularly c-Jun) exhibited reduced exhaustion characteristics (including lower exhaustion markers, increased memory formation, and increased cytokine production), and T cells modified to overexpress and / or contain elevated (e.g., hyperphysiological) levels of one or more AP-1 transcription factors were shown to exhibit enhanced clinical efficacy across multiple malignancies (see, e.g., Examples 1–6). Furthermore, CD19 CAR T cells modified to overexpress c-Jun and CD22 CAR T cells modified to overexpress c-Jun both showed increased CAR T cell recognition of leukemia target cells with low levels of surface antigens (see, e.g., Example 6). CAR T cells modified to overexpress AP-1 transcription factors showed reduced T cell exhaustion and enhanced function in three separate in vivo tumor models using cJUN-modified CAR T cells (see Example 8). Therefore, overexpression of AP-1 transcription factors (particularly c-Jun) prevented the development of T cell exhaustion and maintained T cell function. Taken together, these findings indicate that the compositions and methods of the present invention are widely applicable and address many of the existing barriers to successful CAR T cell therapy.

[0086] Accordingly, the present invention provides modified T cells (e.g., genetically and / or functionally) that maintain function under conditions in which unmodified T cells exhibit T cell exhaustion. In this case, the compositions and methods of the present invention may be used to prevent exhaustion of remodeled T cells (e.g., remodeled to express a specific T cell receptor or a chimeric antigen receptor (CAR)), and to prevent exhaustion of unmodified T cells (e.g., natural or spontaneous T cells (e.g., isolated from a subject)), thereby enhancing the function (e.g., activity against cancer or infectious diseases) of both remodeled and unmodified T cells.

[0087] T cell modifications, such as overexpressing and / or containing elevated levels of one or more AP-1 transcription factors, may prevent the depletion of AP-1 transcription factors in T cells that occurs, for example, when T cells are exhausted (see, e.g., Example 1), and / or result in elevated (e.g., hyperphysiological) levels of AP-1 transcription factors. AP-1 transcription factors (e.g., c-Jun) are factors induced after T cell activation and are involved in the production and secretion of cytokines (e.g., interleukin-2) by T cells. T cells expressing CAR experience sustained antigen-independent signaling via the receptor clusterin, repeating the basic biology of T cell exhaustion, as indicated by high levels of PD-1, TIM-3, and LAG-3 expression, reduced antigen-induced cytokine production, and excessive programmed cell death. One transcription factor was identified and characterized as being reduced in exhausted T cells (see, e.g., Example 1). Modification of T cells to include overexpression and / or inclusion of one or more elevated levels of AP-1 transcription factor significantly enhanced the function of T cells exposed to conditions that induce T cell exhaustion (see, e.g., Examples 2-5). While understanding the mechanism is not required to carry out the present invention, and the invention is not limited to any particular mechanism of action, maintaining and / or increasing AP-1 transcription factor levels (e.g., c-Jun levels) in T cells prevents T cell dysfunction associated with T cell exhaustion (e.g., the minimal effect of AP-1 transcription factor overexpression was observed in non-exhausted T cells, and related or absolute deletion of c-Jun was shown to be a significant factor in T cell exhaustion-related dysfunction).

[0088] In some embodiments, T cells are modified to overexpress and / or contain elevated levels of one or more mutated and / or cleaved AP-1 transcription factors to prevent AP-1 transcription factor depletion in T cells. Experiments performed in the development of the embodiments herein demonstrate that some AP-1 family proteins (e.g., c-Jun) can be mutated and / or cleaved without affecting the ability of mutated / cleaved AP-1 factors to mediate the rescue of dysfunctional T cells. For example, c-Jun polypeptides with N-terminal deletions and mutations (e.g., in the transactivation domain) retain their ability to rescue the function of HA-28z-exhausted CAR T cells and maintain an equivalent increase in cytokine production, compared to c-Jun. In some embodiments, AP-1 (e.g., c-Jun) polypeptides include mutations or deletions in the transactivation and / or δ domain. In some embodiments, AP-1 (e.g., c-Jun) polypeptides include mutations or deletions that inactivate or omit the transactivation and / or δ domain. In some embodiments, the mutant / cleaved AP-1 (e.g., c-Jun) polypeptide contains 70% or more sequence identity (e.g., 70%, 75%, 100%, 150%, 200%, 250%, or a range between these), C-terminal portion (e.g., one-quarter, one-third, one-half), or C-terminal domain (e.g., ε, bZIP and its C-terminal amino acids) of the wild-type AP-1 transcription factor (e.g., c-Jun). In some embodiments, the N-terminal amino acid residues (e.g., 50, 75, 100, 150 residues or a range between them), the N-terminal portion (e.g., one-quarter, one-third, one-half), or the N-terminal domain (e.g., δ, the transactivation domain, and its N-terminal amino acids) of the wild-type AP-1 transcription factor (e.g., c-Jun) are deleted, mutated, or otherwise inactivated. Any embodiment described herein relating to the AP-1 transcription factor may include a mutated / cleaved AP-1 (e.g., c-Jun) polypeptide, which is not inconsistent with, for example, those described above.

[0089] Enhanced expression of AP-1 transcription factors such as c-Fos and c-Jun increased the function of modified T cells, but there are other inhibitory AP-1 family members that are expressed in exhausted activated T cells. Inhibition / knockdown of inhibitory AP-1 complex members (e.g., to increase the availability of canonical AP-1 factors) also reduced T cell exhaustion (see, e.g., Example 7). In particular, inhibition / knockdown of inhibitory AP-1 complex members resulted in a significant enhancement of T cell function (e.g., increased cytokine production and / or expression) in exhausted T cells, rather than in healthy T cells (see, e.g., Figures 8A-D). Accordingly, in some embodiments, the present invention provides compositions and methods for inhibiting T cell exhaustion by inhibiting the expression and / or activity of inhibitory AP-1 complex members (e.g., BATF3 and other BATF family members (BATF 1 and 2), IRF4, IRF8, and other IRF family members (IRF 1, 2, 3, 5, 6, 7, or 9), and ATF family members (ATF 1, 2, 3, 4, 5, 6, or 7)). The present invention is not limited to means of inhibiting the expression and / or activity of AP-1 inhibitory complex members (e.g., genes). For example, in some embodiments, the expression and / or activity of AP-1 inhibitory complex members occurs at the genomic level (e.g., by interference with gene expression). In other embodiments, the inhibition of the expression and / or activity of AP-1 inhibitory complex members occurs at the level of protein expression and / or activity (e.g., by interference with protein expression and / or activity). Exemplary compositions and methods for inhibiting the expression and / or activity of AP-1 inhibitory complex members include, but are not limited to, nuclease disruption, CRISPR-Cas9 systems, zinc finger nuclease targeting, TALEN genome editing, shRNA, siRNA, miRNA targeting, degron controllable promoter, protein inhibitors (e.g., chemoinhibitors, small molecule inhibitors, antibodies, etc.), and dominant-negative expression. Exemplary compositions for inhibiting the expression and / or activity of AP-1 inhibitory complex members are shown in Table 1 below.CAS stands for Chemical Abstracts Service and is a unique numerical identifier assigned to each chemical substance.

[0090] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0091] Accordingly, the present invention provides compositions and methods for reducing T cell exhaustion, comprising T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors, and / or modified to reduce the expression and / or activity of one or more AP-1 inhibitory complex members. The present invention is not limited to diseases or conditions that can be treated using the modified T cells of the present invention. In fact, the compositions and methods provided herein may be useful in treating any disease in which increased T cell activity may provide therapeutic benefits.

[0092] Accordingly, the present invention provides compositions comprising T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors, and / or modified (e.g., genetically) to reduce the expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB, BATF3 and other BATF family members, IRF4, IRF8 and other IRF family members, and ATF family members). As described in detail herein, the T cells may be remodeled or non-remodeled T cells (e.g., tumor-infiltrating lymphocytes (TILs)). Furthermore, the present invention is not limited to the type of T cells modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors. In some embodiments, the T cells are CD3+ T cells (e.g., a combination of CD4+ and CD8+ T cells). In some embodiments, the T cells are CD8+ T cells. In other embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are natural killer (NK) T cells. In some embodiments, the T cells are γδ T cells. In some embodiments, T cells are a combination of CD4+ and CD8 T+ cells (e.g., CD3+). In some embodiments, T cells are memory T cells. In some embodiments, T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and / or γδ T cells. In some embodiments, T cells are cytokine-induced killer cells. In some embodiments, T cells are remodeled to express chimeric antigen receptors. In other embodiments, T cells are remodeled to express specific T cell receptors (e.g., specific to tumor antigens or infectious disease antigens). In some embodiments, T cells are antitumor T cells. The composition may comprise a pharmaceutically acceptable carrier (e.g., a buffer). The composition may further comprise one or more other agents (e.g., chemotherapeutic agents (e.g., chemotherapeutic agents described herein) and / or antibacterial agents).Examples of antimicrobial agents include, but are not limited to, antibodies, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercury nitrate, thimerosal, and / or combinations thereof. The composition may optionally include one or more further agents, such as other drugs for treating T-cell exhaustion (e.g., anti-PD-1 checkpoint inhibitors such as nivolumab), or other pharmaceuticals used to treat a subject for an infection or disease associated with T-cell exhaustion (e.g., antiviral agents, antibiotics, antimicrobial agents, or anticancer drugs).

[0093] Antimicrobial therapeutic agents may be used as therapeutic agents in the compositions of the present invention. Any agent capable of killing, inhibiting, or otherwise weakening microorganisms, and any agent intended to have such activity, may be used. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptors, etc., used alone or in combination. In fact, but are not limited to, any type of antibiotic, including antimicrobial agents, antiviral agents, antifungal agents, etc., may be used.

[0094] Several strategies for targeting tumor antigens are known in the art. For example, GD2-targeted immunotherapy is currently in clinical and preclinical trials for several diseases, including neuroblastoma, osteosarcoma, and melanoma (see, e.g., Thomas et al., PLoS One, 2016. 11(3): p. e0152196; Long et al., Nature Medicine, 2015. 21(6): p. 581-590; Long et al., Cancer Immunology Research, 2016. 4(10): p. 869-880; Yu et al., N Engl J Med, 2010. 363(14): p. 1324-34; Perez Horta et al., Immunotherapy, 2016. 8(9): p. 1097-117; Heczey et al, Molecular Therapy). Unlike mAbs, which do not efficiently cross the blood-brain barrier, activated CAR T cells efficiently enter the CNS after adoptive transfer. Therefore, in one embodiment, any CAR T cells can be modified according to the compositions and methods of the present invention (e.g., modified to overexpress and / or contain elevated levels of one or more AP-1 transcription factors (e.g., thereby enhancing CAR T cell function and / or inhibiting CAR T cell exhaustion)).

[0095] For example, as described herein, modified T cells of the present invention (e.g., modified to overexpress and / or contain one or more AP-1 transcription factors at elevated levels, and / or modified (e.g., genetically) to reduce the expression and / or activity of one or more AP-1 inhibitory complex members (e.g., JunB and BATF3 and other BATF family members, IRF4, and ATF family members)) may also be reconstructed to contain a CAR including a target-specific binding element, otherwise referred to as an antigen-binding moiety. The selection of the moiety depends on the type and number of ligands that delimit the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigenic moiety domain in the CAR of the present invention include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0096] For example, a CAR can be modified to target a desired tumor antigen by altering the antigen-binding portion that specifically binds to the antigen on tumor cells. As used herein, “tumor antigen,” “hyperproliferative disorder antigen,” “antigen associated with hyperproliferative disorder,” or “cancer antigen” refers to an antigen common to certain hyperproliferative diseases, such as cancer. Illustrative antigens mentioned herein are given by example. This list is not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0097] Tumor antigens are proteins produced by tumor cells that exert an immune response, particularly a T-cell-mediated immune response. Therefore, the antigen-binding moiety can be selected based on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigens (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0098] Tumor antigens may include one or more antigenic cancer antigens / epitopes associated with malignant tumors. Malignant tumors express several proteins that can act as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphoma.

[0099] Tumor antigens can also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and do not occur on other cells in the body. TAAs are not specific to tumor cells and are instead expressed on some normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at considerably high levels on tumor cells.

[0100] Examples of TSAs or TAAs include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, and TRP-2, and tumor-specific multisystem antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, and HER-2 / neu; specific tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.291 / BCAA, CA 195, CA Examples include 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0101] Depending on the desired antigen to be targeted, the CAR can be remodeled to include an appropriate antigen-binding moiety specific to the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding moiety for incorporation into the CAR of the present invention.

