RU2804282C2 - Compositions and methods for inhibiting t-cell deputy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions discloses a composition for treating a disease or pathological condition in a subject treatable by administration of T-cells, where the composition contains isolated T-cells and where the isolated T-cells contain an introduced nucleic acid for c-Jun overexpression. Also a method of treating a disease or condition in a subject, comprising administering to a subject having a disease or condition an effective amount of a composition wherein the disease or condition is treatable by administering T-cells is disclosed.

EFFECT: technical result of the group of inventions is to reduce the depletion of isolated T-cells.

31 cl, 2 tbl, 33 dwg, 33 ex

Description

Link to related applications

This application claims the benefit and benefit of 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 in their entirety. incorporated into this document by reference.

Field of technology to which the present invention relates

The present invention relates to T cell compositions and methods of using them in the context of therapy and treatment. In particular, the present invention relates to T cells that are modified (eg, 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 the exhaustion of engineered (e.g., chimeric antigen receptor (CAR) T cells) as well as non-engineered T cells, thereby enhancing T cell function (e.g., activity against cancer or infectious disease). 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 OF THE INVENTION

T cells are immune cells that are activated through T cell receptor (TCR) signaling and costimulation after interaction with an antigen. Physiological activation through the T cell receptor makes T cells capable of exerting potent antitumor and/or anti-infective effects. During resolution of the acute inflammatory response, a subset of activated effector T cells differentiate into long-lived memory cells. In contrast, in patients with chronic infections or cancer, T cells often undergo pathological differentiation toward a state of dysfunction called T-cell exhaustion. T cell exhaustion is characterized by profound changes in metabolic function, transcriptional programming, loss of effector function (eg, cytokine secretion, killing ability), and coexpression of multiple surface inhibitory receptors. The main cause of T cell exhaustion is chronic exposure to antigen leading to continuous TCR signaling. Preventing or reversing T cell exhaustion has long been sought as a means to improve T cell efficiency (eg, in patients with cancer or chronic infections).

Chimeric antigen receptor (CAR) T cells demonstrate impressive response rates in B-cell malignancies, but long-term disease control is observed in only approximately 50% of patients with B-ALL1 and large B-cell lymphoma (ref. 2; included in full). the present description by reference), and is even less commonly seen in CLL (Ref. 3; incorporated herein by reference in its entirety). Moreover, despite numerous trials, CAR T cells have not mediated durable antitumor effects in solid tumors (Ref. 4; incorporated herein by reference in its entirety). Numerous factors limit the effectiveness of CAR T cells, including heterogeneous antigen expression and the requirement for high antigen density for optimal CAR function, allowing for rapid selection of antigen elimination options (refs. 5-7; incorporated herein by reference in their entirety), suppressive the tumor microenvironment (ref. 8; incorporated herein by reference in its entirety); and true T cell dysfunction resulting from T cell exhaustion (refs. 3, 9, 10; incorporated herein by reference in its entirety). T cell exhaustion is increasingly considered as a cause of T cell dysfunction in CAR T cells. Tonic antigen-independent signaling, due to scFv aggregation, typically occurs in CAR-expressing T cells and can cause rapid exhaustion (Ref. 9; incorporated herein by reference in its entirety). Integration of the CD28 endodomain into second-generation CAR T cell receptors enhances expansion but also predisposes CAR T cells to exhaustion in both tonically signaling receptor and CD19-28z CAR T cells exposed to high tumor loads (ref. 9 ; incorporated herein by reference in its entirety). Recently, it was demonstrated that the increased frequency of T cells with exhaustion characteristics contained in CD19-BBz CAR grafts distinguishes non-responders from responders in CLL3-treated patients. A broad body of evidence from a variety of studies points to true T cell dysfunction due to T cell exhaustion as a major factor limiting the effectiveness of CAR T cell therapy and raises the possibility that the development of exhaustion-resistant CAR T cells could significantly improve clinical outcomes.

Brief Disclosure of the Present Invention

The present invention relates to compositions and methods of use for preventing the depletion of engineered (e.g., T cells engineered to express a synthetic receptor, such as engineered T cell receptor or chimeric antigen receptor (CAR)) as well as non-engineered (e.g., native) cells. T cells. T cells modified (eg, to prevent T cell exhaustion) according to the present invention, compositions containing them, and methods of use thereof enhance T cell functionality (eg, activity against cancer or infectious disease).

CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, experiments were conducted to develop embodiments of the present invention using a model system using tonically signaling CAR, which causes the features of exhaustion described in other settings. The results demonstrate that depletion was associated with a profound defect in IL-2 production along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of numerous bZIP and IRF transcription factors that were implicated in inhibitory activity. Engineering CAR T cells to overexpress AP-1 factor (eg, c-Jun) increased expansion potential, increased functional capacity, decreased terminal differentiation, and improved antitumor activity in numerous in vivo tumor models.

Experiments conducted during the development of embodiments herein further demonstrate that functional deficiency of AP-1 factor (eg, c-Jun) mediates dysfunction in exhausted human T cells, and engineering CAR T cells to overexpress AP-1 factor. 1 (eg, c-Jun) renders them resistant to depletion, thereby removing a major barrier to progress for this new class of therapeutics.

Experiments conducted during the development of embodiments herein further demonstrate that knockdown of IRF4 dramatically increases the functional activity of HA-28z CAR T cell-depleted cells, which enhanced in vivo function of c-Jun-modified HA-28z CAR T cells cannot be recapitulated by ex vivo provision of IL-2, which c-Jun enhanced Her2-BBz CAR T cell activity in a suppressive solid tumor microenvironment, that this c-Jun overexpression enhances resistance to TGFβ-mediated suppression of exhausted HA-28z CAR T cells, and that transcriptional changes in c-Jun-modified cells are consistent with decreased exhaustion and increased memory formation.

As described herein, engineered T cells (e.g., T cells engineered to express a synthetic receptor, such as engineered T cell receptor or CAR) as well as unengineered (e.g., native, natural) T cells are provided. that are modified to overexpress and/or contain increased levels (e.g., designed to have physiologically increased levels) of one or more activator protein a 1 (AP-1) transcription factors (e.g., c-Fos, c-Jun, activating transcription factor (ATF) and Jun dimerization protein (JDP)) and/or modified (e.g., genetically) to reduce expression and/or activity of one or more members of the AP-1 inhibitory complex (e.g., JunB and BATF3 and other members of the BATF family , IRF4, and members of the ATF family).

Accordingly, in one aspect, the present invention provides T cells modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors. For example, in one embodiment, c-Jun is expressed in T cells that are engineered to express a synthetic receptor, such as an engineered T cell receptor or chimeric antigen receptor (CAR). In another embodiment, c-Jun is expressed in T cells with a native, natural T cell receptor. The present invention is not limited to means for expressing one or more AP-1 transcription factors. In one embodiment, when coexpressed with an engineered TCR or CAR, c-Jun (and/or another AP-1 transcription factor) and the engineered receptor are coexpressed from different viral vectors. In another embodiment, they are expressed from a single vector construct using a bicistronic vector. c-Jun (and/or other AP-1 transcription factor) may be expressed constitutively or in a regulated manner (eg, using a small molecule remote expression regulation system or using an endogenously regulated system). In another embodiment, the genes for c-Jun and/or other AP-1 transcription factors can be genetically integrated into cellular DNA using a retroviral, lentiviral or other viral vector or through a CRISPR/Cas9-based system. In yet another embodiment, c-Jun and/or other AP-1 transcription factors are/are expressed by RNA or oncolytic virus or other transient expression system known in the art. c-Jun and/or other AP-1 transcription factors can be delivered ex vivo to T cells for adoptive transfer or delivered via genetic transfer in vivo.

Likewise, the present invention is not limited to a type of T cell modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors and/or modified (e.g., genetically) to decrease the expression and/or activity of one or more members of the AP-1 inhibitory complex (for example, JunB and BATF3 and other members of the BATF family, members of the IRF4 and ATF family). In some embodiments, the T cells are CD3+ T cells (eg, a combination of CD4+ and CD8+ T cells). In certain 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 alpha beta T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the T cells are a combination of CD4+ and CD8+ T cells (eg, which are CD3+). In certain embodiments, the T cells are memory T cells. In certain embodiments, the memory T cells are central memory T cells. In certain embodiments, the memory T cells are effector memory T cells. In some embodiments, the T cells are tumor-infiltrating lymphocytes. In certain embodiments, the T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and/or gamma delta T cells. In some embodiments, the T cells are cytokine-induced killer cells.

In some embodiments, the T cell is an antitumor T cell (eg, a T cell with activity against a tumor (eg, an autologous tumor) that becomes activated and expands in response to an antigen). In one embodiment, the antitumor T cells (eg, those useful for T cell adoptive transfer) include T cells derived from peripheral blood that are genetically modified with receptors that recognize and respond to tumor antigens. Such receptors typically consist of extracellular domains containing a tumor antigen-specific single-chain antibody (scFv) associated with intracellular T cell signaling motifs (see, e.g., Westwood, J. A. et al, 2005, Proc. Natl Acad. Sci., USA, 102(52): 19051-19056). Other antitumor T cells include T cells derived from resected tumors or tumor biopsies (eg, tumor infiltrating lymphocytes (TILs)). In another embodiment, the T cell is a polyclonal or monoclonal tumor-reactive T cell (e.g., apheresis-derived, ex vivo expanded against tumor antigens presented by autologous or artificial

antigen presenting cells). In another embodiment, the T cell is engineered to express a T cell receptor of human or murine origin that recognizes a tumor antigen. The present invention is not limited to the type of tumor antigen recognized in this manner. Indeed, any T cell containing a receptor that recognizes a tumor antigen finds use in the compositions and methods of the present invention. Examples include, without limitation, the following: T cells expressing a receptor (e.g., a native or naturally occurring receptor, or a receptor engineered to express a synthetic receptor, such as an engineered T cell receptor or CAR) that recognizes an antigen selected from the following: CD19, CD20, CD22, orphan receptor tyrosine kinase-like 1 (ROR1), disialoganglioside 2 (GD2), Epstein-Barr virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (HCA), CD33/ IL3Rα, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate specific membrane antigen (PSMA), glycolipid F77, epidermal growth factor receptor variant III (EGFRvIII), NY-ESO-1, A3 family member melanoma antigen genes (MAGE) (MAGE-A3), melanoma antigen 1 recognized by T cells (MART-1), GP1000, p53 or other tumor antigens described herein.

In some embodiments, the T cell is engineered to express a CAR. The present invention is not limited to the type of CAR. Indeed, 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 contain increased levels (e.g., have physiologically increased levels) of one or more AP-1 transcription factors (eg c-Jun). In certain embodiments, the CAR comprises an antigen binding domain. In certain embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising heavy 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 comprising one or more immunoreceptor tyrosine activation motifs (ITAMs) (e.g., a T signaling domain -cell receptor (eg, TCR zeta chain). In some embodiments, the CAR contains one or more costimulatory domains (eg, domains that provide a second signal to stimulate T cell activation). The present invention is not limited to the type of costimulatory domain. Indeed, any co-stimulatory domain known in the art may be used, including but not limited to the following: CD28, OX40/CD134, 4-1 BB/CD137/TNFRSF9, high-affinity immunoglobulin Ε receptor gamma subunit (FcERIγ, ICOS/CD278, beta interleukin 2 subunit (ILRβ) or CD122, common cytokine receptor gamma subunit (IL-2Rγ) or CD132 and CD40. In one embodiment, the co-stimulatory domain is a 4-1 BB.

In one aspect, the present invention provides a method of treating a disease or condition in a subject comprising administering to the subject (e.g., a patient) having the disease or condition an effective number of T cells modified to express and/or contain increased levels of one or more transcription factors AP-1 and/or modified (eg, genetically) to reduce the expression and/or activity of one or more members of the AP-1 inhibitory complex (eg, JunB and BATF3 and other members of the BATF family, IRF4 and members of the ATF family). The present invention is not limited to the type of disease or condition being treated. Indeed, any disease or condition that is treatable (for example, for which the severity of signs or symptoms of the disease is reduced by treatment) through the administration of T cells can be treated in an improved and more effective manner using the compositions and methods of the present invention (for example, containing and /or using T cells modified to express and/or contain increased 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 to the type of cancer or the type of infectious disease. Indeed, any cancer known in the art that is treated with T cell therapy can be treated with the compositions and methods of the present invention. Likewise, any infectious disease known in the art for which T cell therapy is used for treatment can be treated using the compositions and methods of the present invention. In one embodiment, administering to a subject (eg, a patient) characterized by the presence of a disease or condition, an effective number of T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors and/or to reduce expression and/or activity of one or more members of the AP-1 inhibitory complex, inhibits T cell exhaustion (e.g., compared to a subject receiving the same number of engineered T cells (e.g., CAR T cells or recombinant TCR-containing T cells) does not modified to express and/or contain increased levels of one or more AP-1 transcription factors or to have reduced expression and/or activity of one or more members of the AP-1 inhibitory complex).

Thus, in one embodiment, the present invention provides a method of inhibiting T cell exhaustion (e.g., maintaining the functionality of T cells exposed to excess antigen (e.g., in the context of treating a disease or condition)) by modifying the T cells to express and /or contain increased levels of one or more AP-1 transcription factors and/or for reduced expression and/or activity of one or more members of the AP-1 inhibitory complex (for example, compared to control T cells not so modified). In one embodiment, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors (e.g., c-Jun) exhibit increased functionality and/or activity (e.g., increased antigen-induced cytokine production , enhanced killing capacity (eg, increased recognition of tumor targets with low surface antigen), increased formation of memory cells and/or increased proliferation in response to antigen), and/or reduced signs of wasting (eg, lower levels of markers indicating wasting ( e.g. PD-1, TIM-3, LAG-3) and/or lower levels of programmed cell death). In some embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors and/or to decrease the expression and/or activity of one or more members of the AP-1 inhibitory complex described herein , significantly enhance clinical efficacy (eg, engineered T cells (eg, CAR T cells) and/or non-engineered natural T cells).

In certain embodiments, the present invention demonstrates that treating a subject having cancer with a therapeutically effective amount of a composition comprising T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors is superior to treatment in a subject having characterized by the presence of cancer, using T cells expressing normal amounts of one or more AP-1 transcription factors. In some embodiments, treating animals (e.g., humans) suffering from cancer with therapeutically effective amounts of immunotherapeutic compositions comprising T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors inhibits development or growth cancer cells or renders the cancer cell population more susceptible to other treatments (eg, the cell death-inducing activity of anticancer drugs or radiation therapy). Accordingly, the compositions and methods of the present invention can be used as monotherapy (for example, to kill cancer cells and/or reduce or inhibit cancer cell growth, induce apoptosis and/or cell cycle arrest in cancer cells) or when administered in combination with one or several additional agents, such as other anticancer agents (eg, anticancer therapeutic drugs that induce cell death or disrupt the cell cycle, or radiation therapy) to render a larger proportion of cancer cells susceptible to killing, inhibit cancer cell growth, induce apoptosis, and/or cell cycle arrest compared with the corresponding proportion of cells in an animal treated with an anticancer drug alone or radiation therapy alone.

Accordingly, in certain embodiments, the present invention provides methods for treating or delaying the progression of cancer in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising T cells modified (e.g., genetically) to express and/or contain increased levels of one or more AP-1 transcription factors (eg, c-Jun) and/or modified (eg, genetically) to reduce expression and/or activity of one or more members of the AP-1 inhibitory complex (eg, JunB and BATF3 and other members of the BATF family, IRF4 and members of the ATF family). In certain embodiments, a therapeutically effective amount of the modified T cell composition reduces the number of cancer cells in a patient following such treatment. In certain embodiments, a therapeutically effective amount of the modified T cell composition reduces and/or eliminates the tumor burden in a patient following such treatment. In certain embodiments, the method further comprises administering radiation therapy to the patient. In certain embodiments, radiation therapy is administered before, concurrently, and/or after the patient receives a therapeutically effective amount of the modified T cell composition. In certain embodiments, the method further comprises administering to the patient one or more anticancer agents and/or one or more chemotherapeutic agents. In certain embodiments, one or more anticancer agents and/or one or more chemotherapeutic agents are administered before, simultaneously with, and/or after the patient receives a therapeutically effective amount of the modified T cell composition. In certain embodiments, combining treatment of a patient with a therapeutically effective amount of modified T cells and a course of an anticancer agent produces greater tumor response and clinical benefit in such patient compared to those patients receiving modified T cells or anticancer drugs/radiation therapy alone . Since the doses for all approved anti-cancer drugs and radiation therapy are known, the present invention provides for various combinations thereof with modified T cells.