[0102] Treatment method In another aspect, the present invention provides a method for treating a disease or condition in a subject (e.g., a patient) having the disease or condition, comprising the step of administering to the subject an effective amount of T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors. The present invention is not limited by the type of disease or condition being treated. In fact, any disease or condition that is treatable by T cell administration (e.g., the signs or symptoms of the disease are improved upon treatment) can be treated in an improved and more effective manner using the compositions and methods of the present invention (including and / or using T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors). In one embodiment, the disease or condition is cancer. In another embodiment, the disease or condition is an infectious disease. The present invention is not limited by the type of cancer or the type of infectious disease. In fact, any cancer known in the art to which T cell therapy is used for treatment can be treated using the compositions and methods of the present invention. Similarly, any infectious disease known in the art to which T cell therapy is used for treatment can be treated using the compositions and methods of the present invention. In one embodiment, administering an effective amount of T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors to a subject (e.g., a patient) with a disease or condition inhibits T cell exhaustion in the subject (e.g., what would otherwise occur if the same amount of unmodified T cells were administered to the subject). For example, in one embodiment, the method includes a method for treating a subject with a disease or condition responsive to adoptive cell therapy. In one embodiment, the method for treating a subject with a disease or condition responsive to adoptive cell therapy includes the step of administering a composition comprising an effective amount of T cells genetically modified to express elevated levels of one or more AP-1 transcription factors, wherein the T cells genetically modified to express elevated levels of one or more AP-1 transcription factors exhibit a reduced level of T cell exhaustion compared to T cells expressing normal levels of one or more AP-1 transcription factors. In one embodiment, the disease or condition includes cancer.

[0103] In one aspect, the present invention provides a method for treating cancer / tumors using T cells modified / remodeled to express one or more AP-1 transcription factors. In various aspects of the present invention, a method for treating cancer / tumors is provided, comprising the step of administering an effective amount of T cells modified / remodeled to express one or more AP-1 transcription factors to a patient having such cancer or tumor. The present invention is not limited by the type of cancer and / or tumor being treated. As used herein, the term “cancer” means any type of malignant neoplasm, most of which may invade surrounding tissues and metastasize to different sites (see, for example, the PDR Medical Dictionary, 1st edition (1995), incorporated herein by reference in its entirety for all purposes). The terms “neoplasm” and “tumor” mean abnormal tissue that grows by cell proliferation faster than normal and continues to grow after the stimulus that initiated proliferation has been removed. Such abnormal tissue exhibits structural organization and a partial or complete lack of functional coordination with normal tissue, which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor). Examples of common cancer categories include, but are not limited to, carcinomas (e.g., malignant tumors of epithelial cell origin, such as common forms of breast cancer, prostate cancer, lung cancer, and colon cancer), sarcomas (e.g., malignant tumors of connective tissue or mesenchymal cell origin), lymphomas (e.g., malignant diseases of hematological cell origin), leukemias (e.g., malignant diseases of hematological cell origin), germ cell tumors (e.g., tumors of totipotent cells, most frequently found in the testes or ovaries in adults; and in the midline of the body, especially at the tip of the coccyx, in fetuses, infants, and young children), and blastoblastic tumors (e.g., malignant tumors typically resembling immature or fetal tissue). Further examples of tumors and / or neoplasms that can be treated using the compositions and methods of the present invention include, but are not limited to, neoplasms associated with cancers of nerve tissue, hematopoietic tissue, breast, skin, bone, prostate, ovary, uterus, cervix, liver, lung, brain, pharynx, gallbladder, pancreas, rectum, parathyroid gland, thyroid gland, adrenal gland, immune system, head and neck, colon, stomach, bronchi, and / or kidneys. In some embodiments, the cancer is latent cancer, previously diagnosed primary cancer, or metastatic cancer.

[0104] In one embodiment, the presence of elevated levels of one or more AP-1 transcription factors in modified T cells (e.g., CAR T cells (compared to, e.g., unmodified T cells)) results in more effective treatment of cancer / tumors (e.g., killing and / or inhibiting progression) than treatment using T cells (e.g., CAR T cells) that are not modified to contain elevated levels of one or more AP-1 transcription factors.

[0105] In one embodiment, the present invention provides a method for treating cancer and / or tumors (e.g., inhibiting growth and / or killing) using T cells (e.g., CD3+ T cells) that are genetically modified to overexpress and / or contain elevated (e.g., hyperphysiological) levels of one or more AP-1 transcription factors, express a receptor that recognizes tumor surface antigens (e.g., CD19, CD20, CD22, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000 and / or p53), and have been remodeled to transmit signals that activate T cells to induce T cell expansion and / or tumor killing. Non-limiting examples of receptors include mAb-derived scFvs that recognize GD2, as well as chimeric antigen receptors (CARs) that incorporate a transmembrane domain and one or more intracellular signaling domains. CAR can further be remodeled to incorporate other signaling elements that facilitate the expansion of remodeled cells after encounter with tumor cell antigens (e.g., GD2 antigen), and elements that enable the long-term survival of remodeled cells. The present invention further provides compositions comprising genetically remodeled cells (e.g., immunotherapy compositions produced and administered in amounts sufficient to reach cancer and / or tumors).

[0106] Construction of modified T cells. As described herein, the present invention provides compositions comprising T cells that are genetically modified (e.g., transduced) to express one or more AP-1 transcription factors (e.g., heterologously). For example, the present invention provides transduced T cells. A “transduced cell” is a cell into which a nucleic acid molecule has been introduced using molecular biological techniques. T cells modified / transduced to express one or more heterologous AP-1 transcription factors can be transduced by any technique that can introduce a nucleic acid molecule into such cells, including, but not limited to, transfection with a viral vector (e.g., retrovirus, lentivirus or other viral vector), transformation with a plasmid vector via a CRISPR / Cas9-based system, and / or introduction of naked DNA by electroporation, lipofection and particle gun acceleration. Viral and / or plasmid vectors may be used for in vitro, in vivo and / or ex vivo expression. AP-1 transcription factors may be co-expressed with a remodeled TCR or CAR, and both the TCR and / or CAR may be co-expressed from the transcription factor as well as another viral vector. In another embodiment, they are expressed from a single vector construct using a bicistronic vector. C-Jun (and / or other AP-1 transcription factors) may be expressed constitutively or in a controlled manner (e.g., using a system that remotely controls expression via small molecules or using an endogenously controlled system). In another embodiment, the c-Jun and / or other AP-1 transcription factor genes may be genetically incorporated into cellular DNA using retrovirus, lentivirus or other viral vectors or by a CRISPR / Cas9-based system. In yet another embodiment, c-Jun and / or other AP-1 transcription factors are expressed via RNA or tumor-disintegrating viruses or other transient expression systems known in the art. C-Jun and / or other AP1 transcription factors (transcription fact) may be delivered to T cells ex vivo (e.g., said T cells used for adoptive transfer) or via in vivo gene transfer.

[0107] The present invention is not limited to chimeric antigen receptors (CARs) expressed in T cells (e.g., CAR constructs used in the methods of the present invention). In one embodiment, the CAR comprises a fusion protein (e.g., a single-stranded variable fragment (scFv)) of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin that binds specifically to a tumor antigen (e.g., GD2). Those skilled in the art will know that scFv is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin linked by a linker peptide (e.g., about 10 to about 25 amino acids). The present invention is not limited to the type of linker. In some embodiments, the linker is glycine-rich (e.g., for flexibility). In some embodiments, the linker contains serine and / or threonine (e.g., for solubility). In some embodiments, the linker contains a glycine-rich portion and a portion containing serine and / or threonine.

[0108] Any antibody / immunoglobulin that specifically binds to a tumor antigen (e.g., GD2) may be used to construct a CAR (e.g., using the VH and VL regions to construct a fusion protein scFv) for expression in immune cells used in the therapeutic methods of the present invention. Examples of such antibodies / immunoglobulins include, but are not limited to, GD2: 14G2a, ch14.18, hu14.18K322A, m3F8, hu3F8-IgG1, hu3F8-IgG4, HM3F8, UNITUXIN, DMAb-20, or any other antibody that specifically binds to GD2 (e.g., known, described, or subsequently identified in the art). A tumor antigen (e.g., GD2) CAR may include a receptor that incorporates a variant within the scFv of a tumor antigen (e.g., GD2) antibody, which is constructed to increase affinity and / or reduce persistent signaling. Tumor antigen (e.g., GD2)CARs may incorporate variable-length hinge regions and / or different transmembrane domains (e.g., between the scFv and the signaling domain). The present invention is not limited by the transmembrane domains used. In fact, any transmembrane domains, including all or part of the transmembrane domains of TCRζ chain (CD3ζ), CD28, OX40 / CD134, 4-1BB / CD137 / TNFRSF9, FcERIγ, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, or CD40, may be used.

[0109] The CAR construct of the present invention may include an intracellular signaling domain that transmits a ligand-binding event to an intracellular signal that (e.g., partially) activates T cells (e.g., the CD3ζ and / or other signaling domains of the innate T cell receptor complex (e.g., the MyD88 signaling domain)). In the absence of a co-stimulatory signal, receptor-ligand binding is sometimes insufficient for adequate activation and proliferation of T cells. Therefore, the CAR construct may include one or more co-stimulatory domains (e.g., providing a second signal to stimulate adequate T cell activation). In one embodiment, a co-stimulatory domain that increases CAR immune T cell cytokine production is used. In another embodiment, a co-stimulatory domain that facilitates T cell replication is used. In yet another embodiment, a co-stimulatory domain that prevents CAR T cell exhaustion is used. In yet another embodiment, a co-stimulatory domain that increases T cell antitumor activity is used. In yet another embodiment, a co-stimulatory domain that enhances CAR T cell survival (e.g., after injection into a patient) is used.Examples of proteins or their domains or parts that may be used to provide co-stimulatory signals include, but are not limited to, B7-1 / CD80;CD28;B7-2 / CD86;CTLA-4;B7-H1 / PD-L1;ICOS / CD278;ILRB / CD122;IL-2RG / CD132;B7-H2;PD-1;B7-H3;PD-L2;B7-H4;PDCD6;BTLA;4-1BB / TNFRSF9 / CD137;FcERIγ;CD40 ligand / TNFSF5;4-1BB ligand / TNFSF9;GITR / TNFRSF18;BAFF / BLyS / TNFSF13B;GITR ligand / TNFSF18;BAFF R / TNFRSF13C;HVEM / TNFRSF14;CD27 / TNFRSF7;LIGHT / TNFRSF14;CD27 ligand / TNFRSF7;OX40 / TNFRSF4;CD30 / TNFRSF8;OX40 ligand / TNFRSF4;CD30 ligand / TNFRSF8;TACl / TNFRSF13B;CD40 / TNFRSF5;2B4 / CD244 / SLAMF4;CD84 / SLAMF5;BLAME / SLAMF8;CD229 / SLAMF3;CD2 CRACC / SLAMF7;CD2F-10 / SLAMF9;NTB-A / SLAMF6;CD48 / SLAMF2;SLAM / CD150;CD58 / LFA-3;CD2;Ikaros;CD53;Integrin α4 / CD49d; Examples include CD82 / Kai-1;integrin α4β1;CD90 / Thy1;integrin α4β7 / LPAM-1;CD96;LAG-3;CD160;LMIR1 / CD300A;CRTAM;TCL1A;DAP12;TIM-1 / KIM-1 / HAVCR;dectin-1 / CLEC7A;TIM-4;DPPIV / CD26;TSLP;EphB6;TSLP R and HLA-DR. In one embodiment, CAR constructs expressed in T cells used in the compositions and methods of the present invention include the CD28 endodomain, the 4-1BB endodomain and / or the OX40 endodomain.In one embodiment, the tumor antigen (e.g., GD2) specific CAR construct of the present invention comprises an antibody scFv that specifically binds to the tumor antigen (e.g., GD2), a transmembrane domain (e.g., CD8), a T cell receptor intracellular signaling domain (e.g., TCRζ chain (CD3ζ)), and at least one costimulatory domain (e.g., 4-1BB).

[0110] The present invention is not limited to means of genetically expressing TCR, CAR, and / or one or more AP-1 transcription factors in T cells. In fact, any means known in the art and / or described herein may be used. Non-limiting examples of methods for genetically remodeling T cells include, but are not limited to, retroviral or lentiviral transduction, transduction using transposase-based systems for gene integration, Crispr / Cas9-mediated gene integration, non-integration vectors such as RNA or adeno-associated viruses, or other methods described herein. Compositions comprising remodeled T cells may incorporate non-remodeled T cells or other immune cells, or T cell subsets selected for greater expansion or survival capacity. To reduce toxicity, the incorporation of elements that enable the killing of remodeled cells may be incorporated. To reduce toxicity and / or increase efficiency, the incorporation of elements that enable the control of protein expression in remodeled cells may be incorporated.

[0111] Cancer treatment agents, compositions, and combination therapies. In some embodiments, the present invention provides a method for treating or slowing the progression of cancer or an infectious disease in an individual, comprising the step of administering an effective amount of the modified T cells of the present invention to the individual. In some embodiments, the treatment produces a sustained response in the individual after discontinuation of the treatment. The methods described herein may find use in the treatment of conditions where enhanced immunogenicity, such as increased oncoimmunogenicity for the treatment of cancer, is desirable. Methods for enhancing immune function in an individual with cancer are also provided herein, comprising the step of administering an effective amount of the modified T cells of the present invention to the individual. Any type of T cells genetically modified to express CARs and / or TCRs known in the art or described herein may be used in these methods.

[0112] In some embodiments, an individual has cancer that is resistant (e.g., indicated to be resistant) to one or more other forms of anticancer treatment (e.g., chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes recurrent or refractory cancer. Recurrent may mean the reappearance of cancer at the site of origin or a new site after treatment. In some embodiments, resistance includes the progression of cancer during treatment with chemotherapy. In some embodiments, resistance includes cancer that does not respond to conventional or traditional treatment with chemotherapeutic agents. Cancer may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, cancer is in the early or late stages.

[0113] In one embodiment, the present invention provides that exposure of animals suffering from cancer / tumors (e.g., humans) to a therapeutically effective dose of an immunotherapy composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors completely inhibits the growth of such cancer cells and / or makes such cancer cells a population more sensitive to cancer drugs or radiotherapy (e.g., their cell death-inducing activity). The immunotherapy compositions and methods of the present invention may be used for the treatment, improvement or prevention of any type of disorder such as cancer.

[0114] In one embodiment, an immunotherapy composition comprising T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors is used to treat, improve, or prevent cancers characterized by resistance to one or more conventional cancer therapies (e.g., cancer cells that are chemically resistant, radioactively resistant, or hormonally resistant). Any T cells genetically modified to express tumor-specific CARs as described herein may be used in the immunotherapy compositions and methods of the present invention.

[0115] The immunotherapy compositions and methods of the present invention (including, for example, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors) may be used to induce cytotoxic activity against tumor cells and / or promote cell survival and function (e.g., survival and function of modified immune cells). For example, the immunotherapy compositions and methods of the present invention may be used to induce interleukin-2 (IL-2) to promote T cell survival; to induce Fas ligand (FasL) and / or tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) (e.g., to induce tumor cell apoptosis); and / or to induce interferon (IFN)γ (e.g., to activate an innate immune response (e.g., against cancer)). In some embodiments, the compositions and methods of the present invention may be used to induce cell cycle arrest and / or apoptosis, and also to enhance the induction of cell cycle arrest and / or apoptosis, either alone or in response to further apoptosis-inducing signals. In some embodiments, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors sensitize cancer cells, including cells that are normally resistant to such inducible stimuli, to cell cycle arrest and / or apoptosis induction.

[0116] In some embodiments, the compositions and methods of the present invention are used to treat diseased cells, tissues, organs, or pathological conditions and / or disease states in animals (mammalian patients, including, but not limited to, humans and companion animals). In this regard, various diseases and conditions respond sensitively to treatment or prevention using the methods and compositions. In some embodiments, the cancer cells to be treated are metastatic. In other embodiments, the cancer cells to be treated are resistant to anticancer agents.

[0117] Some aspects of the present invention provide an effective amount of T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors, as well as a method for administering at least one further therapeutic agent (including, but not limited to, chemotherapeutic antineoplastic agents, apoptosis regulators, antibacterial agents, antiviral agents, antifungal agents, and anti-inflammatory agents) and / or therapeutic techniques (e.g., surgical intervention and / or radiotherapy). In certain embodiments, the further therapeutic agent (one or more) is an anticancer agent.

[0118] Several suitable anticancer agents are intended for use in the methods of the present invention. In fact, the present invention intends to administer many anticancer agents, including, but is not limited to, apoptosis-inducing agents; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biomimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., anticancer drugs, toxins, antibodies conjugated with defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all trans-retinoic acids); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; vascularization inhibitors; proteasome inhibitors; NF-KB modulators; anti-CDK compounds; HDAC inhibitors, etc. Many other examples of chemotherapy compounds and anticancer therapies suitable for co-administration with the disclosed possible substances are known to those skilled in the art.

[0119] In one embodiment, an anticancer agent includes a drug that induces or stimulates apoptosis. Drugs that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, antibodies against TRAIL, TRAIL-R1, or TRAIL-R2); and kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitors, vascular growth factor receptor (VGFR) kinase inhibitors, fibroblast growth factor receptor (FGFR) kinase inhibitors, platelet-derived growth factor receptor (PDGFR) kinase inhibitors). and Bcr-Abl kinase inhibitors (such as Gleevec); antisense molecules; antibodies (e.g., Herceptin, Rituxan, Zevalin and Avastin); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398 and non-steroidal inhibitors). Non-steroidal anti-inflammatory drugs (NSAIDs); anti-inflammatory drugs (e.g., butazolidin, decadron, deltazone, dexamethasone, dexamethasone intensol, dexone, hexadrol, hydroxychloroquine, meticorten, oradexone, orasone, oxyfenbutazone, pediapred, phenylbutazone, plaquenil, prednisolone, prednisone, prelon) Examples include E) and TANDEARIL); and cancer chemotherapy drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, Mylotarg, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitib, bevacizumab, taxotere or taxol); cell signaling molecules; ceramides and cytokines; staurosporine, etc.

[0120] In yet another embodiment, the compositions and methods of the present invention are used in conjunction with at least one anti-overgrowth agent or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and / or animal-derived compounds).

[0121] Suitable alkylating agents for use in this composition and method include, but are not limited to, 1) nitrogen mustards (e.g., mechloretamine, cyclophosphamide, ifosphamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethyleneimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozosin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimide-azolecarboxamide).

[0122] In some embodiments, suitable antimetabolites for use in the present composition and method include, but are not limited to: 1) folate analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), phloxuridine (fluorodeoxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).

[0123] In further embodiments, suitable chemotherapeutic agents for use in the compositions and methods of the present invention include, but are not limited to, 1) vinca alkaloids (e.g., vinblastine (VBL), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon α); 6) platinum coordination complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracendiones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyureas); 9) methylhydrazine derivatives (e.g., procarbazolone). Examples include (N-methylhydrazine; MIH); 10) adrenal cortical inhibitors (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide); and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).

[0124] Any tumor-disintegrating agent commonly used in the context of cancer therapy may also be used in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration maintains a formulary of tumor-disintegrating agents approved for use in the United States. The international counterparts of the U.S. FDA maintain similar formulary formularies.

[0125] Further examples of anticancer agents include compounds identified as having anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplaston, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, and BMS. 247550, Bortezomib, Briostatin-1, Buserelin, Calcitriol, CCI-779, CDB-2914, Cefixime, Cetuximab, CG0070, Silengitide, Clofarabine, Combretastatin A4 Phosphate, CP-675, 206, CP-724, 714, CpG 7909, Curcumin, Decitabine, DENSPM, Doxelcalciferol, E7070, E7389, Ectinacidin 743, Efaproxial, Eflornithine, EKB-569, Enzastaurin, Erlotinib, Exisulind, Fenretinide, Flavopyridol, Fludarabine, Flutamide, Fotemustine, FR901228, G17DT, Galiximab, Gefitinib, Genistein, Gluphosphamide, GTI-2040, Histrelin, HKI-272, Homohalintin, HSPPC-96, hu14.18-Interleukin-2 fusion protein, HuMax-CD4, Iloprost, Imiquimod, Infliximab, Interleukin-12, IPI-504, Ilofluben, Ixabepirone, Lapatinib, Lenalidomide, Restaurtinib, Leuprolide, LMB-9 immunotoxin, Ronafarnib, Luniliximab, Maphosphamide, MB07133, MDX-010, MLN2704, Monoclonal antibody 3F8, Monoclonal antibody J591, Motexafine, MS-275, MVA-MUC1-IL2, Niltamide, Nitrocamptothecin, Nolatrexed dihydrochloride, Nolvadex, NS-9, O6-Benzylguanine, Oblimersen sodium, ONYX-015, Olegovomab, OSI-774, Panitumumab, Paraplatin, PD-0325901, Pemetrexed, PHY906, Pioglitazone, Pirfenidone, Pixantrone, PS-341, PSC 833, PXD101, Pyrazoloacridine, R115777, RAD001, Ranpirinase, Rebeccamycin analog, Rhu angiostatin protein, RhuMab 2C4, Rosiglitazone, Lubitecan, S-1, S-8184, Satraplatin, SB-, 15992, SGN-0010, SGN-40, Sorafenib, SR31747A, ST1571, SU011248, Suberoylanilide Hydroxamic Acid, Suramin, Talabostat, Tarampanel, Tariquidar, Temsirolimus, TGFa-PE3 These include 8 immunotoxins, thalidomide, simalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimethrexate glucuronide, TroVax, UCN-1, valproic acid, vinflunin, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, diloiton, and zoskidal trihydrochloride.

[0126] The present invention provides methods for administering the compositions and methods of the present invention in conjunction with (for example, before, during, or after) radiotherapy. The present invention is not limited by the type, amount, or delivery and administration system used to deliver the therapeutic dose of radiation to the animal. For example, the animal may receive photoradiotherapy, particle beam radiotherapy, other types of radiotherapy, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In yet another embodiment, the radiation is delivered using a gamma knife.

[0127] The source of radiation can be external or internal to the animal. External radiotherapy is the most common and involves directing a beam of high-energy radiation through the skin to the tumor site, for example, using a linear accelerator. Although the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals. Internal radiotherapy involves implanting a radiation source, such as beads, wires, pellets, capsules, or particles, inside the body at or near the tumor site, including the use of a delivery system that specifically targets cancer cells (for example, using particles attached to cancer cell binding ligands). Such implants can be removed after treatment or left inactive in the body. Types of internal radiotherapy include, but are not limited to, close-range radiotherapy, gap radiotherapy, cavitation brachytherapy, and radioimmunotherapy.

[0128] The animal may optionally be given radiosensitizers (e.g., metronidazole, misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5-substituted-4-nitroimidazole, 2H-isoindoledione, [[(2-bromomethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol, nitroaniline derivatives, DNA-affinity hypoxia-selective cytotoxins, halogenated DNA ligands, 1,2,4-benzotriazine oxide, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalators, 5-thiotretrazole derivatives, 3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivatives, texaphrins hydroxide, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, fever (high fever), etc., and radioprotective agents (e.g., cysteamine, aminoalkyl dihydrogen phosphorothioates, amiphostine (WR 2721), IL-1, IL-6, etc.) may be used. Radiosensitizers enhance the killing of tumor cells. Radioprotective agents protect healthy tissue from the harmful effects of radiation.

[0129] Any type of radiation may be administered to animals, insofar as the dose of radiation is tolerable by the animal without having unacceptable negative side effects. Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiotherapy (e.g., high linear energy radiation). Ionizing radiation is defined as radiation containing particles or photons that produce ionization, i.e., gain or lose electrons (as described, for example, in US 5,770,581, which is incorporated herein by reference in whole). The effects of radiation can be controlled, at least in part, by a clinician. In one embodiment, the dose of radiation is subdivided to reduce maximum target cell exposure and toxicity.

[0130] In one embodiment, the total dose of radiation administered to an animal is approximately 0.01 Gray (Gy) to approximately 100 Gy. In another embodiment, approximately 10 Gy to approximately 65 Gy (e.g., approximately 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) is administered during the course of treatment. In some embodiments, the entire dose of radiation may be administered over a single day, although the total dose is ideally subdivided and administered over several days. Preferably, radiotherapy is administered over a course of at least approximately 3 days, for example, at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (approximately 1 to 8 weeks). Therefore, the daily dose of radiation includes approximately 1–5 Gy (e.g., approximately 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy) or 1–2 Gy (e.g., 1.5–2 Gy). The daily dose of radiation should be sufficient to induce the destruction of targeted cells. In one embodiment, when extended over a period of time, radiation is not administered daily, thereby allowing the animal to rest and achieve the therapeutic effect. For example, radiation is preferably administered for 5 consecutive days and then withheld for 2 days between each week of treatment, thereby resulting in 2 days of rest per week. However, depending on the animal's responsiveness and any possible side effects, radiation may be administered 1 day / week, 2 days / week, 3 days / week, 4 days / week, 5 days / week, 6 days / week, or a total of 7 days / week. Radiotherapy may be initiated at any time during the treatment period. In one embodiment, radiation may be initiated in week 1 or week 2 and administered for the remainder of the treatment period. For example, radiation may be administered in weeks 1-6 or 2-6 of a treatment period including 6 weeks to treat, for example, a solid tumor. Alternatively, radiation may be administered in weeks 1-5 or 2-5 of a treatment period including 5 weeks. However, these exemplary radiotherapy administration schedules are not intended to limit the invention.