In certain embodiments, the present invention provides a therapeutically effective amount of a composition (eg, an immunotherapeutic composition) containing T cells modified according to the present disclosure (eg, for use in treating or delaying the progression of cancer in a subject). As described herein, the composition may further contain one or more anticancer agents, for example, one or more chemotherapeutic agents. The present invention also relates to the use of a composition for inducing cell cycle arrest and/or apoptosis. The present invention also relates to the use of compositions for sensitizing cells to additional agent(s), such as inducers of apoptosis and/or cell cycle arrest and chemoprotection of normal cells by inducing cell cycle arrest prior to treatment with chemotherapeutic agents. The compositions of the present invention are useful for the treatment, amelioration, or prevention of disorders, such as any type of cancer or infectious disease, and additionally any cells susceptible to the induction of apoptotic cell death (e.g., disorders characterized by dysregulation of apoptosis, including hyperproliferative diseases, such as cancer). In certain embodiments, the compositions can be used to treat, ameliorate, or prevent cancer that is further characterized by resistance to anticancer therapy (eg, those cancer cells that are resistant to chemotherapy, radiation therapy, hormonal therapy, and the like). The present invention also relates to pharmaceutical compositions containing a composition (eg, immunotherapeutic compositions) containing modified T cells according to the present invention in a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides a composition in which T cells are modified by introducing a nucleic acid encoding c-Jun, and wherein c-Jun and the recombinant receptor are expressed from separate expression vector constructs.

In another embodiment, the present invention provides a composition in which T cells are modified by introducing a nucleic acid encoding c-Jun, and wherein c-Jun and the recombinant receptor are co-expressed from a single expression vector construct.

In another embodiment, the present invention provides a method of treating or delaying the progression of cancer in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising T cells modified (e.g., genetically) to express and/or contain increased levels of one or more transcription factors AP-1 (eg, c-Jun) in combination with a therapeutically effective amount of a TCR signaling inhibitor (eg, to prevent T cell exhaustion). In certain embodiments, the TCR signaling inhibitor is a tyrosine kinase inhibitor. In another embodiment, the tyrosine kinase inhibitor inhibits Lck kinase. The TCR signaling inhibitor can be administered by any suitable route of administration, but is typically administered orally. The subject may be administered multiple cycles of treatment. In certain embodiments, the TCR signaling inhibitor is administered according to a standard dosing schedule (eg, daily or periodically). In another embodiment, the TCR signaling inhibitor is administered for a period of time sufficient to restore at least partial T cell function, then discontinued.

These and other embodiments of the subject matter will be readily apparent to those skilled in the art in light of the disclosure herein.

Description of graphic materials

In fig. Figures 1A-B show that the AP-1 transcription factors c-Fos and c-Jun are down-regulated in GD2-28Z CAR T cells depleted.

In fig. 2 A-E shows that increased expression of AP-1 reduces signs of exhaustion in CAR T cells.

In fig. 3A-F shows that the functional beneficial effect of AP-1 is mainly associated with the expression of c-Jun.

In fig. 4A-E show that bicistronic expression of c-Jun with CAR enhances the functional activity of CAR T cells.

In fig. 5A-C show that bicistronic expression of c-Jun with CAR enhances the functional activity of CAR T cells and the phenotype of central memory T cells.

FIG. 6A-F show that bicistronic expression of c-Jun with CAR increases CAR T cell production of proinflammatory cytokines and decreases IL-10.

FIG. 7A-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.

FIG. 8A-D show that knockdown of the 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-depleted CAR T cells significantly increases IL2 (top) and IFNg (bottom) production after exposure to GD2+ Nalm6-GD2 cell lines (left), 143B osteosarcoma (center) and Kelly neuroblastoma (right). This increase was even greater than for c-Jun overexpression (OE) alone. Dual JUNB-KO and c-JUN-OE T cells did not show any beneficial effect compared to JUNB-KO alone. (B) CRISPR gene knockout (KO) JunB in a GD2-BBZ CAR T cell significantly increases IL2 (top) and IFNg (bottom) production after exposure to GD2+ cell lines Nalm6-GD2 (left), 143B osteosarcoma (center), and Neuroblastoma Kelly (right), however, c-Jun overexpressing (OE) GD2-BBZ CAR T cells showed the greatest functional benefit. Double JUNB-KO and cJUN-OE T cells did not show any benefit compared to cJUN- OE separately. (C) CRISPR gene knockout (KO) of JunB in CD19-28Z (left) or CD19-BBZ (right) CAR T cells had no effect on IL2 production (top) after exposure to Nalm6-GD2 leukemia cells, suggesting that JunB is potent inhibitor only in tonic signaling/GD2-exhausted CAR T cells. (D) CRISPR gene knockout (KO) of BATF3 in HA-28Z-depleted CAR T cells increases IL2 production (top) after exposure to Nalm6-GD2 (left) and Kelly neuroblastoma (right), while IFNγ production is unchanged. HA-28Z CAR-depleted T cells edited with three independent gRNAs targeting BATF3 showed increased IL2 production compared to control or ZB2-edited controls.

In fig. 9A-B show that c-Jun expressing HA-GD2 CAR T cells exhibit superior therapeutic activity in vivo compared to HA-GD2 CAR T cells modified. The growth of Nalm6-GD2 leukemia cells stably expressing firefly luciferase was monitored in vivo using bioluminescent imaging after adoptive transfer of 2xl06CAR+ T cells or mock T cells (non-transduced). (A) Quantitative bioluminescence over time. (B) Images showing individual mice, n=5 mice per group, (all scales 1×10 ⁴ -1×10 ⁶ except d32 sham scales adjusted).

In fig. 10 shows that c-Jun modified GD2-BBZ CAR T cells exhibit superior in vivo activity in an aggressive solid tumor model of 143 B osteosarcoma. The growth of intramuscularly implanted 143B osteosarcoma tumor cells was monitored in vivo using caliper measurements after adoptive transfer of 1x10 ⁷ CAR+ T cells or mock T cells (non-transduced). (A) Quantitative tumor growth over time for n=5 mice per group.

In fig. 11A-D show that c-Jun modified CD19 CAR T cells exhibit enhanced in vivo activity against CD19low Nalm6 leukemia. 3x16 CAR+ T cells were delivered intravenously to mice with a tumor - leukemia Nalm6, CD19-low clone F. (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 Increased 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 showed reduced tumor growth early, but C019-negative disease eventually grew in both groups and did not provide any beneficial effect on survival (p>0.05).

In fig. 12A-J show that HA-28z CAR T cells exhibit phenotypic, functional, transcriptional and epigenetic signatures of T cell exhaustion. a) Reduced expansion of HA-28z compared to CD19-28z CAR T cells during primary culture expansion. D0 = microparticle activation, D2 = transduction. Error bars represent mean ± SEM from n=10 donors. b) Surface expression of markers associated with wasting (D10). c) CD19-28z mainly contains memory stem T cells (CD45RA+CD62L+) and central memory cells (CD45RA- CD62L+), while HA-28z mainly contains memory effector cells CD45RA-CD62L- (D10). d) Release of IL-2 (left) and IFNg (right) after 24-hour co-culture with CD19+GD2+Nalm6- GD2 leukemia cells. Error bars represent the mean ± standard deviation of triplicate wells. One representative donor is shown for each analysis, f) Principal component analysis (PCA) of global transcriptional profiles derived from naïve and CM CD19 or HA CAR T cells, at days 7, 10 and 14 in culture. PC1 (39.3% of variance) separates CD19 from HA CAR T cells. f) Gene expression of the top 200 genes that control PC1. Genes of interest in each cluster are listed above, g) Differentially accessible chromatin regions in CD8+CD19 and HA-28z CAR T cells (D10). Both N and SM subsets are included for each CAR. h) PCA of ATAC-seq chromatin accessibility in CD19 or HA-28z CAR T cells (D10). PCI (76.9% variance) separates CD 19 from HA CAR samples. i) Global chromatin accessibility profile of CD19 and HA-28z CAR T cells derived from a subset (D10). Top 5000 differentially accessible regions (peaks), j) Differentially accessible enhancer regions in CD 19 and ON CAR T cells at the CTLA4 (top) or IL7R (bottom) loci. N - naive, SM - central memory. *p<.05, **p<.01, ***p<.001.ns p>.05.

In fig. 13A-E show the AP-1 family signature in depleted CAR T cells. a) Top 25 score transcription factor motif aberrations at day 10 compared to CD19-CAR expressing T cells using chromVAR analysis showing multiple AP-1 (bZIP) family members in CD4+ and CD8+ T cells derived from subsets N or CM (D10). b) TF motif enrichment analysis in naïve CD8+HA-28z CAR T cell-derived cells demonstrates AP-1 family motifs (bZIP) as the most significantly enriched, c) Expression bulk RNA sequencing (FPKM) of the indicated AP-1 family members ( bZIP) and IRF in CD 19 and HA-28z CAR T cells. Error bars represent the mean ± SEM of n=6 samples from 3 donors showing pairwise expression of CD 19 versus HA for each gene, p-values were obtained using the Wilcoxon matched pairs signed rank test. d) CD19-28z and HA-28z CAR T cells were lysed and the expression of these AP-1 family proteins was assessed by Western blotting. Increased protein expression of JunB, BATF3 and IRF4 in HA-28z CAR T cells compared to CD 19 CAR T cells was confirmed on days 7, 10 and 14 of culture, f) Correlation network of exhaustion-associated transcription factors in derived native 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 formed nodes of the network. Colors represent log2 fold change (FC) (GD2 versus CD 19 CAR). The thickness of the edge represents the magnitude of the correlation in expression between the corresponding pair of genes in cells. Correlation scores >0.1 were used to construct networks. *p<.05, **p<.01, ***p<.001. ns p>.05.

In fig. 14A-K shows that overexpression of c-Jun enhances the function of exhausted CAR T cells. a) Schematic of the JUN-P2A-CAR expression vector. b) Intracellular flow cytometry demonstrating overall c-Jun expression in control and JUN-modified CAR T cells (D10). c) Western blot for total c-Jun and phospho-c-type8er73 in control and JUN-modified CD 19 and CAR T cells (D10). (d) IL-2 and (e) IFNγ production after 24-h coculture of control (blue) or JUN-modified (red) CD 19 and HA CAR T cells in response to antigen + tumor cells. Error bars represent the mean ± standard deviation of triplicate wells. Data from one representative donor are shown. The fold increase in IL-2 or IFNg production in JUN compared to control CAR T cells from multiple donors can be found in Figure 24. f) Left: Flow cytometry plots showing representative expression of CD45RA/CD62L in control versus JUN-CAR T -cells (D10), Right: relative frequency of effector cells (E, RA+62L-), memory stem cells (SCM, RA+62L+), central memory cells (RA-62L+) and effector memory cells (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-tailed t-tests were performed, g) On day 39 of culture of 1x106 viable T cells from FIG. 24 s reseeded and cultured for 7 days with or without IL-2, hj) Cell surface phenotype of control or JUN-CD19-28z CAR T cells from (g) at day 46, h) CD4 versus CD8 expression . i) CD8+JUN-modified CD19-28z CAR T cells with late expansion are characterized by a memory stem cell phenotype (CD45RA+CD62L+). j) Late-expanding CD8+JUN-modified CD19-28z CAR T cells have reduced exhaustion marker expression compared to controls, j) T cells from g were cryopreserved at D10, thawed, and maintained overnight in IL-2. Healthy NSG mice were administered 5x10 ⁶ control or JUN-modified CD 19-28z or CD 19-BBz CAR T cells by intravenous injection. On day 25 after infusion, the number of peripheral blood T cells was determined by flow cytometry. Error bars represent mean ± SEM of n = 5 mice per group. * p<.05, ** p<.01, *** p<.001. ns p>.05. HTM-hinge/transmembrane. ICD - intracellular domain.

In fig. 15A-J show that functional recovery of HA-28z-depleted CAR T cells requires the presence of c-Jun during both chronic and acute T cell stimulation and is independent of JNP. a) Proposed mechanisms of c-Jun-mediated recovery of T cell exhaustion. AP-1-i indicates the AP-1 inhibitory complex. b) Schematic of the DD-regulated JUN expression vector. c) Scheme of drug stabilization of JUN-DD expression. The yellow diamond is a molecule that stabilizes TMP. d) Total c-Jun expression in control, JUN-WT and JUN-DD HA-28z CAR T cells (D10) by intracellular flow cytometry (left) and Western blotting (right), f) IL-2 production ( left) and IFNg (right) in control, JUN-WT, or JUN-DD (OFF, ON) modified HA-28z CAR T cells 24 hours after stimulation with Nalm6-GD2 target cells or 143 V or medium alone (initial level) (D10). In d-e) OFF indicates the absence of TMP, ON indicates T cells cultured in the presence of 10 μM TMP from D4 and during co-culture. In f-g), TMP was added either during T cell expansion (from D4 onwards) or only during co-culture with tumor cells, as indicated in f. For ON-OFF and OFF-ON conditions, TMP was removed/added 18 h before co-culture to ensure complete degradation/stabilization of c-Jun, respectively, prior to antigen exposure, g) IL-2 expression in one representative donor (left, SD for triplicate wells) and fold increase in IL-2 (SEM n=6 independent experiments representing 3 different donors, relative to OFF-OFF condition). h-j) Enhanced functional activity of JUN-CAR T cells is independent of Jun N-terminal phosphorylation (JNP). h) Schematic of the c-Jun protein showing the N-terminal transactivating domain (TAD). Asterisks indicate JNP sites at positions Ser63 and Ser73, which are mutated to alanine in the JUN-AA mutant. i) Western blot of total c-Jun and c-Jun-PSer73 in control, JUN-WT and JUN-AA HA-28z CAR T cells. j) Release of IL-2 (left) and IFNg (right) in control, JUN-WT and JUN-AA HA-28z CAR T cells after 24 hours stimulation with Nalm6-GD2 target cells or 143 V or medium alone (initial level). Error bars represent the mean ± standard deviation of triplicate wells. Representative result of 3 independent experiments. * p<.05, ** p<.01, *** p<.001. ns p>.05. HTM - hinge/transmembrane, ICD - intracellular domain, DD - destabilizing domain from E. coli DHFR, TMP - trimethoprim, WT - wild type.

In fig. 16A-H show that JUN-modified CAR T cells increase in vivo activity against leukemia and solid tumors. In a-c, NSG mice were inoculated with lx Nalm6-GD2 leukemia cells via intravenous injection. 3 x 10 ⁶ mock, HA-28z or JUN-HA-28z CAR+ T cells were administered intravenously on day 3. Tumor progression was monitored using bioluminescence imaging (a-b). Scales are normalized for all time points, c) JUN-HA-28z CAR T cells induced long-term tumor-free survival. Error bars represent mean ±SEM of n=5 mice/group. This finding was replicated in >3 independent experiments, but in some experiments long-term survival was reduced by the growth of GD2(-) Nalm6 clones. d) Scheme of the retroviral vector construct JUN-Her2-BBz. e) Lysis of Her2-BBz CAR T cells of GFP+Nalm6-Her2B target cells at an E:T ratio of 1:8. Error bars represent the mean ± standard deviation of triplicate wells. Representative result of 2 independent experiments. In fh, NSG mice were inoculated with 1×10 ⁶ osteosarcoma 143b-19 cells via intramuscular injection. 1x107 mock, Her2-BBz, or JUN-Her2-BBz CAR T cells were administered IV on day 7 (d7). f) Tumor growth was monitored by caliper measurements, g) Mice treated with JUN-Her2-BBz CAR T cells maintained long-term tumor-free survival, h) At d20 after tumor cell implantation, peripheral blood T cells were quantified in mice, treated as in f. Error bars represent mean ± SEM of n=5 mice/group. Representative result of 2 independent experiments with similar results. * p<.05, ** p<.01, *** p<.001. NTM - hinged/trans membrane. ICD - intracellular domain.

In fig. 17A-1 shows that JUN-CAR T cells enhance T cell function upon suboptimal stimulation, a) IL-2 and b) IFNg production after 24 hours stimulation of control or JUN-modified HA-28z CAR T cells with immobilized idiotypic antibody 1A7 to CAR. Each curve corresponded to nonlinear dose-response kinetics to determine the EC50. The smaller graphs on the right visualize the curve where the antibody concentration is 0-1 μg/ml. c) Vector diagram of the retroviral vector construct JUN-CD22-BBz. d) Surface expression of CD22 on parental Nalm6, Nalm6-22KO and Nalm6-22KO+CD221ow. f) Release of IL-2 (left) and IFNg (right) after co-culture of control or JUN CD22-BBz CAR T cells exposed to Nalm6 and Nalm6-221ow. Error bars represent the mean ± standard deviation of triplicate wells. Representative result of 3 independent experiments. In fi) NSG mice were inoculated with ^1x106 Nalm6-221ow leukemia cells on day 0. On day 4, ^3x106 control cells or JUN-CD22-BBz CAR+ T cells or ^3x106 mock-transduced T cells transferred i.v. Tumor growth was monitored using bioluminescent imaging (f) with images (i). g) Mice receiving JUN-22BBz CAR T cells show an increase in peripheral blood T cells at day 23, h) JUN expression significantly improves long-term survival of mice treated with CAR. In fg, error bars represent the mean ± SEM of n = 5 mice per group. Representative result of 2 independent experiments with similar results. * p<.05, ** p<.01, *** p<.001. NTM - hinged/transmembrane. ICD - intracellular domain.