[0131] In some embodiments of the present invention, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors, and one or more therapeutic or anticancer agents, are administered to animals under one or more conditions, such as by different periods, different durations, different concentrations, or different routes of administration. In some embodiments, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are administered before the therapeutic or anticancer agent, for example, 0.5, 1, 2, 3, 4, 5, 10, 12, 18 hours or more, 1, 2, 3, 4, 5, 6 days or more, or 1, 2, 3, 4, 5, 6 weeks or more before the administration of the therapeutic or anticancer agent. In some embodiments, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are administered after a therapeutic or anticancer agent, for example, 0.5, 1, 2, 3, 4, 5, 10, 12, 18 hours or more, 1, 2, 3, 4, 5, 6 days or more, or 1, 2, 3, 4, 5, 6 weeks or more, following the administration of the anticancer agent. In some embodiments, the modified T cells, modified to express and / or contain elevated levels of one or more AP-1 transcription factors, and the therapeutic or anticancer agent are administered simultaneously but on different schedules, for example, the modified immune cells are administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, once every four weeks or more. In another embodiment, T cells modified to express and / or contain elevated levels of one or more AP-1 transcription factors are administered once a week while a therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, once every four weeks, or more frequently.

[0132] The compositions within the scope of this invention include all compositions, wherein T cells modified to express and / or contain one or more AP-1 transcription factors at elevated levels are included in amounts effective to achieve their intended purpose. While individual needs differ, determining the optimal range of effective amounts of each component is within the scope of the art of this field. In one non-limiting example, T cells modified to express and / or contain one or more AP-1 transcription factors at elevated levels are administered to humans at a dose of 1000-1000 per day (e.g., to treat cancer). 10 To provide T cells, it can be administered to mammals, such as humans. In another embodiment, 1000-10 10 Modified T cells are administered to treat, improve, or prevent cancer (e.g., to prevent metastasis, recurrence, and / or progression of cancer). A unit dose may be administered once or more daily in one or more administrations (e.g., over 1, 2, 3, 4, 5, 6 days or more, or over a week or more).

[0133] T cells may be administered as part of a pharmaceutical formulation that includes a suitable pharmaceutically acceptable carrier, which includes excipients and adjuvants to facilitate the processing and / or administration of modified cells into a pharmaceutically usable formulation. T immune cells and / or pharmaceutical formulations containing said cells may be administered intravenously, intramuscularly, subcutaneously, intratumorally, intraperitoneally, intrasacrally, or intravenously. An effective dose of T cells and / or pharmaceutical formulations containing said cells may be administered for the prevention or treatment of disease. The appropriate dose may be determined based on the type of disease being treated, the type of modified T cells, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0134] The efficacy of any of the methods described herein (e.g., treatment using T cells alone or in combination with one or more chemotherapeutic agents described herein) may be tested in various methods known in the art, such as clinical or preclinical models. Appropriate preclinical models are illustrated herein. In any exemplary model, after the development of a tumor, mice are randomly placed into a treatment group receiving either treatment or a control treatment. Tumor size (e.g., tumor volume) is measured during the course of treatment, and overall survival is also monitored.

[0135] In some embodiments, a sample is obtained as a baseline to measure the response to treatment prior to treatment with T cells (e.g., alone or in combination with other therapies described herein). In some embodiments, the sample is a tissue sample (e.g., formalin-fixed and paraffin-embedded (FFPE), archival, fresh or frozen). In some embodiments, the sample is whole blood. In some embodiments, the whole blood contains immune cells, circulating tumor cells, and any combination thereof.

[0136] Response to treatment can mean one or more of the following: extending survival (including overall survival and survival without progression); producing a desired response (including a complete or partial response); or improving the signs or symptoms of cancer. In some embodiments, responsiveness can mean improvement in one or more factors according to the published set of RECIST guidelines for determining the tumor state in cancer patients, i.e., response, stabilization, or progression. For a more detailed description of these guidelines, see Eisenhauer et al., Eur J Cancer 2009;45: 228-47; Topalian et al., N Engl J Med 2012;366:2443-54; Wolchok et al., Clin Can Res 2009;15:7412-20; and Therasse, P., et al. J. Natl. Cancer Inst. 92:205-16 (2000). A responsive subject is a subject whose cancer(s) show improvement according to one or more factors, for example, based on RECIST criteria. A non-responsive subject is a subject whose cancer(s) do not show improvement according to one or more factors, for example, based on RECIST criteria.

[0137] Conventional response criteria may not be adequate for characterizing the antitumor activity of immunotherapies, as they can produce delayed responses that may be preceded by the first apparent radiographic progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that account for the possible appearance of new lesions and allow for the confirmation of radiographic progression in subsequent evaluations. Thus, in some embodiments, responsiveness may refer to an improvement in one or more factors according to the Immune-Related Response Criteria 2 (irRC). See, for example, Wolchok et al., Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are added to a given tumor burden and lead to, for example, radiographic progression in subsequent evaluations. In some embodiments, the presence of non-target lesions is included in the evaluation of the complete response but not in the evaluation of radiographic progression. In some embodiments, radiographic progression may be determined based solely on measurable disease criteria and / or confirmed by consecutive evaluations for >4 weeks from the first recorded date.

[0138] This specification is considered sufficient to enable those skilled in the art to carry out the invention. In addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. All publications, patents and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0139] Examples The following examples illustrate, but are not limiting, the compounds, compositions, and methods of the present invention. Other appropriate modifications and applications of various conditions and parameters commonly encountered in clinical therapy and obvious to those skilled in the art are within the spirit and scope of the invention.

[0140] material and method Viral vector construction MSGV retroviral vectors encoding the following CARs:CD19-28z, CD19-BBz, GD2-28z, GD2-BBz, Her2-BBz, and CD22-BBz have been previously described. To construct the HA-28z CAR, a point mutation was introduced into the 14G2a scFv of the GD2-28z CAR plasmid to create the E101K mutation. To reduce Fc receptor recognition for in vivo use of HA-28z CAR T cells, the "4 / 2NQ" mutation 46 was introduced into the CH2CH3 domain of the IgG1 spacer region. Codon-optimized cDNAs encoding c-Jun (JUN), c-Fos (FOS), and cleaved NGFR (tNGFR) were synthesized by IDT and cloned into lentiviral expression vectors to create JUN-P2A-FOS, which co-expresses tNGFR under a different PGK promoter, as well as JUN and FOS single-expression vectors. Next, JUN-P2A was subcloned upstream of the CAR reader sequence at the XhoI site of the MSGV CAR vector using the In-Fusion HD Cloning Kit (Takara) to create the JUN-P2A-CAR retroviral vector. Point mutations were introduced into JUN-AA, converting Ser63 and Ser73 to Ala. The E. coli DHFR-DD sequence was inserted upstream of Jun to create the JUN-DD construct. In some cases, GFP cDNA was subcloned upstream of the CAR to create a GFP-P2A-CAR vector control.

[0141] Viral vector creation Retroviral supernatants were prepared in 293GP packaging cell lines as previously described. Briefly, 20ug of MSGV vector plasmid and 10ug of RD114 envelope plasmid DNA were co-transfected with Lipofectamine 2000 into 70% confluent 293GP 20cm plates. The medium was changed 24 and 48 hours after transfection. The 48HR and 72HR viral supernatants were collected, centrifuged, and cell residue removed, and frozen at -80C for future use. Third-generation, self-inactivated lentiviral supernatants were prepared in 293T packaging cell lines as previously described. In short, 18ug pELNS vector plasmid and 18ug pRSV-Rev, 18ug pMDLg / pRRE (Gag / Pol), and 7ug pMD2.G (VSVG envelope) packaging plasmid DNA were co-transfected in 70% confluent 293T 20cm plates using Lipofectamine 2000. The medium was changed 24 hours after transfection. The 24HR and 48HR viral supernatants were collected and combined and concentrated by ultracentrifugation at 28,000 RPM for 2.5 hours. The concentrated lentivirus stock was frozen at -80C for future use.

[0142] T cell isolation Primordial human T cells were isolated from healthy donors using the RosetteSep Human T Cell Enrichment Kit (Stem Cell Technologies). Buffycoat cells were purchased from Stanford Blood Center and processed using Lymphoprep density gradient medium and SepMate-50 tubes according to the manufacturer's protocol. The isolated T cells were stored in CryoStor CS10 cryopreservation medium (Stem Cell Technologies) at a rate of 2 x 10⁶ cells per vial. 7 T cells were cryopreserved.

[0143] CAR T cell production Cryogenically stored T cells were thawed and activated on the same day using Human T-Expander CD3 / CD28 Dynabeads (Gibco) in T cell medium (AIMV supplemented with 5% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U / mL penicillin, and 100 ug / mL streptomycin (Gibco)) at a bead:cell ratio of 3:1. Recombinant human IL-2 (Peprotech) was provided at 100 U / mL. Two and three days after activation, T cells were transduced with a retroviral vector and administered 0.5–1 x 10¹⁶ cells per mL of T cell medium containing IL-2. 6 Cells were maintained. Unless otherwise indicated, CAR T cells were used for in vitro assays or transplanted into mice 10-11 days after activation.

[0144] Retroviral trait introduction Non-tissue culture-treated 12-well plates were coated overnight at 4C with 1 mL of Retronectin (Takara) at 25 ug / mL in PBS. The plates were washed with PBS and blocked with 2% BSA for 15 minutes. Thawed retrovirus supernatant was added at approximately 1 mL per well, and the plates were centrifuged at 32C and 3200 RPM for 2 hours, after which cells were added.

[0145] cell line Kelly neuroblastoma, EW8 Ewing sarcoma, 143b, and TC32 osteosarcoma cell lines were obtained originally from ATCC. Several cell lines were stably transduced with GFP and firefly luciferase (GL). The CD19+CD22+ Nalm6-GL B-ALL cell line was provided by David Barrett. Nalm6-GD2 was prepared by co-transducing Nalm6-GL with cDNA for GD2 and GD3 synthesis. Single-cell clones were then selected for high GD2 expression. Nalm6-22KO and 22low have been previously described and kindly provided by Terry Fry. All cell lines were cultured in complete medium (RPMI (Gibco) supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U / mL penicillin, and 100 U / mL streptomycin).

[0146] Flow cytometry CD22 and Her2 CARs were detected using human CD22-Fc and Her2-Fc recombinant proteins (R&D). Idiotype antibodies and Fc fusion proteins were conjugated using the Dylight488 and / or 650 antibody labeling kit (Thermo Fisher). T cell surface phenotypes were evaluated using the following antibodies. From BioLegend: CD4-APC-Cy7 (clone OKT4), CD8-PerCp-Cy5.5 (clone SK1), TIM3-BV510 (clone F38-2E2), CD39-FITC or APC-Cy7 (clone A1), CD95-PE (clone DX2), CD3-PacBlue (clone HIT3a), from eBioscience: PD1-PE-Cy7 (clone eBio J105), LAG3-PE (clone 3DS223H), CD45RO-PE-Cy7 (clone UCHL1), CD45-PerCp-Cy5.5 (clone HI30), from BD: CD45RA-FITC or BV711 (clone HI100), CCR7-BV421 (clone 150503), CD122-BV510 (clone Mik-b3), CD62L-BV605 (clone DREG-56), CD4-BUV395 (clone SK3), CD8-BUV805 (clone SK1).

[0147] Cytokine production 1x10 5 CAR+ T cells and 1x10 5 Tumor cells were cultured for 24 hours in 200 uL of CM in a 96-well flat-bottom plate. For idiotype stimulation, serial dilutions of 1A7 were cross-linked overnight at 4°C in 1X Coating Buffer (BioLegend) on a Nunc Maxisorp 96-well ELISA plate (Thermo Scientific). The wells were washed once with PBS, and 1x10 5 CAR+ T cells were plated in 200 uL of CM and cultured for 24 hours. Triplicate wells were plated for each condition. The culture supernatants were harvested and analyzed for IFNγ and IL-2 by ELISA (BioLegend).

[0148] Lysis assay 5x10 4 GFP+ leukemia or 2.5x10 4GFP+ adherent tumor cells were co-cultured with CAR T cells in 200 μL CM in 96-well flat-bottom plates for up to 96 hours. Triple wells were cultured for each condition. Plates were imaged every 2-3 hours using the IncuCyte ZOOM Live-Cell analysis system (Essen Bioscience). Four images per well were collected at 10X zoom at each time point. Total integrated GFP intensity per well was evaluated as the quantitative standard for live and GFP+ tumor cells. Values ​​were standardized against the initial measurement and plotted over time. The E:T ratio is shown in the figure caption.

[0149] Western blot and immunoprecipitation Whole-cell protein lysates were obtained in non-denaturing buffer (150 mmol / L NaCl, 50 mmol / L Tris-pH8, 1% NP-10, 0.25% sodium deoxycholate). Protein concentration was estimated by Bio-Rad colorimetric assay. 20 μg of protein was loaded onto an 11% PAGE gel for immunoblotting, and then transferred to a PVF membrane. Signal detection was performed by enhanced chemiluminescence (Pierce) or using the Odyssey imaging system. Representative blots are shown. The following primary antibodies were purchased from Cell Signaling: c-Jun (60A8), Pc-JunSer73 (D47G9), JunB (C37F9), BATF (D7C5), and IRF4 (4964). The BATF3 (AF7437) antibody was from R&D. Immunoprecipitation was performed using 100 mg of whole-cell protein lysate and 7.5 mg of agar conjugate antibody c-Jun (G4) or JunB (C11) (Sant Cruz Biotechnology) in 150 μL of non-denaturing buffer. After incubation at 4°C overnight, the beads were washed three times with non-denaturing buffer, the proteins were eluted in Laemmli sample buffer, boiled, and loaded onto PAGE gels. Detection of the immunoprecipitated proteins was performed using the reagents and antibodies described above.

[0150] mouse Immunocompromised NOD / SCID / IL2Rg- / -(NSG) mice were purchased from JAX and housed in the room. All mice were housed, contained, and treated according to the Stanford University IACUC (APLAC) approved protocol. 6-8 week old mice were administered 1x10⁶ intravenously (IV). 6 Nalm6-GL leukemia or intramuscular (IM) injection of 0.5-1x10 6 One of the 143B osteosarcoma tumors was inoculated. All CAR T cells were IV injected. Time and therapeutic dose are shown in the figure caption. Leukemia progression was measured by bioluminescence imaging using the IVIS imaging system. Values ​​were analyzed using bioimaging software. Solid tumor progression was followed using calipas measurement of injected leg area. Five mice were treated per group in each experiment, and each experiment was repeated two or three times as shown. Mice were randomized to ensure equal pre-treatment tumor load before CAR T cell therapy.

[0151] Blood and tissue analysis At the indicated time points, peripheral blood samples were collected posteriorly orbitally under isoflurane anesthesia. 50 μL of blood was labeled with CD45, CD3, CD4, and CD8, lysed with BD FACS Lysing Solution, and quantified using CountBright Absolute Counting beads (Thermo Fisher) on a BD Fortessa flow cytometry system.

[0152] ATAC-seq ATAC-seq library preparation was performed as previously described48. Briefly, 100,000 cells from each sample were classified into CM by FACS, centrifuged at 500 g and 4°C, and then resuspended in ATAC-seq Resuspension Buffer (RSB) (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin. After separating the samples into two replicates, all subsequent steps were performed. The samples were incubated on ice for 3 minutes and then washed with 1 mL RSB supplemented with 0.1% Tween-20. The nuclei were pelleted at 500 g for 10 minutes at 4°C. The nuclear pellet was resuspended in 50 μL of transposition mixture (25 μL 2x TD buffer, 2.5 μL transposase (Illumina), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween-20, 5 μL H2O) and incubated in a thermomixer at 37°C for 30 minutes with shaking at 1000 RPM. The reaction was cleaned using the Qiagen MinElute PCR Purification Kit. The library was PCR amplified using NEBNext Hi-Fidelity PCR Master Mix and custom primers (IDT) as previously described.20 After 5 cycles of PCR, the library was sufficiently amplified as shown by the qPCR fluorescence curve.20 The library was purified using the Qiagen MinElute PCR Purification Kit and quantified using the KAPA Library Quantification Kit. At the Stanford Functional Genomics Facility, libraries were sequenced on Illumina NextSeq using paired-end 75bp readouts. Adapter sequences were trimmed using SeqPurge and aligned with the hg19 genome using bowtie². Mitochondrial readouts were then filtered using Picard tools for low mapping quality (Q>=20) and PCR duplicates.Next, the inventors converted the bam file to a bed file to obtain the Tn5 modification insertion site ("+" strand +4bp, "-" strand -5bp). To identify the peaks, the inventors determined the peaks for each sample using MACS2 with the insertion bed file: "--shift -75 --extsize 150 --nomodel --call-summits --nolambda --keep-dup all -p 0.00001". To obtain a union peak set, the inventors (1) expanded all vertices to 500bp, (2) merged all vertex bed files, and then (3) used bedtools clustering to select the vertices (summits) with the highest MACS2 scores. This was then filtered using the ENCODE hg19 blacklist (https: / / www.encodeproject.org / annotations / ENCSR636HFF / ) to remove peaks that extended beyond the ends of the chromosome. Next, the inventors annotated these peaks using HOMER and computer-calculated the occurrence of TF motifs using motifmatchr in R with the chromVARMotifs HOMER set. To create sequencing tracks, the inventors loaded the Tn5 modification insertion sites into R and created a binned coverage pileup using rtracklayer, binned every 100 bp. The inventors then counted all insertions within each peak to obtain a count matrix (peak x sample). To determine differential peaks, the inventors first used peaks annotated as "TSS" or "Housekeeping Peaks" for DESeq2 as control genes, and then calculated differential peaks by standardization. All clustering was performed using regularized log-transformed values ​​derived from DESeq2. Transcription factor motif bias analysis was performed using chromVAR as previously described.21TF motif enrichment was calculated using a hypergeometric test in R to test motif representation (from the motifmatchr described above) for a subset of peaks versus all peaks.

[0153] subset RNA-seq For T cell subset-specific RNA-seq, T cells were isolated from healthy donor baffycote as described above. Prior to activation, naive and central memory CD4+ or CD8+ subsets were isolated using the BD FACSAria cell classifier (Stem Cell FACS Core, Stanford University School of Medicine) with the following markers: naive (CD45RA+CD45RO-, CD62L+, CCR7+, CD95-, and CD122-) and central memory (CD45RA-CD45RO+, CD62L+, CCR7+). The classified starting populations were activated, transduced, and cultured as described above. On days 7, 10, and 14 of culture, CAR+ CD4+ and CD8+ cells were classified, and RNA was isolated using the Qiagen mRNEasy kit. Samples were library-prepped and sequenced via the Illumina NextSeq pair-end platform by the Stanford Functional Genomics Core.

[0154] bulk RNA-seq For bulk RNA isolation, healthy donor T cells were prepared as described. Total mRNA was isolated from 2x10⁶ bulk CAR T cells using the Qiagen RNEasy Plus mini isolation kit on day 10 or 11 of culture. Bulk RNA-seq was performed by BGI America (Cambridge, MA) using a BGISEQ-500 platform with a single-end 50bp read length at 30x10⁶ reads per sample. Principal component analysis was performed using plots with the stats package and ggplot2 package in R (version 3.5)49. Gene set enrichment analysis was performed using GSEA software (Broad Institute) as described50,51.

[0155] Single-cell RNA-seq To compare gene expression in single CD19-CAR and GD2-CAR T cells, we classified a naive T cell subset on day 0 for subsequent single-cell analysis on day 10 using the Chromium platform (10x Genomics) and the Chromium Single Cell 3' v2 Reagent Kit, according to the manufacturer's instructions. cDNA libraries were prepared separately for CD19-CAR and GD2-CAR cells, and CD4+ and CD8+ cells were combined in each run and bioinformatically separated downstream. Sequencing was performed on an Illumina NextSeq system (paired ends, 26 bp for read 1 and 98 bp for read 2) to a depth of >100,000 reads per cell. Single-cell RNA sequencing reads were aligned to the Genome Reference Consortium Human Build 38 (GRCh38) to normalize for batch effects and filtered for cellular events using Cell Ranger software (10X Genomics). The total number of T cells, consisting of 804 CD19-CARs and 726 GD2-CARs, was sequenced up to an average of 350,587 readouts per cell after standardization. The cell-gene matrix was further processed using Cell Ranger R Kit software (10X Genomics) as described.52 Briefly, the inventors first selected genes with a unique molecular identifier (UMI) number ≥ 1 in any given cell. Then, for each cell, the UMI number was standardized against the total UMI and multiplied by the median UMI number of all cells. The data was then transformed by adopting the natural logarithm of the resulting data matrix.

[0156] statistical analysis Unless otherwise noted, statistical analysis for significant differences between groups was performed using unpaired (upaired) two-sided t-tests without estimating consistent standard deviations using GraphPad Prism.7 For bulk RNA-seq in Figure 2C, a nonparametric Wilcoxon paired signed-rank test was used. Survival curves were compared using the log-rank Mantel-Cox test. Tables containing complete statistical analyses, including exact p-values, t-ratios, and dof, may be found in the supplementary materials.

[0157] Example 1 Gene expression analysis of T cell exhaustion Antigen-independent sustained signaling by chimeric antigen receptors (CARs) can increase T cell differentiation and exhaustion, thereby limiting the capacity of these cells. For example, GD2-specific CARs have been described to autoaggregate in the absence of antigen, leading to activation of the chronic downstream T cell activation signaling cascade. GD2-CARs incorporating the CD28 costimulatory domain rapidly develop prominent features of T cell exhaustion, while GD2-CARs incorporating the 4-1BB costimulatory domain, despite similarly agglutinating and signaling, show less evidence of T cell exhaustion and maintain greater function. The most prominent features of T cell exhaustion in GD2-28z-expressing T cells include increased surface expression of inhibitory receptors (e.g., PD1, TIM3, LAG3, CD39) upon antigen stimulation, decreased expression of memory markers (e.g., CD62L and CCR7), and decreased cytokine production (particularly IL2).

[0158] To evaluate gene transcription analysis in exhausted versus non-exhausted T cells, experiments were conducted during the development of embodiments of the present invention. Decreased expression of AP-1 family members was found in GD2-28Z CAR T cells compared to GD2-BBZ (non-exhausted CAR) cells (see Figure 1a). Classical AP-1 partners FOS and JUN were also among the higher-level, downregulated genes in GD2-28Z CAR T cells compared to healthy CD19-28Z CAR T cells, as determined by RNA sequencing (see Figure 1b). The AP-1 family of transcription factors is activated downstream of TCR signaling and regulates a broad and diverse array of important T cell functions, including growth, apoptosis, cytokine production, and effector function. Further experiments were conducted during the development of embodiments of the present invention to evaluate and characterize the functional roles of AP-1 family members in CAR T cells (e.g., whether the absence of AP-1 family members in CAR T cells contributes to their CAR T cell phenotype).

[0159] Example 2 Construction of CAR T cells with forced expression of c-Jun and c-Fos To determine whether AP-1 substitution could alleviate symptoms of exhaustion in GD2-28Z CAR T cells, we constructed lentiviral expression constructs with forced expression of c-Jun and c-Fos under a constitutive promoter (see Figure 2a). This construct also encoded a truncated nerve growth factor receptor (NGFR (tNGFR)) expression cassette, which acts as a surface marker for T cell transduction. Subsequently, activated primary human T cells were transduced with or without the AP-1 expression vector (AP-1) to CD19, GD2-BBZ, or high-affinity (HA) GD2-28Z CAR.

[0160] Example 3 Expression of c-Jun and c-Fos in CAR T cells On day 8 of T cell culture, AP-1 transducible CD4 or CD8 CAR T cells were classified using NGFR. Constitutive expression of AP-1 reduced the frequencies of exhaustion-related inhibitory receptors PD1, TIM3, LAG3, and CD39, and increased the memory marker CD62L in both CD4 (see Figure 2b) and CD8 (see Figure 2c) CAR T cells. To assess functional changes in AP-1 transducible CAR T cells, CAR T cells with or without AP-1 cotransduction were co-cultured with CD19 and GD2 antigen-expressing tumor cells. Under most conditions, AP-1 transducible CAR T cells released more IL2 (see Figure 2d) and IFN□ (see Figure 2e) compared to those without.