In fig. 18A-C show that 14g2a-GD2E101K CAR high affinity (HA) T cells exhibit an excess exhaustion signature compared to the parent 14g2a-GD2 CAR. a) Expression of inhibitory surface receptor in CD 19, GD2, and HA-GD2E101K CAR T cells on day 10 of culture. The high affinity mutation E101K results in increased expression of the inhibitory receptor in CD4+ and CD8+ CAR T cells compared to the parental GD2 CAR. b) IL-2 secretion after 24-hour co-culture of HA-GD2E101K or parental GD2-28z CAR T cells with GD2+ target cells. The profile of increased HA-GD2E101K CAR T cell exhaustion is consistent with decreased functional activity, as measured by the ability to produce IL-2 upon stimulation. Error bars represent the mean ± standard deviation of triplicate wells. Representative result of at least 4 independent experiments with similar results, c) PCA bulk RNA-seq shows large variance between HA-GD2E101K and CD 19 CAR T cells, while GD2-28z(sh) CAR T cells are intermediate. On the left are CD4+ T cells. Right - naïve-derived CD8+ CAR T cells, d-f) Expression of HA-GD2E101K CAR causes increased inhibitory receptor expression (d) and decreased memory formation (e) in CD4+ CAR T cells. (CD8+ data in Figure 12). f) PCA RNA-seq in FIG. 12e showing that the separation of PC2 is driven by the separation of SM versus N and PC3 driven by CD4 versus CD8. g) GSEA: sets of genes up-regulated at day 10 of culture in HA-28z CAR T cells versus CD19-28z CAR T cells showed significant overlap with genes up-regulated in exhausted versus memory CD8+ cells ( left), depleted relative to effector CD8+ (right), and depleted relative to naïve CD8+ cells (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 - normalized enrichment index.

In fig. 19A-C show that GD2-28z CAR T cells show a signature of exhaustion at the single cell level, a) Venn diagram showing overlapping genes in single cell differential expression analysis (red) and the top 200 scoring genes driving CD 19 and CD separation HA-28z CAR T cells in bulk RNA sequencing. 79 of the top 200 genes from bulk RNA-seq are differentially expressed by DESeq2 analysis in GD2-28z versus CD19-28z single cells. Highlighted genes from the intersection include inhibitory receptors (CTLA4, LAG3, GITR, effector molecules CD25, IFNG, GZMB and cytokines IL13 and ILIA and AP-1/bZIP family transcription factors BATF3 and IRF4. B) Heat map clustering the top 50 differentially expressed genes in single cell transcriptomic analysis of GD2-28z versus CD19-28z. Each row represents one cell, c) Violin plots depicting individual gene expression in CD8+ GD2-28z and CD19-28z single CAR T cells. Genes upregulated in GD2 CAR T cells include inhibitory receptors, effector molecules, and AP-1 family transcription factors, while CD19 CAR T cells have increased expression of memory-related genes. P -values, which are displayed for each gene above the individual graphs, were calculated using an unpaired two-tailed Wilcoxon-Mann-Whitney U test.

In fig. 20A-D show quality control of ATAC-seq data. a) Insertion dynes and b) insertion distance from transcription start site (TSS) for combined (top) and individual samples (bottom), c) Correlation between samples across replicates. d) Location of matched peaks in each sample by total number of peaks (top) and frequency of occurrence of the total (below).

In fig. 21A-D shows that the AP-1 family contains the most significantly enriched transcription factor motifs in HA-28z-depleted CAR T cells. a) Differentially accessible chromatin regions in CD4+ CD 19 and ON CAR T cells (D10). Both N and SM subsets are included for each CAR. b) PCA from Figure 1h showing that PC2 partitioning is driven by CM partitioning versus N and PC3 partitioning is driven by CD4 versus CD8. c) The top performing transcription factor motifs enriched in chromatin regions differentially accessible in HA-28z CAR T cells contain AP-1/bZIP family factors in all parental T cell subsets. A naïve subset of CD8+ cells is shown in Figure 2. d) Clustering of peaks by common regulatory motif (left) and heatmap of transcription factor motif enrichment (right) within each cluster. 10 different clusters, including clusters associated with exhausted (EX1-EX4) or healthy (HLT1-HLT2) CAR T cells, SM (SM) or N (naïve) parental subset, and CD4 or CD8 T cell subset. Genes of interest in each cluster are highlighted on the right. (N naive cells, SM - central memory cells).

In fig. 22A-C show that AP-1/bZIP family transcription factors are activated in HA-28z CAR T cells and form immunoregulatory complexes, a) Fold change in gene expression (HA/CD19) for the indicated AP-1/bZIP family genes and IRF from RNA-seq data in Figure 2. Error bars represent mean ± SEM of n=6 samples from 3 independent donors. b) Fold changes in protein expression (HA/CD19) for the indicated AP-1/bZIP and IRF family proteins were determined by densitometric Western blot analysis. Error bars represent the mean ± SEM of n = 4 experiments on 3 independent donors. * p<.05, ** p<.01, *** p<.001. c) Western blotting for the indicated AP-1/bZIP family proteins and IRFs upstream (left columns) or after immunoprecipitation for c-Jun (middle columns) or JunB (right columns) in CD19 and HA-28z CAR T- cells. The numbers below represent the fold increase in protein expression for HA compared to CD19 under each condition, and the colored shapes represent the complexes defined to scale. IP Western blots demonstrate 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 in a similar ratio to JunB, while BATF and BATF3 show preferential complexation with JunB.

In fig. 23A-E show that the enhanced activity of API-modified CAR T cells is dependent on c-Jun, but not on c-Fos. a-c) CAR T cells were co-transduced with (API) or without (control) a lentiviral vector encoding both API transcription factors, Fos and Jun, and a truncated surface selection marker NGFR (tNGFR). a) Scheme of the lentiviral construct. b) Representative transduction efficiency of API-modified CAR T cells as measured by surface expression of NGFR in the indicated CD4+ and CD8+ CAR T cells. c) IL-2 production in control or API-modified CAR T cells after 24-hour stimulation with 143B-19 target cells. API-modified HA-28z CAR T cells exhibit increased IL-2 production compared to control CAR T cells. Representative experiment 2 independent experiments with similar results, d-e) CAR T cells were co-transduced with lentiviral vectors encoding either of the API transcription factors, Fos or Jun, and a truncated surface selection marker NGFR (tNGFR). d) Schemes of the Fos and Jun lentiviral constructs. f) IL-2 production in control, Fos-modified or Jun CAR T cells after 24-hour stimulation with Nalm6-GD2 target cells. Error bars represent the mean ± standard deviation of triplicate wells. Representative experiment of 2 independent experiments with similar results. B a and d* denotes a stop codon. *p<.05, **p<.01, ***p<.001, ns p>0.05.

In fig. 24A-C show extended functional assessment of JUN-modified CAR T cells. a-b) Fold increase in IL-2 (a) and IFNg (b) release after 24-hour co-culture with the indicated target cells in JUN compared to control CD19 and HA-28z CAR T cells. Each point represents one independent experiment from different donors, c) Extended expansion of control or JUN-modified CAR T cells in vitro in 3 independent experiments from 3 different healthy donors. At the indicated time points, T cells were reseeded in fresh T cell medium + 100 IU/ml IL2. T cells were counted and quantified to maintain cells at a concentration of 0.5×10 ⁶ /ml every 2-3 days. For donor-1, 5×10 ⁶ viable T cells were replated on days 14 and 28. For donor-2, 5×10 ⁶ viable T cells were replated for 14, 28, 42, and 56 days. For donor-3, 5×10 ⁶ viable T cells were replated on days 10, 17, 24, and 31.

In fig. 25A-E show that overexpression of c-Jun reduces the abundance and complexation of inhibitory members of the AP-1 family: JunB, BATF and BATF3. a) Kinetics of drug-induced c-Jun stability in JUN-DD CAR T cells assessed by Western blotting. At time 0, 10 μM TMP was either added to untreated cells (ON) or washed from pretreated cells (OFF). Cells were removed from each condition after 1, 2, 4, 8, 24, and 48 h and prepared for Western blot analysis of c-Jun expression. The observed band corresponds to the JUN-DD size. b) Densitometric analysis was performed on blots from (a) and normalized to the loading control. Expression was plotted against time and first-order kinetics curves were fitted to the data to determine t1/2 for OFF and ON kinetics. c) Western blot analysis for the indicated AP-1/bZIP family members and IRFs in control and JUN-CAR T cells (D10). The numbers below represent the fold change in protein expression compared to CD19. d) Corresponding decrease in BATF, BATF3 and JUNB mRNA expression in JUN-HA-28z CAR T cells compared to HA-28z. n=3 donors normalized for CD19 mRNA. f) Overexpression of c-Jun reduces the JunB/BATF and JunB/BATF3 inhibitory complexes by IP-Western blot analysis. Input (left columns), immunoprecipitation for c-Jun (middle columns) or JunB (right columns) in control or JUN-HA-28z CAR T cells. IRF4 protein, mRNA, and c-Jun complexation were not affected.

In fig. 26A-E show that JUN-CAR T cells enhance the function of GD2-BBz CAR T cells in solid tumors, a) Vector diagram - 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 following 24-hour stimulation with Nalm6-GD2 or 143B target cells. c) GD2-BBz CAR T cell lysis of GFP+Nalm6-GD2 target cells at an E:T ratio of 1:1 (left) or 1:4 (right). In a–c, error bars represent the mean ± standard deviation of triplicate wells. Representative result of at least 3 independent experiments. In de, NSG mice were inoculated with 0.5 x 10 ⁶ osteosarcoma 143B-19 cells via intramuscular injection. 1x10 ⁷ mock, GD2-BBz, or JUN-GD2-BBz CAR T cells were administered IV on day 3, d) Tumor growth was monitored by caliper measurements, e) CD4+ (higher) or CD8+ (lower) counts Peripheral blood T cells on day 14 after tumor engraftment. Error bars represent mean ± SEM of n = 5 mice per group. Representative result of two independent experiments, although early deaths (unrelated to tumor size) excluded the survival curves in both models. *p<.05, **p<.01, ***p<.001. NTM - hinged/transmembrane. ICD - intracellular domain.

In fig. 27A-E show that N-terminal c-Jun mutations are able to restore HA-28z-depleted CAR T cells. a) Various c-Jun mutations cloned into the HA-28z CAR T cell vector. b) Secretion of IL-2 (top) and IFNg (bottom) after 24-hour co-culture with GD2+ 143B osteosarcoma target cells. c) In vitro lysis of GFP+ Nalm6-GD2 or 143B target cells was measured for 5 days at effector:target ratios of 1:1, 1:2, or 1:4 (E:T). At low E:T ratios and late time points, JUN-WT, JUN-AA, JUN-Dd, and JUN-DTAD demonstrate improved tumor growth control compared with JUN-De, JUN-Dbasic, JUN-DLeu, and JUN-DbZIP 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 after 5 hours Nalm6-GD2 stimulation. f) Quantification of peripheral T cell numbers 12 days after T cell infusion into Nalm6-GD2 leukemia NSG mice.

In fig. 28 shows that knockdown of IRF4 dramatically increases the functional activity of exhausted HA-28z CAR T cells.

In fig. 29A-C show that the transcription mutant (JUN-AA) also restores functional activity and proliferative capacity in CD19 CAR T cells.

In fig. 30 shows that the enhanced in vivo function of c-Jun-modified HA-28z CAR T cells cannot be reproduced by ex vivo provision of IL-2.

In fig. 31A-E show that c-Jun enhanced Her2-BBz CAR T cell activity in a suppressive solid tumor microenvironment.

In fig. 32 shows that overexpression of c-Jun increases resistance to TGFβ-mediated suppression of exhausted HA-28z CAR T cells.

In fig. 33A-D show that transcriptional changes in c-Jun modified cells are consistent with reduced exhaustion and increased memory formation.

Definitions

For purposes of interpretation of this specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

As used herein, the terms “disease” and “pathological condition” are used interchangeably, unless otherwise specified herein, to describe a deviation from a condition considered normal or average for members of a species or group (e.g., humans), and which is harmful to the affected individual under conditions that are not harmful to the majority of individuals of that species or group. Such abnormality may manifest itself as a condition, signs and/or symptoms (eg, diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any disturbance in the normal condition of the subject or any from its organs or tissues, which interrupts or alters the performance of normal functions. A disease or condition may be caused by or result from exposure to a microorganism (e.g., a pathogen or other infectious agent (e.g., virus or bacteria)), may be a response to environmental factors (e.g., malnutrition, occupational hazards, and/or climate ), may be a reaction to a congenital or hidden defect in the body (for example, genetic abnormalities) or to a combination of these and other factors.

The terms “host,” “subject,” or “patient” are used interchangeably herein to refer to an individual being treated (eg, administered) with the compositions and methods of the present invention. Subjects include, without limitation, the following: mammals (eg, humans, mice, rats, monkeys, horses, cows, pigs, dogs, cats and the like). In the context of the present invention, the term “subject” generally refers to an individual who will be or has been administered one or more compositions of the present invention (eg, the modified or engineered (eg, genetically) T cells described herein).

"T cell exhaustion" refers to the loss of T cell function that can result from infection (eg, chronic infection) or disease.

T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, apoptosis, and decreased cytokine secretion. Accordingly, the terms “alleviate T cell exhaustion,” “inhibit T cell exhaustion,” “reduce T cell exhaustion,” and the like refer to a state of restored T cell functionality characterized by one or more of the following: reduced expression and/or content of one or more of PD-1, TIM-3 and LAG-3; increased memory cell formation and/or maintenance of memory markers (eg, CD62L); prevention of apoptosis; increased antigen-induced production and/or secretion of cytokines (eg, IL-2); enhanced ability to destroy; increased recognition of tumor targets with low surface antigen density; increased proliferation in response to antigen.

The terms "buffer" or "buffering agents" refer to materials that, when added to a solution, cause the solution to resist changes in pH.

As used herein, the terms “cancer” and “tumor” refer to tissue containing cells that have lost the ability to control the growth and proliferation or growth of such cells. Cancer and tumor cells typically exhibit loss of contact inhibition, may be invasive, and may exhibit the ability to metastasize (eg, they have lost the ability to adhere to other cells/tissues). The present invention is not limited to the type of cancer or the type of treatment (eg, prophylactic and/or therapeutic treatment). Indeed, various forms of cancer can be treated using the compositions and methods described herein, including, without limitation, brain cancer or other forms of central nervous system cancer, melanoma, lymphoma, bone cancer, epithelial cancer, breast cancer, ovarian cancer , endometrial cancer, colon and rectal cancer, lung cancer, kidney cancer, melanoma, kidney cancer, prostate cancer, sarcomas, carcinomas and/or combinations thereof.

As used herein, the term “metastasis” refers to the process by which cancer spreads or moves from its site of origin to other areas of the body to develop a similar cancerous lesion in the new location. A "metastatic" or "metastatic" cell is a cell that loses adhesive contacts with neighboring cells and migrates through the bloodstream or lymph from the primary site of disease, invading tissues in other parts of the body.

As used herein, the term "anticancer agent" refers to any therapeutic agent (eg, chemotherapeutic compounds and/or molecular therapeutic compounds), antisense therapy, radiation therapy, and the like, used in the treatment of hyperproliferative diseases such as cancer (eg, in mammals , for example in humans).

"Effective amount" refers to an amount of a pharmaceutical composition, anticancer agent, or other drug effective in dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result (eg, alleviation of some or all of the symptoms of the disease being treated).

As used herein, the term “therapeutically effective amount” refers to that amount of a therapeutic agent that is sufficient to result in an amelioration of one or more symptoms of a disorder or to prevent the development of a disorder or to cause regression of a disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount will refer to 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 burden, reduces the number of metastases, reduces progression tumor or increases survival by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least by 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.

As used herein, the terms “sensitize” and “sensitizing” refer to making, by administration of the former agent, an animal or an animal cell more susceptible or more sensitive to biological effects (e.g., promoting or inhibiting an aspect of cellular function, including but not limited to cell division cells, cell growth, proliferation, invasion, angiogenesis, necrosis or apoptosis) of the second agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or inhibition of an aspect of cellular function, including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed when a second agent is administered with with and without the introduction of the first agent. The response of the sensitized cell 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 by 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% by compared to the response in the absence of the first remedy.

As used herein, the terms “purified” or “purify” refer to the removal of impurities or unwanted compounds from a sample or composition. As used herein, the term “substantially purified” refers to the removal of from about 70 to 90%, up to 100%, of impurities or unwanted compounds from a sample or composition.

As used herein, the terms “administration” and “administering” refer to the act of providing the composition to a subject. Exemplary routes of administration to the human body include, but are not limited to, the eye (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lung (inhalation), oral mucosa (buccal), ear, rectal, by injections (for example, intravenously, subcutaneously, intraperitoneally, intratumorally, etc.), topically, and the like. In one embodiment, the administration of T cells according to the present invention is carried out by intravenous infusion.