[0161] Example 4 CAR T cells with forced expression of c-Jun or c-Fos To evaluate whether the functional benefits of AP-1 expression require both c-Fos and c-Jun expression, individual lentiviral vectors encoding either c-Jun or c-Fos alone were constructed (see Figure 3a). CAR T cells were transduced with either the c-Fos or c-Jun encoding vector.

[0162] Upon CAR T cell stimulation in antigen-positive tumor cells, only CAR T cells expressing c-Jun showed an increase in IL2, while c-Fos expression alone was insufficient (see Figure 3b). IFN□ secretion was similar, but the effect of c-Jun overexpression was dramatically lower (see Figure 3c). To confirm the increase in cytokine expression in c-Jun-expressing CAR T cells at the individual cell level, intracellular cytokine staining (ICS) was performed by flow cytometry 6 hours after T cell stimulation in antigen-positive tumor cells. In most cases, c-Jun-transduced CAR T cells showed increased frequency and / or mean fluorescence intensity (MFI) of IL2 (see Figure 3d), IFN□ (see Figure 3e), and TNFa (see Figure 3f) compared to control cells (wo), while c-Fos transduction generally resulted in decreased cytokine production compared to the control. These data suggest that c-Jun overexpression can increase both the frequency of CAR T cells responding to antigen stimulation and the levels of cytokines produced by individual CAR T cells upon antigen encounter.

[0163] Example 5 Construction of a bicistronic vector co-expressing c-Jun and CAR derived from the same vector. Since c-Jun has been shown to be a major cause of increased activity in AP-1-transduced CAR T cells, we constructed bicistronic retroviral vectors co-expressing c-Jun and CAR from the same vector, isolated by the viral 2A ribosome skipping peptide sequence (see Figure 4a). These constructs ensure that any CAR+ T cells also co-express c-Jun, eliminating the need for co-transduction and classification to achieve a purely double-positive population. To test the functional effects of c-Jun re-expression across multiple antigen specificities and tumor indicators, eight different CAR vectors: CD19-28Z, CD19-BBZ, CD22-28Z, CD22-BBZ, GD2-28Zshort, GD2-28ZLong, HA(GD2)-28ZLong, and GD2-BBZ were separately cloned into the c-Jun backbone.

[0164] c-Jun expression in combination with most CARs resulted in decreased expression of surface fatigue markers (see Figures 4b and 5a). Enhanced cytokine secretion was observed in GD2 CAR T cells co-expressing c-Jun compared to control CAR T cells co-expressing GFP (see Figures 4d-e, 5c, and 6a-b).

[0165] c-Jun mediated increased cytokine secretion in response to both leukemia (Nalm6-GD2) and GD2+ pediatric solid tumors: Ewing sarcoma (EW8), osteosarcoma (143B), and neuroblastoma (Kelly), highlighting the broad clinical applicability of c-Jun-enhanced GD2 CAR T cells. Furthermore, c-Jun overexpression promoted an increase in the frequency of central memory CAR T cells (see Figure 5b). Using intracellular cytokine staining (ICS), increased pro-inflammatory cytokine production was also observed at the individual cell level in both CD4+ and CD8+ c-Jun-CAR T cells (Figures 4c and 6c-e). We also noted that a decrease in the production of the anti-inflammatory cytokine IL10 in c-Jun CAR T cells compared to controls (see Figure 6f) suggests that enhanced c-Jun expression may contribute to the enhancement of the Th1 cytokine profile.

[0166] Example 6 c-Jun replacement and functional activity in different CAR T cells As detailed herein, c-Jun substitution in exhausted CAR T cells (e.g., GD2 CAR T cells) can improve exhaustion in CAR T cells. However, lower exhaustion markers and a trend toward increased functional activity were also observed in healthy CD19 CAR T cells. While CD19 CAR T cells mediated a significant clinical response in B-ALL patients, an increase in relapses occurred in up to 30% of patients with CD19-low or negative disease. CD22 CARs, an alternative strategy for targeting B-cell malignancies, can also be limited by low CD22 antigen density in some patient leukemia cells. Therefore, experiments were conducted during the development of embodiments of the present invention to evaluate and characterize the activity of c-Jun-CD19 and c-Jun-CD22 CAR T cells against normal (Nalm6) or low-antigen-expressing tumor cells (Nalm6-F and Z or Nalm6-22low for CD19) (see Figure 7).

[0167] c-Jun did not enhance CD19 CAR activity in response to high antigen levels on Nalm6, but there was a significant improvement in IL2 (see Figure 7a) and IFN□ (see Figure 7b) in response to CD19-low Nalm6 clones Z and F. In the INCUCYTEImmune Cell Killing Assay (see Figure 7c), three GFP+ Nalm6 cell lines were co-cultured with CAR T cells for 92 hours. All four CD19 CARs killed the original Nalm6 tumor (measured by the decline in GFP intensity over time). Both CD19-28Z and c-Jun-CD19-28Z were able to kill CD19-low tumor cell clones F and Z, but only CD19-BBZ CAR T cells had a moderate effect. The addition of c-Jun-CD19-BBZ CAR T cells showed a significant increase in killing CD19-low clones compared to 19-BBZ alone. Enhanced cytokine secretion by CD22 CAR T cells co-expressing c-Jun (particularly CD22-BBZ) was also observed in response to both parental Nalm6 and Nalm622low (see Figures 7d-e).

[0168] Example 7 Inhibition of AP-1 inhibitory complex members reduces T cell exhaustion. While enhanced c-Fos and c-Jun expression may increase the function of modified T cells, other inhibitory AP-1 family members are expressed in exhausted and activated T cells. Therefore, experiments were conducted during the development of embodiments of the present invention to determine and characterize whether inhibition / knockdown of inhibitory AP-1 complex members (e.g., to increase the availability of canonical AP-1 factor) could reduce T cell exhaustion (e.g., increase T cell function). The CRISPR-Cas9 gRNA system was designed to target potent inhibitory AP-1 members. Cytokine production from JUNB and BATF3 gene-edited (knockout, KO) CAR T cells was evaluated. JUNB knockdown significantly enhanced IL2 and IFNg production from exhausted HA-GD2-28Z and GD2-BBZ CAR T cells, but had no effect on CD19 CAR T cells (see Figures 8A-C). This indicates that JUNB knockdown had a positive effect on exhausted T cells rather than healthy T cells. Similarly, BATF3 knockout also increased IL2 (not IFNg) production from exhausted HA-28Z CAR T cells compared to control edited T cells (see Figure 8D).

[0169] Example 8 In vivo efficacy of c-Jun-modified CAR T cells The in vivo efficacy of c-Jun-modified CAR T cells was evaluated in several different tumor models. Exhausted HA-GD2 CAR T cells expressing c-Jun showed superior therapeutic in vivo activity compared to unmodified HA-GD2 CAR T cells in a Nalm6 leukemia model (N6-GD2) that was remodeled to express GD2 (see Figure 9). c-Jun-modified GD2-BBZ CAR T cells showed superior in vivo activity in an aggressive osteosarcoma solid tumor model (see Figure 10). Finally, CD19 CAR T cells expressing c-Jun showed enhanced in vivo activity against low antigen density Nalm6 clone F (see Figure 11).

[0170] Example 9 Expression of HA-28z CAR in human T cells rapidly induces prominent characteristics of T cell exhaustion. The GD2-28z CAR is a fatigued phenotype in human T cells following the expression of a CAR incorporating the GD2-specific 14g2a scFv, TCRζ, and CD28 endodomains as a result of sustained signaling mediated by antigen-independent aggregation (Reference 9, which is incorporated herein by reference in its entirety). Experiments conducted during the development of the embodiments herein have shown that the expression of a CAR containing 14g2a scFv with the E101K point mutation that gives high affinity (HA) interaction with GD219 (HA-28z CAR) similarly induces fatigue in human T cells, although it exhibits a more severe phenotype (Figures 12 and 18a-c). In contrast to CD19-28z CAR T cells, HA-28z CAR T cells, when stimulated with CD19+GD2+ Nalm6 leukemia, exhibited pronounced phenotypic and functional features of exhaustion, including decreased expression in culture (Figure 12a), increased surface expression of inhibitory receptors PD-1, TIM-3, LAG-3, and CD39 (Figures 12b and 18d), impaired effector differentiation and poor memory formation (Figures 12c and 18e), and decreased IFN-γ and significantly reduced IL-2 production (Figure 12d).

[0171] To better understand the molecular basis of T cell exhaustion in this system, the transcriptome of HA-28z was compared to CD19-28z CAR T cells. Purified naive (N) and central memory (CM) T cells were transduced with HA or CD19-28z CAR, and then RNA was isolated on days 7, 10, and 14 of culture. Classification of a previously selected subset allowed for evaluation of the impact of T cell differentiation state and the distinction between CD4 and CD8 exhaustion in the development of T cell exhaustion in this model. Principal component analysis (PCA) across all 24 samples revealed that the most powerful driver of variation was the presence of HA- vs CD19-28z CAR (PC1, 39.3% variation, Figure 12e), and the model that persistent signaling in HA-28z CAR T cells drives exhaustion in all T cell subsets tested was not contradicted. However, the distinction was observed based on the starting differentiation state, as N vs CM was reflected between the CD4 vs CD8 populations driving PC2 (22.88% variation) (Figure 18f) and PC3 (11.9% variation) (Figure 12e and 18f).

[0172] Genes involved in activation (IFNG, GZMB, IL2RA), inhibitory receptors (LAG3, CTLA4), and several inflammatory chemokines / cytokines (CXCL8, IL13, IL1A) are among the top 200 genes driving PC1 (most differentially expressed in HA- vs. CD19-28z CAR T cells across all subsets) (Figure 12f), while genes downregulated in HA-28z CAR T cells include genes associated with naive and memory T cells (IL7R, TCF7, LEF1, and KLF2). Using GSEA, we showed that genes upregulated in HA-28z vs. CD19-28z CAR T cells on day 10 overlap with a previously described set of exhaustion-related genes in the chronic LCMV mouse model 13 (Figure 18g). Although the degree of exhaustion was less pronounced in GD2-28z CAR T cells, differential gene expression analysis of single-cell GD2-28z versus CD19-28z CAR T cells revealed similar gene expression profiles (Figure 19). In summary, these data qualify HA-28z and GD2-28z expressing T cells as models for testing human T cell exhaustion.

[0173] T cell exhaustion is associated with changes in chromatin accessibility in murine models and human patients with chronic viral infection and cancer (References 12, 17; incorporated herein by reference in their entirety). Chromatin accessibility analysis using ATAC-seq20 (Figure 20) of N or CM-driven CD4+ and CD8+ HA-28z vs CD19-28z CAR T cells showed significant changes in the epigenetic signature on day 10 of culture (Figure 12g), with CD8+ HA-28z CAR T cells showing >20,000 unique chromatin accessible regions (peaks) (FDR < 0.1 and log2FC > 1) compared to <3,000 unique peaks in CD8+ CD19-28z CAR T cells. These patterns of changes in exhaustion-induced chromatin accessibility were similar in CD4+ T cells (Figure 21a). Similar to transcriptome analysis, PCA revealed HA- vs CD19-CAR as the most powerful driver of differential chromatin states (PC1 variance 79.6%, Figure 12h), with weaker but significant differences observed between N vs CM cells (PC2 variance 7.4%), and between CD4 vs CD8 subsets (PC3 variance 6.5%) (Figure 21b). Clustering of the top 5,000 differentially accessible regions (peaks) revealed overall similar chromatin accessibility in HA-28z CAR T cells regardless of the starting subset (Figure 12i). HA-28z CAR T cells showed increased chromatin accessibility at proximal regulatory sites of exhaustion-related genes such as CTLA4, and decreased accessibility at proximal regulatory sites of memory-related genes such as IL7R (Figure 12j).

[0174] Example 10 Powerful AP-1 signature in exhausted CAR T cells revealed by epigenetic and transcriptional analyses To identify transcriptional programs predicted to be dysregulated by epigenetic changes induced in exhausted T cells, we compared the bias of transcription factor (TF) motifs between the open chromatins of exhausted and healthy CAR T cells. Using ChromVAR analysis (reference 21; incorporated herein by reference in its entirety), we identified 25 most differential motifs across all eight samples, many of which were found to belong to the AP-1(bZIP) family (Figure 13a). Similarly, TF motif enrichment analysis revealed that AP-1 / bZIP and bZIP / IRF-binding motifs were among those most significantly enriched in exhausted CAR T cells (Figures 13b and 21c).

[0175] Clustering of differentially accessible peaks based on shared TF motif enrichment identified four clusters associated with exhausted HA-28z CAR T cells (Figure 21d, EX1-EX4). The exhaustion-associated clusters contained peaks proximal to genes such as BTLA, CD39, IFNG, and CTLA4, suggesting common TF regulation of exhaustion-associated genes. All four exhaustion-associated clusters showed strong enrichment for AP-1 and AP-1-related family TFs, naturally demonstrating widespread AP-1 TF regulation by exhaustion-associated gene control. Strong enrichment for NFkB, NFAT, and RUNX TF family motifs was also observed in some of the exhaustion clusters, indicating that a subset of exhaustion-associated genes may be regulated by these transcriptional programs and can reproduce the epigenetic signature of exhaustion observed in other models (references 12, 17, 22; all of which are invoked herein by reference). Clusters associated with healthy CD19-28z CAR T cells (HLT1-2) showed a similar profile to clusters strongly associated with the naive initiation subset. This observation is consistent with the idea that healthy CAR T cells maintain an epigenetic signature more closely resembling naive-derived T cells, a subset associated with increased persistence and efficacy in adoptive T cell therapy (reference 23; incorporated herein by reference in its entirety), but is not inconsistent with the idea that chronic antigen stimulation leads to a wide range of diverse epigenetic reprogramming.

[0176] AP-1-related TFs harmonize to form diverse sets of homodimers and heterodimers via interactions within a common bZIP domain and can dimerize with IRF transcription factors (references 14, 24; all of which are incorporated herein by reference). AP-1 factor complexes compete for binding to DNA elements containing the core TGA-G / C-TCA consensus motif. IL-2 transcription is driven by activation of complexes, such as those containing the classically described AP-1 heterodimers c-Fos and c-Jun. Conversely, other AP-1 and IRF family members can directly attenuate c-Jun activity and / or drive immunoregulatory gene expression in T cells (references 14, 24-29; all of which are incorporated herein by reference). To assess whether altered AP-1 binding chromatin accessibility is associated with increased availability of activating and inhibitory bZIP and IRF TFs, experiments comparing transcript levels of bZIP and IRF family members using RNA-seq in exhausted HA-28z versus healthy CD19-28z CAR T cells were conducted during the development of the embodiments herein. Paired RNA-seq analysis across three different donors revealed consistent patterns of overexpression of bZIP and IRF family members, with JUNB, FOSL1, BATF, BATF3, ATF3, ATF4, and IRF4 being the most significant (Figures 13c and 22a). Western blot analysis confirmed sustained protein overexpression of JunB, IRF4, and BATF3 in HA- versus CD19 CAR T cells (Figures 13d and 22b), with immunoregulatory BATF / IRF TF showing higher levels of expression compared to c-Jun. The increased levels of inhibitory bZIP / IRF family members were further demonstrated by Western blotting of Jun immunoprecipitation (IP) or by the explanation that several inhibitory family members are in direct complex with c-Jun and JunB in HA-28z-exhausted CAR T cells (Figure 22c).Single-cell RNA-seq analysis of CD8+ T cells expressing CD19-28z versus GD2-28z CARs revealed that bZIP family members JUN, JUNB, JUND, and ATF4 were among the most differentially expressed and were widely involved in the exhausted GD2-28z CAR T cell network (Figures 13e and 19).

[0177] Example 11 c-Jun overexpression (OE) reduces functional exhaustion in CAR T cells. Exhausted CAR T cells exhibit very poor IL-2 production (Figure 12d), and based on evidence of preferential overexpression of bZIP and IRF transcription factors that drive immune regulation and exhaustion-related programs, it was hypothesized that T cell dysfunction in exhausted cells might be due to a relative deletion in the c-Jun / c-Fos heterodimer necessary to drive IL-2 transcription. HA-28z and CD19-28z CAR T cells were co-transfected with bicistronic lentiviral vectors to overexpress c-Jun and c-Fos. HA-28z CAR T cells remodeled to overexpress AP-1 showed increased IL-2 production upon antigen stimulation (Figures 23a-c). However, enhancement of functionality using a single expression vector was observed only in HA-28z CAR T cells with c-Jun OE, and transduction with a c-Fos single expression vector did not produce a consistent improvement in functionality (Figures 23d-e).

[0178] To further investigate c-Jun's ability to enhance the function of exhausted CAR T cells and to ensure constitutive c-Jun expression in all CAR-expressing T cells, we constructed a bicistronic vector co-expressing c-Jun and a CAR transgene isolated by a viral P2A skipping peptide (JUN-CAR, Figure 14a). These expression vectors increased c-Jun expression in both CD19 and HA-28z CAR T cells (Figure 14b), but c-Jun was preferentially activated (phosphorylated) in JUN-HA CAR T cells (Figure 14c), which was consistent with c-Jun N-terminal phosphorylation (JNP) by the JNK protein activated downstream of the persistent TCR signaling cascade propagated via HA-28z CAR30. Upon stimulation with GD2+ tumor cell lines, JUN-HA-28z CAR T cells showed a significant increase in IL-2 and IFNg production compared to control HA-28z CAR T cells (Figures 14d-e). The multiplier increase in cytokine production in the c-Jun OE setting was substantially greater for JUN-HA CARs compared to JUN-CD19 CAR T cells (Figure 24a-b). Similarly, JUN-HA CAR T cells showed an increased frequency of SCM / CM vs. E / EM subsets compared to HA-28z CAR T cells (Figure 14f), but no significant difference was observed between CD19 and JUN-CD19 CAR T cells in subset composition at day 10 of culture. In summary, these data are consistent with a model in which c-Jun OE is functionally more significant in exhausted T cells overexpressing inhibitory bZIP and IRF TF.

[0179] To evaluate whether c-Jun OE enhances long-term proliferative capacity associated with antitumor effects in solid tumors (reference 31; incorporated herein by reference in its entirety), and to test whether c-Jun OE can increase function in CAR T cells lacking sustained signaling (CD19-28z, CD19-BBz) or CAR T cells with lower levels of sustained signaling (GD2-BBz), in vitro expansion of JUN-CAR T cells from three different healthy donors was measured over a long period (Figure 24c). A consistent pattern of enhanced long-term proliferative capacity in the presence of c-Jun OE was observed, which remained IL-2-dependent, as these cells immediately ceased expansion in the absence of IL-2 (Figure 14g). Consistent with c-Jun's ability to induce resistance to exhaustion, subsequent expansion of CD8+ JUN-CD19-28z CAR T cells showed reduced expression of exhaustion markers compared to control CD19-28z CAR T cells tested at the same time point, and maintained a robust subset of cells with the stem cell memory (SCM) phenotype (CD45RA+CD62L+) (Figure 14h-j). We evaluated the constitutive expansion of JUN-CAR T cells adopted into tumor-free NSG mice. Peripheral blood T cell counts increased at 25 days post-infusion in mice treated with both JUN-CD19-28z and JUN-CD19-BBz CAR T cells compared to controls (Figure 14k), which resulted in accelerated GVHD in JUN-CD19-BBz CAR T cell-treated mice. In summary, the data shows that c-Jun OE mitigates T cell exhaustion in many tested CARs, including those incorporating CD28 or 4-1BB costimulatory domains, regardless of whether the exhaustion is driven by enhanced, long-term expansion or sustained signaling.

[0180] Example 12 Molecular requirements for c-Jun-mediated rescue of CAR T cell exhaustion To determine whether c-Jun OE can rescue exhausted T cells through two non-exclusive mechanisms, experiments were conducted during the development of the embodiments described herein. c-Jun OE may directly enhance AP-1-mediated gene transcription by increasing the availability of Fos / Jun or Jun / Jun dimers, or it may act indirectly by disrupting inhibitory AP-1 / IRF transcriptional complexes (AP-1-i)14,24 that drive exhaustion-related gene expression (Figure 15a). To better understand the mechanism by which c-Jun OE alleviates T cell exhaustion, c-Jun expression was transiently regulated by fusing a destabilization domain (DD) derived from E. coli dihydrofolate reductase (DHFR) to the N-terminus of c-Jun (JUN-DD, Figure 15b). DD is stabilized in the presence of the cell-permeable small molecule trimethoprim (TMP), leading to stable c-Jun expression. However, in the absence of TMP, DD is destabilized, inducing proteasomal degradation of the total fusion protein (Figure 15c). JUN-DD CAR T cells rapidly increased c-Jun expression in the presence of TMP (1 / 2max at 6.76 hours after drug exposure), while JUN-DD rapidly became undetectable in the absence of TMP (t1 / 2 at 1.84 hours) (Figures 15d and 25a-b). JUN-DD-CAR T cells mediated increased IL-2 and IFNg production only in the presence of TMP, confirming a decisive role for c-Jun levels in regulating CAR T cell function (Figure 15e). This was because the direct effect of c-Jun on transcription could be rapidly affected; therefore, we tested whether functional rescue occurred when c-Jun OE in HA-28z CAR T cells was limited to a period of acute antigen stimulation (off-on). In contrast, if c-Jun OE was required for competition with the inhibitory AP-1 complex (AP-1i) during the induction of exhaustion, a longer exposure (on-off) during T cell expansion may be required (Figure 15f). Compared to HA-28z CAR T cells that did not experience c-Jun OE (off-off), both conditions mediated partial rescue, but complete rescue of IL-2 function required c-Jun OE (on-on) during both T cell expansion and antigen stimulation (Figure 15g).These findings are consistent with a model in which c-Jun overexpression directly enhances gene transcription during acute stimulation downstream of antigen encounter, and indirectly regulates molecular reprogramming during the progression of exhaustion. Furthermore, reductions in both protein and mRNA expression of JUNB and BATF / BATF3 family members were observed upon c-Jun overexpression (Figures 25c-d) and reduction of the JunB / BATF complex by IP (Figure 25e).

[0181] An indirect model of c-Jun-mediated disruption of the inhibitory AP-1 complex is independent of direct c-Jun transcriptional activation. To test whether direct c-Jun-mediated gene activation is necessary for functional rescue of T cell exhaustion, we constructed a JNP-deficient c-Jun with alanine substitutions at Ser63 and Ser73 in the c-Jun transactivation domain, which has been shown to be important for c-Jun-mediated gene transcription (references 33, 34; all of which are incorporated herein by reference), thereby preventing phosphorylation at these sites (c-JunAA) (Figure 15h~i). JUNAA-HA-28z CAR T cells showed comparable increases in IL-2 and IFNg production compared to wild-type JUN-CAR T cells (Figure 25j). In summary, these data were consistent with a model in which c-Jun-mediated rescue of exhaustion does not require direct gene activation.

[0182] Example 13 JUN-CAR T cells mediate the enhancement of antitumor activity in vivo. To determine whether JUN-CAR T cells exhibit enhanced activity in vivo, experiments were conducted during the development of the embodiments described herein. Nalm6-GD2+ leukemia cells were implanted in mice and treated with control HA-28z or JUN-HA-28z CAR T cells on day 3. While HA-28z CAR T cells showed some antitumor activity, the treatment was ultimately unsuccessful as all mice died due to the disease (median survival d59). In contrast, JUN-HA-28z CAR T cells mediated complete tumor regression by day 24 and resulted in prolonged tumor-free survival (Figure 16a-c). To address whether c-Jun OE can enhance the function of CARs targeting solid tumors, the effects of JUN-CAR were evaluated using Her2 and GD2-targeted CARs incorporating the 4-1BB costimulatory domain, which is a preferred signaling domain for conferring long-term persistence (references 35-37; all of which are invoked herein by reference). In an extended exovivo killing assay of 143b osteosarcoma, JUN-Her2-BBz CAR T cells showed significantly more potent killing activity with an effector:target (E:T) ratio of 1:8, consistent with enhanced capacity per cell unit (Figure 16d-e). Similarly, JUN-Her2-BBz CAR T cells prevented tumor growth in vivo and resulted in a dramatic improvement in long-term survival associated with increased T cell expansion in vivo (Figure 16f-h). Similar results were observed when comparing GD2-BBz and JUN-GD2-BBz CAR T cells against 143b osteosarcoma (Figure 26), confirming the benefits of c-Jun OE in CAR T cells responsive to solid tumors and in CAR T cells incorporating the 4-1BB signaling domain.