As used herein, the terms “co-administration” and “co-administration” refer to the administration of at least two agents (eg, genetically modified immune cells and one or more other agents, eg, anticancer agents) or therapies to a subject. In some embodiments, the co-administration of two or more agents or treatments is simultaneous. In other embodiments, the first agent/therapy is administered before the second agent/therapy. In some embodiments, coadministration can be accomplished by the same or a different route of administration. Those skilled in the art will appreciate that the compositions and/or routes of administration of the various agents or treatments used may vary. A suitable dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or treatments are administered together, the respective agents or treatments are administered at lower dosages than would be appropriate for administering them separately. Thus, coadministration is particularly desirable in embodiments where coadministration of agents or treatments reduces the required dosage of potentially harmful (e.g., toxic) agent(s) and/or where coadministration of two or more agents results in sensitization of the subject to the beneficial effects one of the agents through the co-administration of another agent.

As used herein, the terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to compositions that are substantially free of adverse reactions (eg, toxic, allergic or other immunological reactions) when administered to a subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, including, 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, coatings, sodium lauryl sulfate, isotonic and absorption retarding agents, disintegrants (eg potato starch or sodium starch glycolate), polyethylene glycol and the like. The compositions may also include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and known in the art (see, for example, Martin, Remington's Pharmaceutical Sciences, 15th ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein via link).

As used herein, the term “pharmaceutically acceptable salt” refers to any salt (eg, formed by reaction with an acid or base) of the composition of the present invention that is physiologically tolerated in the target subject. "Salts" of the compositions of the present invention can be obtained from inorganic or organic acids and bases. Examples of acids include, without limitation, the following: hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic , benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid and the like. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, can be used in the preparation of salts useful as intermediates in the preparation of the compositions of the present invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metal hydroxides (eg sodium), alkaline earth metal hydroxides (eg magnesium), ammonia and compounds of the formula NW4+, where W is C1-4 alkyl, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate , chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate and the like . Other examples of salts include the anions of the compounds of the present invention coupled with a suitable cation such as Na+, NH ₄ + and NW ₄ + (where W represents a C1-4 alkyl group), and the like. For therapeutic use, salts of the compounds of the present invention are considered 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 a pharmaceutically acceptable compound.

For therapeutic use, the salts of the compositions according to the present invention are considered 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 a pharmaceutically acceptable composition.

As used herein, the term “at risk for a disease” refers to a subject who is predisposed to experience a particular disease (eg, an infectious disease). This predisposition may be genetic (eg, a particular genetic tendency to experience a disease such as an inherited disorder), or due to other factors (eg, environmental conditions, exposure to harmful compounds present in the environment, etc.). Thus, it is not intended that the present invention 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 it intended that the present invention be limited to any particular risk. or a specific disease (for example, cancer).

As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of immunotherapeutics, such delivery systems include systems that allow the storage, transport or delivery of immunogenic agents and/or ancillary materials (eg, written instructions for use of the materials, etc.) from one location to another. For example, kits include one or more insert containers (eg, boxes) containing appropriate immunotherapies (eg, engineered T cells and/or excipients). As used herein, the term "fragmented kit" refers to delivery systems containing two or more separate containers, each containing a portion of all components of the kit. Containers can be delivered to the intended recipient together or separately. For example, the first container may contain a composition containing an immunotherapeutic composition for a particular use, while the second container contains a second agent (eg, a chemotherapeutic agent). Indeed, any delivery system containing two or more separate containers, each containing a portion of the components of the entire kit, is included in the term "fragmented kit". In contrast, a "combo kit" refers to a delivery system containing all the components required for a particular use in a single container (e.g., a single box containing each of the required components). The term "set" includes both fragmented and combined sets.

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

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

"Single chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, these domains being present on the same polypeptide chain. Typically, the scFv polypeptide further contains 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), p. 269-315.

As used herein, the term “antigen binding protein” refers to proteins that bind to a specific antigen. "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.

As used herein, the term “epitope” refers to that portion of an antigen that comes into contact with a particular immunoglobulin.

The terms "specific binding" or "specifically binding" when used in relation to the interaction of an antibody (or part thereof (eg, scFv)) and a protein or peptide means that the interaction depends on the presence of a specific sequence or structure (eg, an antigenic determinant or epitope) on protein, in other words, the antibody (or part of it (for example, scFv)) recognizes and binds to a specific sequence or structure of a protein, rather than to proteins in general. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

As used herein, the terms “nonspecific binding” and “background binding” when used in relation to an antibody-protein or peptide interaction refer to an interaction that is independent of the presence of a particular structure (i.e., the antibody binds to proteins in general rather than to specific structure, such as an epitope).

As used herein, the term “subject suspected of having cancer” refers to a subject who has one or more symptoms suggestive of cancer (eg, a noticeable lump or mass) or who is being screened for cancer (eg, during a physical examination ). A subject suspected of having cancer may also be characterized by the presence of one or more risk factors for cancer. A subject suspected of having cancer would not typically be tested for cancer. However, a “subject suspected of having cancer” includes an individual who has received a preliminary diagnosis (eg, CT scan showing a mass) but for whom a confirmatory test (eg, biopsy and/or histology) has not been performed or whose type and/or stage of cancer is unknown. The term also includes people who have previously had cancer (eg, an individual in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and sometimes found not to have cancer.

As used herein, the term “subject diagnosed with cancer” refers to a subject who has been tested and found to have cancer cells. Cancer can be diagnosed using any suitable method, including, but not limited to, biopsy, x-ray, blood test, etc.

As used herein, the term “postoperative tumor tissue” refers to cancerous tissue (eg, organ tissue) that has been removed from a subject (eg, during surgery).

As used herein, the term “subject at risk for developing cancer” refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposures and previous cases of cancer, pre-existing non-cancerous diseases and lifestyle.

As used herein, the term “characterizing a cancer in a subject” refers to identifying one or more properties of a cancer sample in a subject, including, but not limited to, the presence of benign, precancerous or cancerous tissue and the stage of the cancer.

As used herein, the term “determining tissue characteristics in a subject” refers to identifying one or more properties of a tissue sample (e.g., including, but not limited to, the presence of cancerous tissue, the presence of precancerous tissue that may become cancerous, and the presence of cancerous tissue that may metastasize ).

As used herein, the term “cancer stage” refers to a qualitative or quantitative assessment of the level of cancer development. 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 (for example, within the same organ or area of the body or to a different organ).

As used herein, the term “primary tumor cell” refers to a cancer cell that is isolated from a tumor in a mammal and has not been extensively cultured in vitro.

As used herein, the terms “treatment,” “therapeutic use,” or “drug use” refer to any and all uses of the compositions and methods of the present invention that treat a disease state or symptoms or otherwise prevent, inhibit, slow, or reverse the progression of a disease or other unwanted symptoms in any way. For example, the terms “cancer treatment” or “tumor treatment” or grammatical equivalents as used herein mean suppression, regression or partial or complete disappearance of a pre-existing cancer or tumor. The definition is intended to include any decrease in the size, aggressiveness, or growth rate of a pre-existing cancer or tumor.

As used herein, the terms “improved therapeutic effect” and “increased therapeutic efficacy” in relation to cancer refer to slowing or reducing the growth of cancer cells or solid tumors or reducing the total number of cancer cells or the total tumor burden. “Improved therapeutic effect” or “increased therapeutic efficacy” means that the individual's condition improves according to any clinically acceptable criteria, including relief of established tumor, increased life expectancy, or improved quality of life.

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

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

As used herein, the term “site-specific recombination target sequences” refers to nucleic acid sequences that provide recognition sequences for recombination factors and the location where recombination occurs.

As used herein, the term “nucleic acid molecule” refers to any molecule containing a nucleic acid, including but not limited to DNA or RNA. The term covers sequences that include any of the known base analogues of DNA and RNA, including without limitation the following: 4-acetyl cytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)-uracil, 5 -fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2 -methyladenine, 2-methylguanine, 3-methyl cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarbonylmethyluracil, 5- methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, oxybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, N-uracil-5-hydroxyacetic acid methyl ester and 2,6-diaminopurine.

As used herein, the term “heterologous gene” refers to a gene that is not found in its natural environment. For example, a heterologous gene involves a gene from one species introduced into another species. A heterologous gene also includes a gene native to the organism that is altered in some way (eg, mutated, added in multiple copies, associated with non-native regulatory sequences, etc.). Heterologous genes differ from endogenous genes in that heterologous gene sequences are typically attached to DNA sequences that are not found naturally associated with gene sequences on a chromosome or are associated with parts of a chromosome that are not found in nature (for example, genes expressed at loci , where the gene is not normally expressed). Cells that contain a heterologous gene are described herein as “modified” or “engineered” cells. For example, T cells that contain a heterologous AP-1 transcription factor gene (e.g., a heterologous AP-1 transcription factor gene expression construct (e.g., used to overexpress the AP-1 transcription factor in T cells)) and/or a heterologous gene receptor (eg, a heterologous T cell receptor gene expression construct or a heterologous chimeric antigen receptor gene expression construct) are described herein as modified and/or engineered T cells.

As used herein, the term “gene expression” refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., through the enzymatic action of RNA polymerase) and for protein-coding genes into protein through "translation" of mRNA. Gene expression can be regulated at many stages of the process. "Positive regulation" or "activation" refers to regulation that increases the production of gene expression products (ie, RNA or protein), whereas "negative regulation" or "repression" refers to regulation that decreases production. Molecules (eg, transcription factors) that are involved in positive or negative regulation are often called "activators" and "repressors", respectively.

In addition to containing introns, the genomic forms of a gene may also include sequences located at both the 5' and 3' ends of sequences that are present in the RNA transcript. These sequences are called "flanking" sequences or regions (these flanking sequences are located 5' or 3' from the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences, such as promoters and enhancers, that control or influence transcription of the gene. The 3' flanking region may contain sequences that control transcription termination, post-transcriptional cleavage and polyadenylation.

As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a DNA sequence encodes an amino acid sequence.

As used herein, the terms “oligonucleotide having a nucleotide sequence encoding a gene” and “polynucleotide having a nucleotide sequence encoding a gene” mean a nucleic acid sequence containing the coding region of a gene, or in other words, a nucleic acid sequence that encodes a gene product . The coding region may be present in the form of cDNA, genomic DNA or RNA. When present in the form of DNA, the oligonucleotide or polynucleotide may be single-stranded (ie, sense strand) or double-stranded. Suitable control elements, such as enhancers/promoters, splice boundaries, polyadenylation signals, etc., can be placed in close proximity to the coding region of the gene, if necessary, to ensure proper transcription initiation and/or proper processing of primary RNA transcription. Alternatively, the coding region used in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice boundaries, insertion sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

As used herein, the terms “in functional combination,” “in a functional order,” and “operably linked” refer to the linking of nucleic acid sequences such that the nucleic acid molecule is capable of directing transcription of a given gene and/or synthesis of the desired protein molecule occurs. The term also refers to the linking of amino acid sequences in such a way that a functional protein is produced.

The term “isolated,” when used with respect to a nucleic acid, as in the phrase “isolated oligonucleotide” or “isolated polynucleotide,” refers to a nucleic acid sequence that has been identified and separated from at least one component or contaminant with which it is normally associated in its natural source. An isolated nucleic acid is one that is found in a form or under conditions different from those in which it occurs naturally. In contrast, unisolated nucleic acids are those nucleic acids, such as DNA and RNA, that are in the state in which they exist in nature. For example, a given DNA sequence (eg, a gene) is found on a host cell chromosome in close proximity to neighboring genes; RNA sequences, such as a particular sequence of mRNA that codes for a particular protein, are found in a cell as a mixture with many other mRNAs that code for many proteins. However, an isolated nucleic acid encoding a given protein includes, by way of example, that nucleic acid in cells that normally express the protein, where the nucleic acid is in a different chromosomal location from that in natural cells or is otherwise flanked by a different sequence nucleic acid than that found in nature. The isolated nucleic acid, oligonucleotide or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be used for protein expression, the oligonucleotide or polynucleotide will contain at least a sense or coding strand (i.e. the oligonucleotide or polynucleotide may be single stranded) but may contain both sense and antisense strands (i.e. i.e. the oligonucleotide or polynucleotide can be double-stranded).

As used herein, the term “native protein” indicates that the protein does not contain the amino acid residues encoded by the vector sequences; that is, the native protein contains only those amino acids that are found in the protein, as they occur in nature. The native protein can be produced by recombinant methods or can be isolated from a naturally occurring source.

As used herein, the term “part” with respect to a protein (as in the phrase “a portion of a given protein”) refers to fragments of that protein. Fragments can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors characterized by a bacterial origin of replication and mammalian episomal vectors). Lentiviral vectors or retroviral vectors can be used (eg, to introduce DNA encoding one or more AP-1 transcription factors and/or a CAR construct into cells (eg, T cells)). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”. In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. In the present description, the terms "plasmid" and "vector" may be used interchangeably, since plasmid is the most commonly used form of vector.

As used herein, the term “expression vector” refers to a recombinant DNA molecule containing the desired coding sequence and corresponding nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often together with other sequences. Eukaryotic cells are known to use promoters, enhancers, and termination and polyadenylation signals.

As used herein, the term “transfection” refers to the introduction of foreign DNA into eukaryotic cells. Transfection can be accomplished by a variety of methods known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and bioballistics.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into genomic DNA.

The term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell where the foreign DNA cannot integrate into the genome of the transfected cell. Foreign DNA remains in the nucleus of the transfected cell (for example, for several days). During this time, foreign DNA is exposed to regulatory control elements that regulate the expression of endogenous genes in the chromosomes. The term "transient transfectant" refers to cells that have taken up foreign DNA but have failed to integrate that DNA.

As used herein, the term “selectable marker” refers to the use of a gene that encodes an enzymatic activity that confers the ability to grow in an environment lacking what would otherwise be an essential nutrient; in addition, the selectable marker may confer antibiotic or drug resistance to the cell in which the selectable marker is expressed. Selectable markers may be “dominant”; a dominant selectable marker encodes an enzymatic activity that can be found in any eukaryotic cell line. Examples of dominant selectable markers include the bacterial aminoglycoside 3'-phosphotransferase gene (also called the neo gene), which confers resistance to the drug G418 in mammalian cells, the bacterial hygromycin G phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin, and a bacterial xanthine guanine phosphoribosyltransferase gene (also called the gpt gene) that confers the ability to grow in the presence of mycophenolic acid. Other selectable markers are non-dominant in the sense that their use must be associated with a cell line that lacks the activity of the corresponding enzyme. Examples of non-dominant selectable markers include the thymidine kinase (tk) gene, which is used in combination with tk-negative (tk-) cell lines, the CAD gene, which is used in combination with CAD-deficient cells, and the mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene. , which is used in combination with hprt-negative (hprt-) cell lines. For a review of the use of selectable markers in mammalian cell lines, see Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), p. 16.9-16.15.

As used herein, the term “in vitro” refers to the artificial environment and processes or reactions that occur in the artificial environment. In vitro media may, without limitation, consist of tubes and cell cultures. The term "in vivo" refers to the natural environment (such as an animal or cell) and the processes or reactions that occur in the natural environment.

As used herein, the term “cell culture” refers to any in vitro culture of cells. Included in this term are continuous cell lines (eg, immortalized phenotype), primary cell cultures, transformed cell lines, continuous cell lines (eg, non-transformed cells), and any other cell population maintained in vitro.

As used herein, the term “sample” is used in its broadest sense. In one sense it is meant to include a specimen or culture obtained from any source, as well as biological and environmental specimens. Biological samples can be obtained from animals (including humans) and include liquids, solids, tissues and gases. Biological samples include blood products such as plasma, serum and the like. Such examples, however, should not be construed as limiting the types of designs applicable to the present invention.

Detailed Disclosure of Embodiments

The present invention is based on the discovery that T cells modified (eg, genetically) to overexpress and/or contain increased (eg, supraphysiological) levels of one or more AP-1 transcription factors (eg, c-Jun), exhibit reduced levels of T cell exhaustion (eg, compared to unmodified T cells expressing normal levels of AP-1 transcription factors). As described in detail herein, expression of the AP-1 transcription factors Fos and c-Jun is reduced/down-regulated in exhausted T cells, and T cells modified to overexpress and/or contain increased levels of c-Jun or other factor AP-1 transcriptions show reduced levels of T cell exhaustion. For example, overexpression of AP-1 transcription factors (specifically c-Jun) prevented the development of T cell exhaustion and maintained T cell functionality (eg, 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) show reduced signs of exhaustion (including lower levels of exhaustion markers, increased memory formation and increased cytokine production), indicating that that T cells modified to overexpress and/or contain increased (e.g., supraphysiological) levels of one or more AP-1 transcription factors demonstrate increased clinical efficacy in multiple malignancies (see, e.g., Examples 1-6) . In addition, both CD19 CAR T cells modified to overexpress c-Jun and CD22 CAR T cells modified to overexpress c-Jun showed increased CAR T cell recognition of target leukemia cells with low levels of surface antigen (see, for example, example 6). CAR T cells modified to overexpress AP-1 transcription factors demonstrated reduced T cell exhaustion and enhanced functionality in three separate in vivo tumor models using cJUN-modified CAR T cells (see Example 8). Thus, overexpression of AP-1 transcription factors (specifically c-Jun) prevented the development of T cell exhaustion and maintained T cell functionality. Taken together, these results indicate that the compositions and methods of the present invention are broadly applicable and address many of the existing barriers to successful CAR T-cell therapy.

Accordingly, the present invention relates to modified T cells (e.g., genetically and/or functionally modified (e.g., to overexpress and/or contain increased levels of one or more AP-1 transcription factors and/or to reduce the expression and/or activity of one or several members of the AP-1 inhibitory complex)) that maintain functionality under conditions in which unmodified T cells exhibit T cell exhaustion. Thus, the compositions and methods of the present invention can be used to prevent the exhaustion of engineered T cells (e.g., those engineered to express a specific T cell receptor or those engineered to express a chimeric antigen receptor (CAR)) and to prevent the exhaustion of non-engineered T cells ( for example, native or natural T cells (eg, isolated from a subject), thereby enhancing the functionality (eg, activity against cancer or infectious disease) of engineered as well as non-engineered T cells.

Modifying T cells to overexpress and/or contain increased levels of one or more AP-1 transcription factors can prevent exhaustion of AP-1 transcription factors in the T cell (e.g., this occurs when T cells are exhausted (see e.g. example 1) and/or lead to increased (for example, supraphysiological) levels of AP-1 transcription factors). AP-1 transcription factors (eg, c-Jun) are factors that are induced following T cell activation and are involved in the production and secretion of cytokines (eg, interleukin-2) by T cells. T cells that express CAR undergo tonic, antigen-independent signaling due to receptor clustering and recapitulate the fundamental biological mechanism of T cell exhaustion, as evidenced by high levels of PD-1, TIM-3 and LAG-3 expression, reduced antigen-induced cytokine production and excessive programmed cell death. AP-1 transcription factors have been identified and characterized as reduced in exhausted T cells (see, for example, example 1). Modification of T cells to overexpress and/or contain increased levels of one or more AP-1 transcription factors significantly increased T cell functionality under conditions that cause T cell exhaustion (see, for example, Examples 2-5). Although an understanding of the mechanism is not required for the practice of the present invention, and although the present invention is not limited to any particular mechanism of action, maintaining and/or increasing AP-1 transcription factor levels (eg, c-Jun levels) within T cells prevents T cell dysfunction. -cells associated with T cell exhaustion (e.g., minimal effect of AP-1 transcription factor overexpression was observed in nonexhausted T cells, indicating that relative or absolute c-Jun deficiency represents an important factor in exhaustion-associated dysfunction T cells).

In some embodiments, the T cells are modified to overexpress and/or contain increased levels of one or more mutated and/or truncated AP-1 transcription factors to prevent depletion of AP-1 transcription factors in the T cell. Experiments conducted during the development of embodiments herein demonstrate that portions of the AP-1 family proteins (eg, c-Jun) can be mutated and/or truncated without affecting the ability of the mutated/truncated AP-1 factors to mediate the restoration of dysfunctional T -cells. For example, c-Jun polypeptides with N-terminal deletions and mutations (eg, in the transactivation domain) retain their ability to restore the function of HA-28z-depleted CAR T cells and maintain an equivalent increase in cytokine production compared to c-Jun. In some embodiments, the AP-1 polypeptide (eg, c-Jun) contains mutations or deletions in the trans and/or delta activation domains. In some embodiments, the AP-1 polypeptide (eg, c-Jun) contains mutations or deletions that render the transactivation and/or delta domains inactive or not present. In some embodiments, the mutated/truncated AP-1 polypeptide (e.g., c-Jun) contains 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or ranges between them) or greater sequence identity to the C-terminal amino acid residues (e.g., 50 residues, 75 residues, 100 residues, 150 residues, 200 residues, 250 residues, or ranges therebetween), the C-terminal portion (e.g., the fourth, third, half) or C-terminal domains (eg, epsilon, bZIP and their C-terminal amino acids) of the wild-type AP-1 transcription factor (eg, c-Jun). In some embodiments, N-terminal amino acid residues (e.g., 50 residues, 75 residues, 100 residues, 150 residues, or intervals therebetween), N-terminal portion (e.g., quarter, third, half), or N-terminal domains (e.g. , delta, transactivation domain and its N-terminal amino acid) of a wild-type AP-1 transcription factor (eg, c-Jun) is deleted, mutated, or otherwise inactivated. Any embodiments described herein with reference to the AP-1 transcription factor may contain a mutated/truncated AP-1 polypeptide (eg, c-Jun), consistent with, for example, the above.

Although enhanced expression of AP-1 transcription factors such as c-Fos and c-Jun increased the functionality of engineered T cells, there are other inhibitory AP-1 family members expressed in exhausted activated T cells. Inhibition/knockdown of members of the AP-1 inhibitory complex (eg, to increase the availability of canonical AP-1 factors) also reduced T cell exhaustion (eg, see Example 7). Specifically, inhibition/knockdown of AP-1 inhibitory complex members resulted in significant improvements in T cell function (e.g., increased cytokine production and/or expression) in exhausted T cells, but not in healthy T cells (e.g., see FIG. 8A-D). Thus, in some embodiments, the present invention provides compositions and methods for inhibiting T cell exhaustion by inhibiting the expression and/or activity of members of the AP-1 inhibitory complex (e.g., BATF3, and other members of the BATF family (BATF 1 and 2), IRF4 , IRF8, and other members of the IRF family (IRF 1, 2, 3, 5,6, 7, or 9), and members of the ATF family (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 members of the AP-1 inhibitory complex (eg, genes). For example, in some embodiments, the expression and/or activity of members of the AP-1 inhibitory complex occurs at the genomic level (eg, by disrupting gene expression). In other embodiments, inhibition of expression and/or activity of members of the AP-1 inhibitory complex occurs at the level of protein expression and/or activity (eg, by disrupting protein expression and/or activity). Exemplary compositions and methods of inhibiting the expression and/or activity of members of the AP-1 inhibitory complex include, but are not limited to, the following: nuclease disruption, CRISPR-Cas9 systems, zinc finger nuclease targeting, TALEN genome editing, shRNA, siRNA, microRNA targeting, degrons , regulated promoters, protein inhibitors (eg, chemical inhibitors, small molecule inhibitors, antibodies, etc.), expression of dominant negatives, and the like. Exemplary compositions for inhibiting the expression and/or activity of members of the AP-1 inhibitory complex are presented in Table 1 below. CAS stands for Chemical Abstracts Service and is a unique numeric identifier assigned to each chemical substance.

Thus, the present invention provides compositions and methods for reducing T cell exhaustion comprising T cells modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors and/or modified to have decreased expression and/or activity one or more members of the AP-1 inhibitory complex. The present invention is not limited to the disease or condition that can be treated using the modified T cells of the present invention. Indeed, the compositions and methods presented herein may be useful in the treatment of any disease for which increased T cell activity may provide a therapeutic benefit.

Accordingly, the present invention provides a composition comprising T cells modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors and/or modified (eg, genetically) to reduce expression and/or activity of one or more several members of the AP-1 inhibitory complex (eg, JunB, BATF3 and other members of the BATF family, IRF4, IRF8 and other members of the IRF family, and members of the ATF family). As described in detail herein, T cells can be engineered or unengineered T cells (eg, tumor infiltrating lymphocytes (TILs)). Moreover, the present invention is not limited to the type of T cell modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors. In some embodiments, the T cells are CD3+ T cells (eg, a combination of CD4+ and CD8+ T cells). In certain 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 T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the T cells are a combination of CD4+ and CD8+ T cells (eg, which are CD3+). In certain embodiments, the T cells are memory T cells. In certain embodiments, the T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and/or gamma delta T cells. In some embodiments, the T cells are cytokine-induced killer cells. In some embodiments, the T cells are engineered to express a chimeric antigen receptor. In another embodiment, the T cells are engineered to express a specific T cell receptor (eg, with specificity for a tumor antigen or an infectious disease antigen). In some embodiments, the T cells are antitumor T cells. The composition may contain a pharmaceutically acceptable carrier (eg, a buffer). The composition may further contain one or more other agents (e.g., a chemotherapeutic agent (e.g., the chemotherapeutic agents described herein) and/or an antimicrobial agent. Exemplary antimicrobial agents include, but are not limited to, antibodies, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thiomersal and/or combinations thereof.The composition may optionally include one or more additional agents, such as other drugs for the treatment of T-cell depletion (for example, a checkpoint inhibitor - an anti-PD-1 antibody such as nivolumab), or other drugs used to treat a subject for an infection or disease associated with T-cell depletion (eg, antivirals, antibiotics, antimicrobials, or anticancer agents).

Antimicrobial therapeutic agents can be used as therapeutic agents in the composition of the present invention. Any agent that can kill, inhibit, or otherwise impair the function of microorganisms may be used, as well as any agent purported to have such activities. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (eg, defensins), antisense nucleic acids, membrane disrupting agents, and the like, used alone or in combination. Indeed, any type of antibiotic can be used, including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.

Several strategies for targeting tumor antigens are known in the art. For example, immunotherapies targeting GD2 are currently in clinical and preclinical studies in several diseases, including neuroblastoma, osteosarcoma and melanoma (see, for example, 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 successfully cross the blood-brain barrier, activated CAR T cells effectively enter the CNS after adoptive transfer. Accordingly, in one embodiment, any CAR T cell can be modified in accordance with the compositions and methods of the present invention (e.g., modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors (e.g., thereby enhancing functionality of CAR T cells and/or inhibition of CAR T cell exhaustion)).

For example, as described herein, a modified T cell of the present invention (e.g., modified to overexpress and/or contain increased levels of one or more AP-1 transcription factors and/or modified (e.g., genetically) to reduce expression and /or activity of one or more members of the AP-1 inhibitory complex (eg, JunB and BATF3 and other members of the BATF family, IRF4 and members of the ATF family) may also be designed to contain a CAR that contains a target-specific binding element, otherwise called antigen-binding fragment. The choice of fragment depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease condition. Examples of cell surface markers that can act as ligands for an antigenic fragment domain in a CAR of the present invention include markers associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

For example, a CAR can be designed to target a tumor antigen of interest by designing a desired antigen-binding moiety that specifically binds to an antigen on a tumor cell. As used herein, the term “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” or “cancer antigen” refers to antigens that are common to specific hyperproliferative disorders such as cancer. Exemplary antigens mentioned herein are included by way of example. This list is not intended to be exclusive, and additional examples will be apparent to those skilled in the art.

Tumor antigens are proteins that are produced by tumor cells that trigger an immune response, particularly T cell-mediated immune responses. Thus, the antigen binding fragment 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 antigen, carcinoembryonic antigen (CEA), human chorionic gonadotropin beta subunit, alphafetoprotein (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 carcinoma tumor antigen 1, MAGE, ELF2M, neutrophil elastase, ephrin-B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin .

A tumor antigen may contain one or more antigenic cancer antigens/epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, the following: tissue-specific antigens such as MART-1, tyrosinase and GP100 in melanoma and prostatic 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 HER-2/Neu/ErbB-2 oncogene. Another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulin is a truly tumor-specific immunoglobulin antigen that is unique to an individual tumor. B cell differentiation antigens such as CD 19, CD20, and CD37 are other candidate target antigens in B cell lymphoma.

The tumor antigen may also be a tumor specific antigen (TSA) or a tumor antigen (TAA). TSA is unique to tumor cells and is not found in other cells in the body. TAA is not unique to the tumor cell and is instead also expressed on some normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. Expression of an antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are typically present at extremely low levels in normal cells but that are expressed at much higher levels. high levels on tumor cells.

Examples of TSA or TAA include, but are not limited to, differentiation antigens such as MART-1/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE- 1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus EBVA antigens and human papillomavirus (HPV) E6 and E7 antigens. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG -72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.291\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM , HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

Depending on the desired antigen to be targeted, the CAR can be designed to include an appropriate antigen binding moiety specific for the desired target antigen. For example, if CD19 is the desired antigen to be targeted, an anti-CD19 antibody can be used as an antigen binding fragment for inclusion in the CAR of the present invention.

Treatment methods

In another aspect, provided herein are methods of treating a disease or condition in a subject comprising administering to the subject (e.g., a patient) having the disease or condition an effective number of T cells modified to express and/or contain increased levels of one or more factors AP-1 transcription. The present invention is not limited to the type of disease or condition being treated. Indeed, any disease or condition that is treatable (eg, for which the signs or symptoms of the disease are reduced in intensity by treatment) by administering T cells can be treated in an improved and more effective manner using compositions and methods of the present invention (eg, containing and/or using T cells modified to express and/or contain increased 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 to the type of cancer or the type of infectious disease. Indeed, any cancer known in the art that is treated with T cell therapy can be treated using the compositions and methods of the present invention. Likewise, any infectious disease known in the art that is treated with T cell therapy can 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 increased levels of one or more AP-1 transcription factors to a subject (e.g., a patient) characterized by the presence of a disease or condition inhibits T cell exhaustion in the subject (eg, what might otherwise happen when the same number of unmodified T cells is administered to a subject). For example, in certain embodiments, the methods provide methods for treating a subject having a disease or condition that is responsive to treatment with adoptive cell therapy. In certain embodiments, methods of treating a subject having a disease or condition responsive to treatment with adoptive cell therapy comprise the step of administering a composition comprising an effective amount of T cells genetically modified to express increased levels of one or more AP-1 transcription factors wherein T cells genetically modified to express increased levels of one or more AP-1 transcription factors exhibit reduced levels of T cell exhaustion compared to T cells expressing normal levels of one or more AP-1 transcription factors. In certain embodiments, the disease or condition includes cancer.

In one aspect, the present invention relates to methods of treating forms of cancer/tumors using T cells modified/engineered to express one or more AP-1 transcription factors. In accordance with various aspects of the present invention, methods are provided for treating cancer/tumors, the methods comprising administering to a patient having such cancer or tumor an effective amount of T cells modified/engineered to express one or more AP-1 transcription factors. The present invention is not limited to the type of cancer and/or tumor to be treated. As used herein, the term “cancer” refers to various types of malignant neoplasms, most of which can invade surrounding tissue and can metastasize to different areas (see, for example, PDR Medical Dictionary, 1st edition (1995), incorporated herein document by reference in its entirety for all purposes). The terms "neoplasm" and "tumor" refer to abnormal tissue that grows through cellular proliferation faster than normal and continues to grow after the stimuli that initiate proliferation are removed. Such abnormal tissue exhibits partial or complete lack of structural organization and functional coordination with normal tissue, which may be either benign (ie, benign tumor) or malignant (ie, malignant tumor). Examples of general categories of cancer include, but are not limited to, carcinomas (e.g., malignant tumors originating from epithelial cells, such as, for example, common forms of breast, prostate, lung, and colon cancer), sarcomas (e.g., malignant tumors originating from connective tissue or mesenchymal cells), lymphomas (for example, malignancies originating from hematopoietic cells), leukemias (for example, malignancies originating from hematopoietic cells), germ cell tumors (for example, tumors originating from totipotent cells). In adults, they most often occur in the testis or ovary; in fetuses, infants and young children are most often found along the midline of the body, especially at the tip of the coccyx), blast tumors (for example, a typically malignant tumor that resembles immature or fetal tissue), and the like. Additional 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 cancer of the nervous tissue, hematopoietic tissue, breast, skin, bone, prostate, ovary, uterus, cervix, liver, lungs, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal glands, immune system, head and neck, colon, stomach, bronchi and/or kidneys. In some embodiments, the cancer is an occult cancer, a previously diagnosed primary cancer, or a metastatic cancer.

In certain embodiments, the presence of increased levels of one or more AP-1 transcription factors in modified T cells (e.g., CAR T cells (e.g., compared to T cells that are not modified)) results in more effective treatment (e.g. , killing and/or inhibiting the progression of) forms of cancer/tumors compared to treatment using T cells (eg, CAR T cells) that are not modified to contain increased levels of one or more AP-1 transcription factors.

In certain embodiments, the present invention relates to methods of treating (eg, inhibiting the growth and/or killing) forms of cancer/tumors using T cells (eg, CD3+ T cells) genetically engineered to overexpress and/or contain elevated (eg, supraphysiological) levels of one or more AP-1 transcription factors and which are engineered to express a receptor that recognizes tumor surface antigen (eg, CD19, CD20, CD22, ROR1, GD2, EBV protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen, CD33/IL3Ra, c-Met, PSMA, F77 glycolipid, EGFRvIII, NY-ESO-1, MAGE-A3, MART-1, GP1000 and/or p53) and transmit a signal that activates the T cell to cause T cell expansion and/or tumor destruction. A non-limiting example of a receptor is a chimeric antigen receptor (CAR), which includes a mAb-derived scFv that recognizes GD2, as well as a transmembrane domain and one or more intracellular signaling domains. The CAR can be further engineered to include other signaling elements that facilitate expansion of the engineered cells upon encounter with a tumor cell antigen (eg, GD2 antigen), as well as elements that ensure long-term persistence of the engineered cells. The present invention further provides compositions containing genetically engineered cells (eg, immunotherapeutic compositions produced and administered in quantities sufficient to produce cancers and/or tumors).