[0183] Example 14 c-Jun overexpression lowers the CAR T cell activation threshold, enabling recognition of tumor cells with lower antigen densities. Elevated levels of inhibitory AP1 family members in exhausted CAR T cells led to the hypothesis that dysfunction in exhausted CAR T cells is associated with a higher threshold for activation, which can be standardized, at least partially, by restoring the balance between activating and inhibitory AP1 family members. To test this, we compared cytokine production in HA-CAR vs. JUN-HA-CAR T cells in response to serial dilutions of platelet-bound 1A7, an anti-idiotype antibody that binds to 14g2a scFv, allowing control of stimulation intensity. c-Jun OE substantially increased the maximum IL-2 and IFNg produced and substantially decreased the amount of 1A7 required to induce IL-2 secretion, which was not inconsistent with the decrease in the activation threshold in JUN-HA-CAR T cells (Figure 17a-b).

[0184] It is increasingly recognized that limiting the expression level of target antigens on tumor cells limits CAR function (references 5, 6, 38; all of which are incorporated herein by reference). Currently, CD22dim relapse has been reported in leukemia patients after the initial response to CD22 CAR therapy. Since c-Jun OE lowers the activation threshold in persistently signaling HA-28z CAR T cells, we evaluated whether JUN-CAR recognizes and kills tumor cells with lower antigen densities that may evade recognition by control CAR T cells. When JUN-CD22-BBz CAR (Figure 17) T cells were challenged in CD22low leukemia (Figure 17d), JUN-CAR T cells showed increased cytokine production in vitro (Figure 17e) and dramatically increased antitumor activity in vivo (Figures 17f-i). Control CD22-BBz CAR T cells showed initial activity when administered at a dose of 3 x 10⁶ CAR T cells, but this treatment ultimately failed to control tumor growth (mean survival d45). In contrast, JUN-CD22-BBz CAR T cells mediated a significant antitumor effect and were entirely therapeutic. Therefore, c-Jun OE exhibits significantly improved antitumor control in four tumor models, associated with improved growth, resistance to exhaustion, and enhanced ability to recognize low antigenic targets.

[0185] Example 15 Cleavage JUN protein To determine the necessary JUN domains for mediating the rescue of dysfunctional T cells, experiments were conducted during the development of the embodiments described herein. A series of JUN mutants with deletions of various domains were constructed (Figure 27a). JUN mutants with N-terminal deletions and cleavage (JUN-AA, JUN-Dd, and JUN-DTAD) retained the ability to rescue HA-28z-exhausted CAR T cells, while C-terminal mutations in the ε and bZIP domains were non-functional in rescue. The JUN-AA, JUN-Dd, and JUN-DTAD constructs retained a comparable increase in cytokine production compared to JUN-WT (Figures 27b, d) and improved long-term killing with a lower E:T ratio compared to JUN-De, JUN-Dbasic, JUN-DLeu, and JUN-DbZIP mutants (Figure 27c). Consistent with their improved in vitro functional activity, HA-28z CAR T cells expressing JUN-WT and JUN-DTAD show increased in vivo proliferation when injected into mice with N6-GD2 leukemia compared to JUN-DLeu and JUN-DbZIP.

[0186] Example 16 Knockdown of IRF4 dramatically increases the functional activity of exhausted HA-28z CAR T cells. We conducted experiments to test the ability of knockdown to rescue the functional activity (cytokine secretion) of exhausted HA-28z CAR T cells by optimizing CRISPR gRNA for nine different bZIP / IRF family components. Summary data showing the results of 3 to 6 independent experiments using healthy donors are shown in Figure 28. As shown in Figure 28, JunB and BATF3 knockouts improved functional activity in some donors, while IRF4 knockout dramatically improved IL-2 secretion in stimulated HA-28z CAR T cells in all donors tested (left and center). IRF4-KO CAR T cells further showed improved baseline IL-2 secretion from sustained signaling.

[0187] Example 17 The transcription variant (JUN-AA) also rescues functional activity and proliferative capacity in CD19 CAR T cells. Experiments were conducted on the generation of JUN-AA mutations in CD19 CAR T cells. JUN-AA retained increased reactivity to low antigen density (Figure 29A) and enhanced long-term proliferation in culture (Figure 29B). In vivo, c-Jun showed improved survival of Nalm6 leukemia mice treated with low-dose "stress test" doses of CD19 CAR T cells (Figure 29C).

[0188] Example 18 The enhanced in vivo function of c-Jun-modified HA-28z CAR T cells cannot be replicated by ex vivo provision of IL-2. IL-2 production is an excellent biomarker for exhaustion-resistant cells, but the enhanced function of JUN-CAR T cells cannot be replicated by IL-2 alone (Figure 30). 250,000 IU / mouse were administered IP on days 7, 9, 11, 13, and 15 after tumor implantation. 1x10 6 CAR+ T cells were administered IV on day 7. A representative of three independent experiments with similar findings is shown in Figure 30.

[0189] Example 19 c-Jun enhances Her2-BBz CAR T cell activity within the suppressive solid tumor microenvironment. JUN-CAR T cells show reduced exhaustion and increased functional activity ex vivo. 143B osteosarcoma xenografts were implanted into NSG mice by intramuscular injection. After the solid tumor mass became measurable (14 days after tumor inoculation), 1x10 7Her2-BBz control (blue) or JUN-Her2-BBz (red) CAR T cells were administered IV. Figure 31A shows that JUN-Her2-BBz CAR T cells mediated regression of large, established 143B solid tumors, but Her2-BBz CAR T cells showed no control compared to mock, untransduced T cells. Two weeks after T cell injection (before complete tumor eradication in JUN mice), mice were euthanized, solid tumor tissue was extracted, and mechanically isolated (n=6-8 mice per group). Figure 31B shows that solid tumor digests from JUN-treated mice contained a significantly higher percentage of CD8+ T cells (total viable cell frequency) and retained a higher frequency of CAR+ CD8+ T cells. Figure 31C shows that tumor-localized CD8+ T cells exhibited reduced expression (and co-expression) of exhaustion-related inhibitory receptors PD-1 and CD39 in JUN-Her2-BBz (n=6 mice per group, left) (representative flow plot on the right).

[0190] Next, we conducted experiments to evaluate the functional capacity of tumor-localized CAR T cells by exovivo restimulation using Nalm6-Her2+ target cells (Figures 31D and E). As shown in Figure 31D, 5x10 4 FACS sort CD45+ T cells, 5x10 4 Target cells were restimulated, and IL-2 secretion was measured by ELISA 24 hours later. As shown in Figure 31E, 3 x 10 5 Single-cell tumor digests, 3 x 10 5 Nalm6-Her2+ isolated T cells were restimulated for 6 hours in the presence of monensin and CD107a antibodies. After 6 hours, the production of CD107a, IL-2, IFNγ, and TNFα in single cells was evaluated by intracellular cytokine staining. Exovivo-stimulated JUN-Her2-BBz CAR T cells showed significantly higher frequencies of cells producing CD107a, IL-2, IFNγ, and TNFα, as well as dual cytokine-producing cells, compared to Her2-BBz controls.

[0191] Example 20 c-Jun overexpression increases resistance of exhausted HA-28z CAR T cells to TGFβ-mediated suppression. Control and JUN-WT or JUN-AA modified HA-28z CAR T cells were stimulated with Nalm6-GD2 target cells in the presence or absence of 5nM TGFβ. IL-2 secretion was measured by ELISA, as shown in Figure 32 left, and one representative donor was identified. The catastrophic decrease in IL-2 levels under TGFβ+ vs. TGFβ-free conditions (n=3 independent donors from 3 independent experiments) is shown in Figure 32 right.

[0192] Example 21 Transcriptional changes in c-Jun-modified cells are consistent with reduced fatigue and increased memory formation. Figure 33A shows a heatmap of genes differentially regulated in exhausted HA-28z CARs upon the addition of c-Jun overexpression (193 genes downregulated by c-Jun, 176 genes upregulated by c-Jun, p(adj)<0.05; n=3 independent donors). The gene of interest is highlighted to the right. Genes downregulated in Jun cells include inhibitory receptors (LAG3, ENTPD1) and other possible inhibitory AP-1 family members (BATF3, JUNB), as well as other genes associated with exhaustion (IL13, GZMB, LTA, TNFRSF9). Genes upregulated by c-Jun are associated with naive and memory T cells (IL7R, SELL (CD62L), CD44, and transcription factors LEF1 and FOXP1).

[0193] Figure 33B shows a Venn schematic diagram illustrating downregulated genes and duplicate genes driving exhaustion in PC1 in c-Jun-HA-28z CAR T cells, while Figure 33C shows upregulated genes in c-Jun with duplicate genes associated with healthy (CD19) CAR T cells (bottom). Approximately 25% of the top genes driving the differences between healthy and exhausted T cells may be regulated by c-Jun overexpression, suggesting that the AP-1 family may account for approximately 25% of the exhausted phenotype in this model.

[0194] Figure 33D shows a heatmap illustrating the gene expression of 50 genes in the overlapping region from b to c across a total of 12 samples (n=3 donors per condition).

[0195] Table 2 provides a complete list of genes altered by c-Jun. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0196] Example 22 Inhibiting IRF4 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF4 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF4 inhibitors compared to control T cells.

[0197] Example 23 Inhibition of IRF8 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF8 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF8 inhibitors compared to control T cells.

[0198] Example 24 Inhibiting BATF reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of BATF inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with BATF inhibitors compared to control T cells.

[0199] Example 25 Inhibiting BATF3 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of BATF3 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with BATF3 inhibitors compared to control T cells.

[0200] Example 26 Inhibiting JUNB reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of JUNB inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with JUNB inhibitors compared to control T cells.

[0201] Example 27 Inhibition of IRF1 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF1 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF1 inhibitors.

[0202] Example 28 Inhibition of IRF2 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF2 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF2 inhibitors.

[0203] Example 29 Inhibition of IRF3 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF3 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF3 inhibitors.

[0204] Example 30 Inhibition of IRF5 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF5 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF5 inhibitors.

[0205] Example 31 Inhibition of IRF6 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF6 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF6 inhibitors compared to control T cells.

[0206] Example 32 Inhibition of IRF7 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF7 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF7 inhibitors.

[0207] Example 33 Inhibiting IRF9 reduces T cell exhaustion. Control and exhausted T cells were exposed to effective doses (0.1–1000 μM) of IRF9 inhibitors shown in Table 1. Samples were collected and processed for cytokine and interferon production assays. Compared to control T cells, increased cytokine IL2 and / or interferon IFNγ production was observed in exhausted T cells treated with IRF9 inhibitors.

[0208] All publications and patents referenced in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the embodiments described for carrying out the present invention, which will be apparent to those skilled in the art in the relevant field, are intended to fall within the scope of the following claims.

[0209] References The following references, some of which are cited above by number (e.g., reference X), are incorporated herein by reference in their entirety. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

Claims

1. An in vitro or ex vivo method for preparing therapeutic T cells that are less prone to exhaustion, comprising the step of modifying T cells and introducing nucleic acid encoding c-Jun.

2. The method according to claim 1, wherein c-Jun is human c-Jun.

3. The method according to claim 1 or 2, wherein c-Jun is wild-type c-Jun.

4. T cells, CD3 + T cells, CD8 + T cells, CD4 + T cells, natural killer (NK) T cells, γδ T cells, CD4 + Cells and CD8 + The method according to any one of claims 1 to 3, selected from the group consisting of combinations of T cells, memory T cells, cytokine-induced killer cells, tumor-infiltrating lymphocytes, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the T cells are derived from natural, naturally occurring T cells obtained from an excised tumor or from leukocyte extraction from a blood sample.

6. The method according to any one of claims 1 to 5, wherein T cells express recombinant receptors.

7. The method according to claim 6, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

8. The method according to claim 6, wherein the recombinant receptor is a T cell receptor (TCR).

9. The method according to any one of claims 6 to 8, wherein the recombinant receptor is specific to the tumor antigen.

10. The method according to any one of claims 6 to 9, wherein c-Jun and a recombinant receptor are co-expressed from a single expression construct.

11. The method according to any one of claims 1 to 10, wherein T cells are further modified to reduce or eliminate the expression and / or activity of one or more AP-1 inhibitory complex members.

12. The method according to claim 11, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, BATF family members, IRF4, ATF family members, or combinations thereof.

13. An expression vector comprising a first nucleotide sequence encoding c-Jun and a second nucleotide sequence encoding a recombinant receptor.

14. The expression vector according to claim 13, wherein c-Jun is human c-Jun.

15. The expression vector according to claim 13 or 14, wherein c-Jun is wild-type c-Jun.

16. The expression vector according to any one of claims 13 to 15, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

17. An expression vector according to any one of claims 13 to 15, wherein the recombinant receptor is a T cell receptor (TCR).

18. An expression vector according to any one of claims 13 to 17, wherein the recombinant receptor is specific to a tumor antigen.

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