Construction of modified T cells.

As described herein, the present invention provides compositions containing T cells that are modified (eg, genetically (eg, transduced)) to express (eg, heterologously) one or more AP-1 transcription factors. For example, the present invention relates to transduced T cells. A "transduced cell" is a cell into which a nucleic acid molecule has been introduced using molecular biology techniques. T cells, which are

modified/transduced to express one or more heterologous AP-1 transcription factors, may be transduced by any technique by which a nucleic acid molecule can be introduced into such a cell, including but not limited to transfection with viral vectors (e.g., retroviral, lentiviral or other viral vector) through a CRISPR/Cas9-based system, transformation with plasmid vectors and/or introduction of naked DNA by electroporation, lipofection and gene gun acceleration. Viral and/or plasmid vectors can be used for in vitro, in vivo and/or ex vivo expression. The AP-1 transcription factor can be coexpressed with an engineered TCR or CAR, with both the transcription factor and the TCR and/or CAR being coexpressed from different viral vectors. In another embodiment, they are expressed from a single vector construct using a bicistronic vector. c-Jun (and/or other AP-1 transcription factor) can be expressed constitutively or in a regulated manner (eg, using a small molecule remote expression control system or using an endogenously regulated system). In another embodiment, the genes for c-Jun and/or other AP-1 transcription factors can be genetically integrated into cellular DNA using a retroviral, lentiviral or other viral vector or through a CRTSPR/Cas9-based system. In yet another embodiment, c-Jun and/or another AP-1 transcription factor is expressed by an RNA or oncolytic virus or other transient expression system known in the art. c-Jun and/or other AP 1 transcription may be delivered ex vivo to T cells (eg, where T cells are used for adoptive transfer) or delivered via genetic transfer in vivo.

The present invention is not limited to chimeric antigen receptor (CAR) expressed in T cells (eg, the CAR construct used in the methods of the present invention). In one embodiment, the CAR comprises a heavy (VH) and light chain (VL) variable region fusion protein (eg, single chain fragment variable (scFv)) of an immunoglobulin that specifically binds to a tumor antigen (eg, GD2). Those skilled in the art will know that scFv is a fusion protein of the heavy (VH) and light chain (VL) variable regions of immunoglobulins linked to a linker peptide (eg, about 10 to about 25 amino acids in length). The present invention is not limited to the type of linker. In some embodiments, the linker is enriched with glycine (eg, for flexibility). In some embodiments, the linker contains serine and/or threonine (eg, for solubility). In some embodiments, the linker contains a glycine-rich portion and a serine and/or threonine portion.

Any antibody/immunoglobulin that specifically binds to a tumor antigen (eg, GD2) can be used to construct a CAR (eg, using the VH and VL regions to construct a scFv fusion protein) 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, for GD2: 14G2a, ch14.18, hu14.18K322A, m3F8, hu3F8-IgG1, hu3F8-IgG4, HM3F8, UNITUXIN, DMAb-20, or any other antibody that binds with specificity to GD2 (eg, known or described in the art, or not yet identified). A tumor antigen (eg, GD2) CAR may contain a receptor including variants within a tumor antigen (eg, GD2) antibody scFv generated to enhance affinity and/or reduce tonic signaling. A tumor antigen CAR (eg, GD2) may include variable length hinge regions (eg, between scFv and signaling domains) and/or different transmembrane domains. The present invention is not limited to the transmembrane domain used. Indeed, any transmembrane domain may be used, including without limitation all or part of the TCR zeta chain transmembrane domain (CD3ζ, CD28, OX40/CD134, 4-1BB/CD137/TNFRSF9, FcERIy, ICOS/CD278, ILRB/CD122, IL -2RG/CD132 or CD40.

The CAR construct of the present invention may include an intracellular signaling domain (e.g., native T cell receptor complex CD3 zeta and/or another signaling domain (e.g., MyD88 signaling domain)) that transduces a ligand binding event to an intracellular signal (e.g., which activates (e.g., partially) a T cell. In the absence of costimulatory signals, receptor-ligand binding is often insufficient to fully activate and proliferate the T cell. Thus, a CAR construct may include one or more costimulatory domains (e.g., that provide a second signal to stimulate full activation of T cells). In one embodiment, a co-stimulatory domain is used that increases cytokine production of immune CAR T cells. In another embodiment, a co-stimulatory domain is used that facilitates T cell replication. In yet another embodiment, a costimulatory domain that prevents CAR T cell exhaustion. In another embodiment, a costimulatory domain is used that enhances the antitumor activity of T cells. In yet another embodiment, a costimulatory domain is used that enhances the survival of CAR T cells (eg, after infusion into patients). Examples of proteins or domains or portions thereof 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/TNFSFS; 4-1 BB ligand/TNFSF9; GITR/TNFRSF18; BAFF/BLyS/TNFSF13B; GITR ligand/TNFSF18; BAFF R/TNFRSF13C; N VEM/TNFRS F14; CD27/TNFRSF7; LIGHT/TNFSF14; CD27 ligand/THP8P7; OX40/TNFRSF4; CD30/TNFRSF8; OX40 ligand/TCR8P4; CD30 ligand/TNFSFS; TAC1/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 alpha 4/CD49d; CD82/Kai-1; integrin alpha 4 beta 1; CD90/Thyl; integrin alpha 4 beta 7/LPAM-1; CD96; LAG-3; CD160; LMIR1/CD300A; CRTAM; TCL1A; DAP 12; TIM-1/KIM-1/HAVCR; Dectin-1/CLEC7A; TIM-4; DPPIV/CD26; TSLP; EphB6; TSLP R and HLA-DR. In one embodiment, the CAR construct expressed in T cells used in the compositions and methods of the present invention includes a CD28 endodomain, a 4-1BB endodomain, and/or an OX40 endodomain. In certain embodiments, a tumor antigen (e.g., GD2) specific CAR construct of the present invention comprises an antibody scFv that specifically binds to a tumor antigen (e.g., GD2), a transmembrane domain (e.g., CD8), an intracellular T signaling domain -cell receptor (eg, TCR zeta chain (CD3-zeta)) and at least one costimulatory domain (eg, 4-1BB).

The present invention is not limited to means of genetic expression of TCR, CAR and/or one or more AP-1 transcription factors in T cells. Indeed, any means known in the art and/or described herein can be used. Non-limiting examples of T cell genetic engineering methods include, but are not limited to, retrovirus- or lentivirus-mediated transduction, transduction using transposase-based gene integration systems, Crispr/Cas9-mediated gene integration, non-integrating vectors such as RNA or adeno-associated viruses, or others methods described herein. Compositions containing engineered T cells may include non-engineered T cells or other immune cells or subclasses of T cells selected for greater expansion or persistence. To reduce toxicity, it may be possible to include elements that allow the engineered cells to be killed. To reduce toxicity and/or enhance efficacy, elements may be included that allow the expression of the protein in the engineered cells to be regulated.

Anticancer therapeutic agents, compositions and combination therapy.

In certain embodiments, the present invention provides methods for treating or delaying the progression of cancer or treating or delaying the progression of an infectious disease in an individual, comprising administering to the individual an effective amount of modified T cells according to the present invention. In some embodiments, the treatment results in a sustained response in the individual after treatment is discontinued. The methods described herein may find use in the treatment of conditions in which increased immunogenicity is desired, for example, increasing the immunogenicity of a tumor for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual having cancer by administering to the individual an effective amount of modified T cells according to the present invention. These methods can use any type of T cells genetically modified to express CARs and/or TCRs known in the art or described herein.

In some embodiments, the individual has a cancer that is resistant (eg, has been demonstrated to be resistant) to one or more other forms of cancer treatment (eg, chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes recurrent or refractory cancer. Recurrence can refer to the reappearance of cancer in the original site or a new site after treatment. In some embodiments, resistance includes progression of cancer during chemotherapy treatment. In some embodiments, resistance includes cancers that do not respond to conventional or conventional treatment with a chemotherapeutic agent. The cancer may be resistant at the start of treatment, or it may become resistant during treatment. In some embodiments, the cancer is at an early stage or at an advanced stage.

According to certain embodiments of the present invention, exposing animals (eg, humans) suffering from cancer/tumors to therapeutically effective amounts of immunotherapeutic compositions containing T cells to express and/or contain increased levels of one or more AP-1 transcription factors , inhibits the growth of such cancer cells directly and/or renders such cells a population more susceptible to anticancer drugs or radiation therapy (eg, their cell death-inducing activity). The immunotherapeutic compositions and methods of the present invention can be used to treat, ameliorate, or prevent disorders such as any type of cancer.

In certain embodiments, immunotherapeutic compositions comprising T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors are used to treat, ameliorate, or prevent cancer that is resistant to one or more conventional therapies. cancer treatment (for example, those cancer cells that are chemoresistant, resistant to radiation therapy, resistant to hormones, etc.). As described herein, any T cells genetically modified to express a tumor-specific CAR can be used in the immunotherapeutic compositions and methods of the present invention.

Immunotherapeutic compositions (e.g., containing T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors) and according to the present invention can be used to induce cytotoxic activities against tumor cells and/or to promote survival and function cells (eg, survival and function of modified immune cells). For example, the immunotherapeutic compositions and methods of the present invention can be used to induce interleukin-2 (IL-2) to promote T cell survival; to induce Fas ligand (FasL) and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (eg, to induce apoptosis of tumor cells); and/or to induce interferon (IFN)-gamma (eg, to activate the innate immune response (eg, against cancer)). In some embodiments, the compositions and methods of the present invention are used to induce cell cycle arrest and/or apoptosis, as well as to enhance the induction of cell cycle arrest and/or apoptosis, either alone or in response to additional apoptosis induction signals. In some embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors sensitize cancer cells to the induction of cell cycle arrest and/or apoptosis, including cells that are normally resistant to such inducing stimuli.

In some embodiments, the compositions and methods of the present invention are used to treat abnormal cells, tissues, organs, or pathological conditions and/or disease states in an animal (eg, a mammalian patient, including but not limited to humans and companion animals). In this regard, various diseases and pathologies can be treated or prevented using the present methods and compositions. In some embodiments, the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are resistant to anticancer agents.

In some embodiments, the present invention provides methods for administering an effective amount of T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastic agents , apoptosis-modulating agents, antimicrobial agents, antiviral agents, antifungal agents and anti-inflammatory agents) and/or therapeutic techniques (eg, surgery and/or radiation therapy). In a specific embodiment, the additional therapeutic agent(s) are an anticancer agent.

A number of suitable anticancer agents are provided for use in the methods of the present invention. Indeed, the present invention provides, without limitation, the administration of numerous antitumor agents, such as the following: agents that induce apoptosis; polynucleotides (eg, antisense, ribozymes, siRNA); polypeptides (eg enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (eg, antibodies conjugated to anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (eg, interferons (eg, IFN-α) and interleukins (eg, IL-2)); means of adoptive immunotherapy; hematopoietic growth factors; agents that induce differentiation of tumor cells (for example, all-trans retinoic acid); gene therapy reagents (eg, antisense therapy reagents and nucleotides); antitumor vaccines; angiogenesis inhibitors; proteasome inhibitors; NF-KB modulators; anti-CDK compounds; HDAC inhibitors; etc. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.

In certain embodiments, anticancer agents include agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, irradiation (eg, X-rays, gamma rays, UV radiation); tumor necrosis factor (TNF)-related factors (eg, TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (eg, epidermal growth factor receptor (EGFR) kinase inhibitor, vascular endothelial growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-kinase inhibitors Abl (such as Gleevec (GLEEVEC)); antisense molecules; antibodies (such as Herceptin, Rituxan, Zevalin and Avastin); antiestrogens (such as raloxifene and tamoxifen); antiandrogens (such as flutamide, bicalutamide, finasteride, aminoglutetamide, ketoconazole and corticosteroids ); cyclooxygenase 2 (COX-2) inhibitors (eg, celecoxib, meloxicam, NS-398 and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (eg, butazolidine, decadron, deltasone, dexamethasone, dexamethasone intensol, dexon, hexadrol, hydroxychloroquine, meticorten, oradexon, orazone, oxyphenbutazone, pediapred, phenylbutazone, plaquenil, prednisolone, prednisone, prelon and tandearyl); and anticancer chemotherapy drugs (eg, irinotecan (Camptosar), CPT-11, fludarabine (Fludara), dacarbazine (DTIC), dexamethasone, mitoxantrone, mylotarg, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine , bortezomib, gefitinib, bevacizumab, taxotere or taxol); cell signaling molecules; ceramides and cytokines; staurosporine and the like.

In other embodiments, the compositions and methods of the present invention are used in conjunction with at least one antihyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (eg, herbs and other compounds of plant and/or animal origin).

Alkylating agents suitable for use in the present compositions and methods include, without limitation, the following: 1) nitrogen mustards (eg, mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysine) and chlorambucil); 2) ethylenimines and methylmelamines (for example, hexamethylmelamine and thiotepa); 3) alkylsulfonates (for example, busulfan); 4) nitrosoureas (eg, carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU) and streptozocin (streptozotocin)); and 5) triazenes (eg, dacarbazine (DTIC; dimethyltriazenoimide-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the present compositions and methods include, without limitation, the following: 1) folic acid analogs (eg, methotrexate (ametopterin)); 2) pyrimidine analogs (eg, fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorooxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogues (eg, mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).

In other embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, without limitation, the following: 1) vinca alkaloids (eg, vinblastine (VBL), vincristine); 2) epipodophyllotoxins (for example, etoposide and teniposide); 3) antibiotics (eg, dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C)); 4) enzymes (for example, L-asparaginase); 5) biological response modifiers (for example, interferon-alpha); 6) platinum coordination complexes (for example, cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (for example, mitoxantrone); 8) substituted ureas (for example, hydroxyurea); 9) methylhydrazine derivatives (eg procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (for example, mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (for example, prednisone); 12) progestins (eg, hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate); 13) estrogens (for example, diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (for example, tamoxifen); 15) androgens (for example, testosterone propionate and fluoxymesterone); 16) antiandrogens (eg, flutamide); and 17) gonadotropin-releasing hormone analogues (eg, leuprolide).

Any oncolytic agent that is commonly used in the context of cancer therapy can also be used in the compositions and methods of the present invention. For example, the US Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International partner agencies of the US FDA maintain similar formularies.

Anticancer agents further include compounds that have been found to have anticancer activity. Examples include, but are not limited to, the following: 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, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasentene, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, B MS 247550, bortezomib , bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximabate CG0070, cilengitide, clofarabine, combretastatin A4-phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doc sercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, Glu phosphamide, GTI-2040, histrelin, HKI -272, homogarringtonine, HSPPC-96, hu14.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, immunotoxin LMB-9, lonafarnib, luniliximab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafine, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9 , Ob-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazole o acridine, R115777 , RAD001, ranpirnase, rebeccamycin analogue, rhu-angiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248 , suberoylanilidehydroxamic acid, suramin, thalabostat, talampanel, tariquidar, temsirolimus, immunotoxin TGFa-PE38, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP401 01M, volociximab, vorinostat , VX-680, ZD1839, ZD6474, zileuton and zosuquidar trihydrochloride.

The present invention relates to methods of administering the compositions and methods of the present invention in conjunction with (eg, before, during or after) radiation therapy. The present invention is not limited to the types, amounts, or delivery and administration systems used to deliver a therapeutic dose of radiation to an animal. For example, the animal may receive photon radiation therapy, particle beam radiation therapy, other types of radiation therapy, and combinations thereof. In some embodiments, radiation is delivered to the animal using a linear accelerator. In other embodiments, radiation is delivered using a gamma knife.

The radiation source may be external or internal to the animal. External beam radiation therapy is the most common and involves directing a beam of high-energy radiation to the tumor site through the skin using, for example, a linear accelerator. Although the radiation beam is localized to the tumor site, it is almost impossible to avoid affecting normal, healthy tissue. However, external radiation is generally well tolerated by animals. Internal radiation therapy involves implanting a radiation source, such as beads, rods, pellets, capsules, particles, and the like, within the body at or near the site of a tumor, including the use of delivery systems that specifically target cancer cells (eg, using particles attached to cancer cell-binding ligands). Such implants may be removed after treatment or left inactive in the body. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (eg, metronidazole, misonidazole, intra-arterial 5-bromodeoxyuridine (Budr), intravenous iododeoxyuridine (IudR), nitroimidazole, 5-substituted-4-nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-amino] methyl]-nitro-1H-imidazole-1-ethanol, nitroaniline derivatives, selective DNA-affinity hypoxia cytotoxins, halogenated DNA ligand, 1,2,4-benzotriazine oxides, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamide, nicotinamide, ACRIDINE-interceptor, derivative of 5-tintrazole, 3-nitro-1,2,4-triazole, derivative of 4.5-dinitromemidazole, hydroxylated teksafrines, cisplatin, mitomycin, thyripazamine, nitrosomochevin, mercaptopurin, methotrexate, fluorurasil, blacksin, vincristin, carbostin , epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia) and the like), radioprotectors (for example, cysteamine, aminoalkyl dihydrophosphorothioates, amifostine (WR2721), IL-1, IL-6 and the like). Radiosensitizers enhance the destruction of tumor cells. Radioprotectors protect healthy tissues from the harmful effects of radiation.

Any type of radiation may be administered to an animal, provided that the dose of radiation is tolerated by the animal without unacceptable negative side effects. Suitable types of radiation therapy include, for example, ionizing (electromagnetic) radiation therapy (eg, X-rays or gamma radiation) or particle beam radiation therapy (eg, high linear energy radiation). Ionizing radiation is defined as radiation containing particles or photons that have sufficient energy to produce ionization, i.e. gain or loss of electrons (as described, for example, in US Pat. No. 5,770,581, incorporated herein by reference in its entirety). The effects of radiation exposure can be at least partially controlled by a physician. In one embodiment, the radiation dose is fractionated to maximize the effect on target cells and reduce toxicity.

In one embodiment, the total radiation dose administered to the animal is from about 0.01 Gray (Gy) to about 100 Gy. In another embodiment, from about 10 Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) is administered over the course of treatment. Although in some embodiments the full dose of radiation can be administered over one day, optimally the total dose is fractionated and administered over several days. It is desirable that radiation therapy be administered for at least about 3 days, such as at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52 or 56 days (approximately 1-8 weeks). Accordingly, the daily radiation dose will be approximately 1-5 Gy (for example, 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 (for example, 1.5-2 Gy). The daily dose of radiation must be sufficient to cause destruction of target cells. When lengthening the period, according to one embodiment, the radiation is not administered every day, which allows the animal to rest and allow the effects of the therapy to be realized. For example, it is desirable to administer radiation for 5 consecutive days, and not administer it for 2 days, during each week of treatment, thereby providing 2 days of rest per week. However, radiation may be administered 1 day per week, 2 days per week, 3 days per week, 4 days per week, 5 days per week, 6 days per week, or all 7 days per week, depending on the animal's response and any potential side effects . Radiation therapy can be started at any time during the therapeutic period. In one embodiment, irradiation begins in week 1 or week 2 and is administered for the remainder of the therapeutic period. For example, radiation is administered at 1-6 weeks or at 2-6 weeks of a therapeutic period of 6 weeks to treat, for example, a solid tumor. Alternatively, radiation is administered at 1-5 weeks or 2-5 weeks of the therapeutic period, which includes 5 weeks. These illustrative radiation therapy administration regimens are not intended, however, to limit the present invention.

In some embodiments of the present invention, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: with various frequency, with different durations, with different concentrations, different routes of administration, etc. In some embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors are administered before 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 or more days, or 1, 2, 3, 4, 5, 6 or more weeks before administration of a therapeutic or anticancer agent. In some embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors are administered after a therapeutic or antineoplastic agent, e.g., 0.5, 1, 2, 3, 4, 5 , 10, 12, 18 or more hours, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6 or more weeks after administration of the anticancer agent. In some embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors and a therapeutic or anticancer agent are administered simultaneously but in different schedules, for example, the modified immune cells are administered daily while 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 other embodiments, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors are administered once per week, while the therapeutic or anticancer agent is administered daily, once per week, once every two weeks, once every three weeks, once every four weeks or more.

Compositions within the scope of the present invention include all compositions in which T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors are contained in an amount that is effective to achieve the intended purpose. Although individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. In one non-limiting example, T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors can be administered to mammals, such as humans, to provide an individual with 1000 to 10 ¹⁰ T cells per day (eg , for the treatment of cancer). In another embodiment, from 1000 to 10 ¹⁰ modified T cells are administered to treat, ameliorate, or prevent cancer (eg, to prevent metastasis, recurrence, and/or progression of cancer). A unit dose may be administered in one or more administrations one or more times per day (eg, over 1, 2, 3, 4, 5, 6 or more days or weeks).

T cells can be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers containing excipients and excipients that facilitate the processing and/or introduction of the modified cells into preparations that can be used pharmaceutically. Immune T cells and/or pharmaceuticals containing them can be administered intravenously, intramuscularly, subcutaneously, intratumorally, intraperitoneally, intrathecally or intraventricularly. An effective amount of T cells and/or pharmaceuticals containing them can be administered to prevent or treat a disease. The appropriate dosage may be determined based on the type of disease being treated, the type of T cell modified, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician.

The effectiveness of any of the methods described herein (e.g., treatment with T cells modified to express and/or contain increased levels of one or more AP-1 transcription factors, alone or in combination with one or more chemotherapeutic agents described herein document) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are provided herein by way of example. For any exemplary model, after tumor development, mice are randomly assigned to treatment or control treatment groups. Tumor size (eg, tumor volume) is measured during the course of treatment, and overall survival is also monitored.

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

Responsiveness to treatment may refer to any one or more of the following: increased survival (including overall survival and progression-free survival); providing an objective response (including a complete response or partial response); or improvement in signs or symptoms of cancer. In some embodiments, susceptibility may refer to improvement in one or more factors according to a published set of RECIST guidelines for determining tumor status in a patient with cancer, i.e. response, stabilization or progression. For a more detailed discussion of these recommendations, 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 susceptible subject may refer to a subject whose cancer shows improvement, for example, according to one or more factors based on RECIST criteria. A non-responsive subject may refer to a subject whose cancer does not show improvement, for example, according to one or more factors based on RECIST criteria.

Conventional response criteria may be insufficient to characterize the antitumor activity of immunotherapies, which may cause delayed responses that may be preceded by initial overt radiologic progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that take into account the possible occurrence of new lesions and allow confirmation of radiological progression at follow-up evaluation. Accordingly, in some embodiments, responsiveness may refer to improvement in one of several factors according to 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 an already defined tumor load and monitored, for example, for radiological progression during subsequent evaluation. In some embodiments, the presence of non-target lesions is included in the assessment of complete response and is not included in the assessment of radiological progression. In some embodiments, radiological progression can be determined based only on measurable disease and/or can be confirmed through serial assessment >4 weeks from the first documented date.

The present description of the invention is considered sufficient to enable one skilled in the art to practice the present invention. Various modifications according to the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, and they come within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

EXAMPLES

The following examples illustrate, but do not limit, the compounds, compositions and methods of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters commonly encountered in clinical therapy and which will be apparent to those skilled in the art are within the spirit and scope of the present invention.

Materials and methods

Viral vector construction

MSGV retroviral vectors encoding the following CARs have been previously described: CD19-28z, CD19-BBz, GD2-28z, GD2-BBz, Her2-BBz, and CD22-BBz. To generate the HA-28z CAR, a point mutation was introduced into the 14G2a scFv of the GD2-28z CAR plasmid to create the E101K mutation. “4/2NQ” 46 mutations were introduced into the CH2CH3 domains of the IgG1 spacer region to reduce Fc receptor recognition for in vivo use of HA-28z CAR T cells. Codon-optimized cDNAs encoding c-Jun (JUN), c-Fos (FOS), and truncated NGFR (tNGFR) were synthesized by IDT and cloned into lentiviral expression vectors to generate JUN-P2A-FOS as well as individual JUN expression vectors and FOS, coexpressing tNGFR under the control of a separate PGK promoter. JUN-P2A was then subcloned into the XhoI site of MSGV CAR vectors using the In-Fusion HD cloning kit (Takara) upstream of the CAR leader sequence to generate JUN-P2A-CAR retroviral vectors. For JUN-AA, point mutations were introduced to convert Ser63 and Ser73 to Ala. The E. coli DHFR-DD sequence was inserted upstream of Jun to generate JUN-DD constructs. In some cases, the GFP cDNA was also subcloned upstream of the CAR to create the GFP-P2A-CAR control vector.

Viral vector production

Retroviral supernatant was prepared in the 293GP packaging cell line as previously described. Briefly, 20 cm 293GP plates at 70% confluency were cotransfected with 20 μg of MSGV vector plasmid and 10 μg of RD114 envelope plasmid DNA using Lipofectamine 2000. The medium was replaced at 24 and 48 h posttransfection. Viral supernatants from 48 h and 72 h were collected, centrifuged to remove cellular debris, and frozen at −80°C for later use. A third generation self-inactivating lentiviral supernatant was prepared in the 293T packaging cell line as previously described. Briefly, 20 cm 293T plates were cotransfected at 70% confluency with vector plasmid 18 μg pELNS vector plasmid DNA and 18 μg pRSV-Rev, 18 μg pMDLg/pRRE (Gag/Pol) and 7 μg pMD2.G DNA (VSVG shell) packaging plasmid with using Lipofectamine 2000. The medium was replaced 24 hours after transfection. At 24 h and 48 h, viral supernatants were collected, pooled, and concentrated by ultracentrifugation at 28,000 rpm for 2.5 h. Concentrated lentiviral starting materials were frozen at -80°C for future use.

T cell isolation

Primary human T cells were isolated from healthy donors using the RosetteSep Human T Cell Enrichment Kit (Stem Cell Technologies). Buffy coats were purchased from the Stanford Blood Center and processed according to the manufacturer's protocol using Lymphoprep density gradient media and SepMate-50 tubes. Isolated T cells were cryopreserved at 2×10 ⁷ T cells per vial in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).

CAR T cell production

Cryopreserved T cells were thawed and activated the same day using paramagnetic Human T-Expander CD3/CD28 Dynabeads (Gibco) at a 3:1 bead-to-cell ratio in T cell medium (AIMV supplemented with 5% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco)). Recombinant human IL-2 (Peprotech) was administered at a concentration of 100 U/ml. T cells were transduced with the retroviral vector on days 2 and 3 after activation and maintained at 0.5-1×10 ⁶ cells per ml in T cell medium with IL-2. Unless otherwise stated, CAR T cells were used for in vitro assays or transferred into mice 10–11 days after activation.

Retroviral transduction

12-well plates not treated with tissue culture were coated overnight at 4°C with 1 ml of retronectin (Takara) at a concentration of 25 μg/ml in PBS. The plates were washed with PBS and blocked with 2% BSA for 15 minutes. Thawed retroviral supernatant was added at ∼1 ml per well and centrifuged for 2 hours at 32°C and 3200 rpm before adding cells.

Cell lines

Neuroblastoma Kelly, Ewing's sarcoma EW8, osteosarcoma 143b, and TC32 cell lines were originally obtained from ATCC. Some 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 generated by co-transducing Nalm6-GL with cDNAs for GD2 synthase and GD3 synthase. A single cell clone was then selected to express GD2 at high levels. Nalm6-22KO and 221ow were previously described and kindly provided by Terry Fry. All cell lines were cultured in complete medium (RPMI supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco)).

Flow cytometry

CAR CD22 and Her2 were detected using recombinant human CD22-Fc and Her2-Fc proteins (R&D). Idiotypic antibodies and Fc fusion proteins were conjugated to Dylight488 and/or 650 antibody labeling kits (Thermo Fisher). T cell surface phenotype was assessed using the following antibodies:

From BioLegend: CD4-APC-Cy7 (clone OKT4), CD8-PerCp-Cy5.5 (clone SKI), 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 (SK3 clone), CD8-BUV805 (SKI clone).

Cytokine production

1x10 ⁵ CAR+ T cells and 1x10 ⁵ tumor cells were cultured in 200 μl of SM in 96-well flat-bottom plates for 24 hours. For idiotype stimulation, serial dilutions of 1A7 were cross-linked in 1× coating buffer (BioLegend) overnight at 4°C in Nunc Maxisorp 96-well ELISA plates (Thermo Scientific). Wells were washed once with PBS and 1 x 10 ⁵ CAR+ T cells were seeded in 200 μl of SM and cultured for 24 hours. Wells were seeded in triplicate for each condition. Culture supernatants were collected and analyzed for IFNγ and IL-2 by ELISA (BioLegend).

Lysis assay

5 x 10 ⁴ GFP + leukemia cells or 2.5 x 10 ⁴ GFP + adherent tumor cells were co-cultured with CAR T cells in 200 μl of CM in 96-well flat-bottom plates for up to 96 hours. Wells were seeded in triplicate for each condition. Plates were imaged every 2–3 hours using the IncuCyte ZOOM Live Cell Analysis System (Essen Bioscience). 4 images per well at 10× magnification were collected at each time point. The total integrated GFP intensity per well was assessed as a quantitative measure of live GFP + tumor cells. Values were normalized to the original measurement and plotted against time. The E:T ratios are indicated on the figure legends.

Western blotting and immunoprecipitation

Whole cell protein lysates were prepared in nondenaturing buffer (150 mmol/L NaCl, 50 mmol/L Tris-pH8, 1% NP-10, 0.25% sodium deoxycholate). Protein concentrations were assessed using a Bio-Rad colorimetric assay. Immunoblotting was performed by loading 20 μg of protein onto 11% PAGE gels followed by transfer to PVF membranes. Signals were detected using enhanced chemiluminescence (Pierce) or an Odyssey imaging system. Representative blots are shown. The following primary antibodies used were purchased from Cell Signaling: c-Jun (60A8), P-c-JunSer73 (D47G9), JunB (C37F9), BATF (D7C5), and IRF4 (4964). BATF3 antibody (AF7437) was purchased from R&D. Immunoprecipitations were performed in 100 mg of whole cell lysates in 150 μl of nondenaturing buffer and 7.5 mg of c-Jun (G4) or JunB (C11) agar-conjugated antibodies (Sant Craz Biotechnology). After overnight incubation at 4°C, microparticles were washed 3 times with nondenaturing buffer, and proteins were eluted in Laemmli sample buffer, boiled, and loaded onto PAGE gels. Detection of immunoprecipitated proteins was carried out using the above-mentioned reagents and antibodies.

Mice

Immunocompromised NOD/SCID/IL2Rg−/− (NSG) mice were purchased from JAX and reared in house. All mice were bred, housed, and treated according to protocols approved by the Stanford University IACUC (APLAC). Mice aged 6-8 weeks were inoculated with either 1×10 ⁶ Nalm6-GL leukemia cells via intravenous (IV) injection or 0.5-1×10 ⁶ osteosarcoma 143B cells via intramuscular (IM) injection. All CAR T cells were administered via intravenous injection. The time and dose of treatment are indicated on the figure legends. Leukemia progression was measured by bioluminescence imaging using the IVIS imaging system. Values were analyzed using Living Image software. Progression of the solid tumor was monitored using a caliper to measure the area of the injected hind paw. 5 mice per group were treated in each experiment, and each experiment was repeated 2 or 3 times as indicated. Mice were randomized to equal pretreatment tumor burden before treatment with CAR T cells.

Blood and tissue analysis

Peripheral blood sampling was performed by retroorbital blood sampling under isoflurane anesthesia at the indicated time points. 50 μl of blood was labeled with CD45, CD3, CD4, and CD8, lysed using BD FACS lysis solution, and quantified using CountBright Absolute Counting beads (Thermo Fisher) on a BD Fortessa flow cytometer.

ATAC-seq analysis

ATAC-seq library preparation was performed as previously described ⁴⁸ . Briefly, 100,000 cells from each sample were sorted by FACS into CM, centrifuged at 500 g at 4°C, then resuspended in ATAC-seq resuspension buffer (RSB) (10 mM Tris-HCl, 10 mM NaCl, 3 mM _MgCl2 ) with the addition of 0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin. Samples were divided into two replicates each before all subsequent steps. Samples were incubated on ice for 3 minutes, then washed with 1 ml RSB supplemented with 0.1% Tween -20. Nuclei were pelleted at 500g for 10 minutes at 4°C. The nuclear pellet was resuspended in 50 μl of transposition mixture (25 μl 2× TD buffer, 2.5 μl transposase (Illumina), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, 5 µl H ₂ O) and incubated at 37°C for 30 minutes in a thermomixer with shaking 1000 rpm. The reaction mixture was purified using the Qiagen MinElute PCR purification kit. Libraries were amplified by PCR using NEBNext High Fidelity PCR Master Mix and custom primers (IDT) as previously described ²⁰ . The libraries were sufficiently amplified after 5 cycles of PCR, as shown by qPCR fluorescence curves ²⁰ . Libraries were purified using the Qiagen MinElute PCR purification kit and quantified using the KAPA Library Quantification Kit. Libraries were sequenced on Illumina NextSeq at the Stanford Functional Genomics Center with paired-end 75-bp reads. Adapter sequences were trimmed using SeqPurge and aligned to the hg19 genome using bowtie2. These reads were then filtered for mitochondrial reads, low mapping quality (Q>=20), and PCR duplicates using Picard tools. We then converted the BAM values to BED and obtained Tn5-corrected insertion sites (“+” stranded + 4 bp, “-” stranded - 5 bp). To identify the peaks, we named the peaks for each sample using MACS2 "--shift -75 --extsize 150 --nomodel --call-summits --nolambda -- keep-dup all - p 0.00001" using insert BED values. To obtain the pooling peak set, we (1) expanded all vertices to 500 bp, (2) merged all BED vertex files, and then (3) used the bedtools cluster and selected the vertex with the highest MACS2 score. It was then filtered using the ENCODE hg19 blacklist ( https://www.encodeproject.org/annotations/ENCSR636HFF/ ) to remove peaks extending beyond the chromosome ends. We then annotated these peaks using HOMER and calculated TF motif occurrence using motifmatchr in R with the HOMER chromVARMotifs set. To generate sequencing tracks, we read Tn5-corrected insertion sites in R and generated overlap clusters clustered every 100 bp using rtracklayer. We then counted all insertions that fell within each peak to obtain a matrix of counts (peak x samples). To determine differential peaks, we first used peaks that were designated as “TSS” as control genes or “service peaks” for DESeq2, and then calculated differential peaks using this normalization. All clustering was performed using regularized log-transformed values from DESeq2. Transcription factor motif divergence analysis was performed using chromVAR as previously described ²¹ . TF motif enrichment was calculated using a hypergeometric test in R, testing motif representation (from motifmatchr above) in a subset of peaks versus all peaks.

RNA sequencing subsets

For T cell subset-specific RNA sequencing, T cells were isolated from buffy coats of healthy donors as described above. Before activation, subsets of naive and central memory CD4+ or CD8+ cells were isolated using a BD FACSAria cell sorter (Stem Cell FACS Core, Stanford University School of Medicine) using the following markers: naive (CD45RA+CD45RO-, CD62L+, CCR7+, CD95- and CD122-), central memory cells (CD45RA-CD45RO+, CD62L+, CCR7+). The sorted starting populations were activated, transduced, and cultured as described above. At days 7, 10, and 14 of cell culture, CAR+CD4+ and CD8+ cells were sorted and RNA isolated using the Qiagen mRNEasy kit. Samples were pre-library prepared and sequenced using the Illumina NextSeq paired-end read sequencing platform using the Stanford Functional Genomics Core.

Bulk RNA sequencing

For total RNA isolation, healthy donor T cells were obtained as described. On day 10 or 11 of culture, total mRNA was isolated from 2 x 10 ⁶ total CAR T cells using the Qiagen RNEasy Plus Mini Isolation Kit. Bulk RNA sequencing was performed by BGI America (Cambridge, MA) using the BGISEQ-500 platform, single-end read length 50 bp, 30 × 10 ⁶ reads per sample. Principal component analysis was performed using the stats package and plots with the ggplot2 package in R (version 3.5) 49. Gene set enrichment analysis was performed using GSEA software (Broad Institute) as described 50, 51.

Single cell RNA sequencing

To compare gene expression in individual CD19-CAR and GD2-CAR T cells, we sorted an initial subset of T cells on day 0 for subsequent single cell analysis on day 10 using the Chromium platform (10× Genomics) and Chromium kit reagents Single Cell 3' v2 according to manufacturer's instructions. cDNA libraries were prepared separately for CD19-CAR and GD2-CAR cells, and CD4+ cells and CD8+ cells were pooled in each experiment for further bioinformative separation. Sequencing was performed on an Illumina NextSeq system (paired-end reads, 26 bp in read 1 and 98 bp in read 2) to a depth of >100,000 reads per cell. Single-cell RNA-seq reads were aligned to the Human Genome Reference Consortium version 38 (GRCh38), normalized for group effects, and filtered for cellular events using Cell Ranger software (10X Genomics). A total of 804 CD19-CAR and 726 GD2-CAR T cells were sequenced to an average of 350,587 reads after normalization per cell. The cellular gene array was further processed using Cell Ranger R Kit software (10X Genomics) as described ⁵² . Briefly, the present inventors first selected genes with a number of unique molecular identifiers (UMI)≥1 in any given cell. UMI scores were then normalized to the UMI sums for each cell and multiplied by the median UMI number across cells. The data was then transformed by taking the natural logarithm of the resulting data matrix.

Statistical analysis

Unless otherwise stated, statistical analysis of significant differences between groups was performed using unpaired two-tailed t tests without regard to SD agreement using GraphPad Prism7. For bulk RNA-seq in Figure 2C, the nonparametric Wilcoxon matched pairs signed rank test was used. Survival curves were compared using the Cox-Mantel log-rank test. A table with complete statistical analyzes including exact p values, t ratios, and dof can be found in the supplemental materials.

Example 1

T cell exhaustion gene expression analysis

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can enhance T cell differentiation and exhaustion, limiting their activity. For example, GD2-specific CARs have been described to self-aggregate in the absence of antigen, leading to activation of a chronic downstream T cell activation signaling cascade. While GD2-CARs incorporating the CD28 costimulatory domain rapidly develop characteristic features of T-cell exhaustion, GD2-CARs incorporating the 4-1BB costimulatory domain show fewer features of T-cell exhaustion and retain greater functionality. despite similar aggregation and signaling. The most characteristic features of T cell exhaustion in GD2-28z expressing T cells include increased surface expression of inhibitory receptors (eg, PD1, TIM3, LAG3, CD39), decreased expression of memory markers (eg, CD62L and CCR7), and reduced cytokine production (especially IL2) when stimulated by antigen.

Experiments were conducted during the development of embodiments of the present invention to evaluate gene transcription analysis in exhausted and non-exhausted T cells. Reduced expression of AP-1 family members was found in GD2-28Z CAR T cells compared to GD2-BBZ (non-depleted CAR) (see Fig. 1a). The classical AP-1 partners FOS and JUN were also among the most down-regulated genes in GD2-28Z CAR T cells compared to healthy CD19-28Z CAR T cells by RNA sequencing analysis (see Fig. 1b). The AP-1 family of transcription factors is activated downstream of TCR signaling and regulates a broad and diverse set of key T cell functions, including growth, apoptosis, cytokine production, and effector functions. Additional 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 cells) cellular phenotype).

Example 2

Construction of CAR T cells with enhanced expression of c-Jun and c-Fos

To determine whether AP-1 replacement could alleviate exhaustion symptoms in GD2-28Z CAR T cells, a lentiviral expression construct was constructed with enhanced expression of c-Jun and c-Fos under the control of a constitutive promoter (see Fig. 2a). This construct also encoded a truncated nerve growth factor receptor (NGFR (tNGFR)) expression cassette as a surface marker for T cell transduction. Subsequently, activated primary human T cells were transduced with CD 19, GD2-BBZ, or high-affinity (HA) GD2-28Z CAR with (AP-1) or without (wo) AP-1 expression vector.

Example 3

Expression of c-Jun and c-Fos in CAR T cells

At day 8 of T cell culture, AP-1 transduced CD4 or CD8 CAR T cells were sorted using NGFR. Constitutive expression of AP-1 decreased the frequency of exhaustion-associated inhibitory receptors PD1, TIM3, LAG3 and CD39 and increased the memory marker CD62L in both CD4 (see Fig. 2b) and CD8 (see Fig. 2c) CAR T cells . CAR T cells were cocultured with (AP-1) or without (wo) AP-1 along with CD19 and GD2 antigen-expressing tumor cells to evaluate functional changes in AP-1-transduced CAR T cells. In most cases, AP-1 transduced CAR T cells released more IL2 (see Fig. 2d) and IFNγ (see Fig. 2e) compared to cells without it.

Example 4

CAR T cells with enhanced expression of c-Jun or c-Fos

To assess whether the functional benefit of AP-1 expression requires both expression of c-Fos and c-Jun, separate lentiviral vectors encoding only c-Jun or c-Fos were generated (see Fig. 3a). CAR T cells were transduced with c-Fos or c-Jun encoding vectors.

After stimulation of CAR T cells with antigen-positive tumor cells, only CAR T cells expressing c-Jun resulted in an increase in IL2, whereas expression of c-Fos alone was insufficient (see Fig. 3b). Overexpression of c-Jun affected IFNγ secretion in a similar but less dramatic manner (see Fig. 3c). To confirm increased cytokine expression in c-Jun CAR-expressing T cells at the single-cell level, intracellular cytokine staining (ICS) was performed by flow cytometry after 6-hour stimulation of T cells with antigen-positive tumor cells. In most cases, an increase in the frequency and/or mean fluorescence intensity (MFI) of IL2 (see Fig. 3d), IFNγ (see Fig. 3e) and TNFα (see Fig. 3f) in c-Jun CAR transduced T cells was detected compared to cells without (wo), while c-Fos transduction generally resulted in decreased cytokine production compared to controls. These data indicate that overexpression of c-Jun can increase both the frequency of CAR T cells responding to antigen stimulation and the content of cytokines produced by an individual CAR T cell upon encountering an antigen.

Example 5

Construction of bicistronic vectors coexpressing c-Jun and CAR from the same vector

Because c-Jun has been shown to be primarily responsible for the increased activity of AP-1 transduced CAR T cells, bicistronic retroviral vectors have been created that coexpress c-Jun and CAR from the same vector separated by a ribosomal slip sequence to excise viral peptides 2A (see Fig. 4a). These constructs ensured that any CAR+T cell would also coexpress c-Jun and eliminated the need for co-transduction and sorting to achieve pure double-positive populations. Eight different CAR vectors were separately cloned into the c-Jun backbone: CD19-28Z, CD19-BBZ, CD22-28Z, CD22-BBZ, GD2-28Zshort, GD2-28ZLong, HA(GD2)-28ZLong and GD2-BBZ, in order to to test the functional impact of c-Jun reexpression on multiple antigen specificities and indications for different tumors.

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

c-Jun mediates increased cytokine secretion in response to both leukemia (Nalm6-GD2) and GD2+ solid tumors in children, Ewing sarcoma (EW8), osteosarcoma (143B) and neuroblastoma (Kelly), highlighting the broad clinical utility of enhanced c -Jun GD2 CAR T cells. In addition, overexpression of c-Jun promotes an increase in the frequency of central memory CAR T cells (see Fig. 5b). Using intracellular cytokine staining (ICS), it was found that increased production of pro-inflammatory cytokines was also observed at the single cell level (Figures 4c and 6c-e) in both CD4+ and CD8+ c-Jun-CAR T cells. We also noted a decrease in the production of the anti-inflammatory cytokine IL10 in c-Jun CAR T cells compared to controls (see Fig. 6f), which also indicates that increased expression of c-Jun may contribute to the enhanced Thl cytokine profile .

Example 6

c-Jun replacement and functional activity in various CAR T cells

As described in detail herein, replacement of c-Jun in exhausted CAR T cells (eg, GD2 CAR T cells) can alleviate exhaustion in CAR T cells. However, trends toward decreased markers of exhaustion and increased functional activity were also observed in healthy CD19 CAR T cells. While CD19 CAR T cells have mediated significant clinical responses in patients with B-ALL, an increasing number of relapses occur in up to 30% of patients with CD19-low expression or CD19-negative disease. CD22 CAR, an alternative strategy for targeting B-cell malignancies, may also be limited by the low density of CD22 antigen in some patient leukemia cells. Thus, during the development of embodiments of the present invention, experiments were conducted to evaluate and characterize the activity of c-Jun-CD19 and c-Jun-CD22 CAR T cells against tumor cells with normal (Nalm6) or low antigen expression (Nalm6-F and Z for CD19 or Nalm6-221ow) (see Fig. 7).

Although c-Jun did not enhance CD19 CAR activity against high levels of antigen on Nalm6, significant improvements in IL2 (see Fig. 7a) and IFNγ (see Fig. 7b) were observed in response to Nalm6 clones Z and F with low CD19 expression. In the INCUCYTE immune cell killing assay (see Figure 7c), 3 GFP+Nalm6 cell lines were co-cultured with CAR T cells for 92 hours. All 4 CD19 CARs eliminated the parent Nalm6 tumor (as measured by loss of GFP intensity over time). Both CD19-28Z and c-Jun-CD19-28Z were able to kill tumor cells with low levels of CD19, clones F and Z, but CD19-BBZ CAR T cells had only a modest effect. Addition of c-Jun-CD19-BBZ CAR T cells showed a significant increase in killing of low CD19 clones compared to 19-BBZ alone. Increased secretion of CD22 CAR cytokines by c-Jun coexpressing CD22 CAR T cells (especially CD22-BBZ) was also observed in response to both naive Nalm6 and Nalm6-22low (see Fig. 7d-e).

Inhibition of AP-1 inhibitory complex members reduces T cell exhaustion

Although increased expression of c-Fos and c-Jun can enhance the functionality of engineered T cells, there are other inhibitory members of the AP-1 family expressed in exhausted activated T cells. Accordingly, experiments were conducted during the development of embodiments of the present invention to determine and characterize whether inhibition/knockdown of members of the AP-1 inhibitory complex (eg, to increase the availability of canonical AP-1 factors) could reduce T cell exhaustion (eg, increase function T cells). CRISPR-Cas9 gRNA systems have been developed to target potential AP-1 inhibitory members. Cytokine production was assessed from JUNB and BATF3 gene-edited CAR T cells (knockout, KO). Knockdown of JUNB significantly increased IL2 and IFNγ production from HA-GD2-28Z and GD2-BBZ CAR T-cell-depleted cells, while it had no effect on CD19 CAR T cells (see Fig. 8A-C). This indicated that JUNB knockdown had a positive effect on exhausted but not healthy T cells. Likewise, BATF3 knockout also increases IL2 (but not IFNγ) production from HA-28Z CAR-depleted T cells (see FIG. 80) compared to control edited T cells.

Example 8

In vivo efficacy of c-Jun-modified CAR T cells

The in vivo efficacy of c-Jun-modified CAR T cells has been assessed in several different tumor models. c-Jun HA-GD2-expressing depleted CAR T cells exhibited superior therapeutic activity in vivo compared to HA-GD2-modified CAR T cells in the Nalm6 leukemia model engineered to express GD2 (N6-GD2) (see FIG. 9) . c-Jun-modified GD2-BBZ CAR T cells exhibit excellent in vivo activity in a solid tumor model of aggressive osteosarcoma (see Fig. 10). Finally, c-Jun-expressing CD19 CAR T cells exhibit enhanced in vivo activity against the low antigen density Nalm6 clone F (see Fig. 11).

Expression of HA-28z CAR in Human T Cells Rapidly Induces Characteristic Signs of T Cell Exhaustion

The GD2-28z CAR is an exhausted phenotype in human T cells following expression of a CAR comprising the GD2-specific 14g2a scFv, TCR zeta, and CD28 endodomain, resulting in tonic signaling mediated through antigen-independent aggregation (ref. 9; presently document incorporated by reference in its entirety). Experiments conducted during the development of embodiments herein demonstrate that expression of a CAR containing a 14g2a scFv carrying the E101K point mutation that confers a higher affinity (HA) interaction with GD219 (HA-28z CAR) similarly induces depletion in human T cells, although with a more severe phenotype (Figs. 12 and 18a-c). In contrast to CD19-28z CAR T cells, HA-28z CAR T cells had significant phenotypic and functional signs of exhaustion, including decreased expansion in culture (Fig. 12a), increased surface expression of the inhibitory receptors PD-1, TIM- 3, LAG-3 and CD39 (Figs. 12b and 18d), overdifferentiation of effectors and poor memory formation (Figs. 12c and 18e), as well as reduced IFN-g and a significant reduction in IL-2 production when stimulated with CD19 leukemia cells +GD2+Nalm6 (Fig. 12d).

To better elucidate the molecular basis of T cell exhaustion in this system, the transcriptome of HA-28z was compared with CD19-28z-CAR T cells. Purified naïve (N) and central memory (CM) T cells were transduced with HA or CD19-28z CAR and then RNA was isolated at days 7, 10, and 14 of culture. Sorting of preselected subgroups allowed assessment of the influence of T cell differentiation state and differences between CD4 and CD8 depletion in the development of T cell exhaustion in this model. Principal component analysis (PCA) across all 24 samples showed that the strongest contributor to variance was the presence of NA compared to CD19-28z CAR (PC1, 39.3% of variance, Fig. 12e), consistent with a model in which tonic transmission signaling in HA-28z CAR T cells causes exhaustion in all T cell subsets studied. Differences, however, were observed based on the initial differentiation state, as N versus SM was reflected in PC2 (22.88% variance) (Fig. 18f) and between the CD4 and CD8 populations that resulted in PC3 (11.9% variance ) (Figs. 12e and 18f).

Among the top 200 PC1 driving genes (most differentially expressed in HA versus CD19-28z CAR T cells across all subsets) (Fig. 12f) were genes associated with activation (IFNG, GZMB, IL2RA), inhibitory receptors (LAG3, CTLA4) and some inflammatory chemokines/cytokines (CXCL8, IL13, ILIA), while genes negatively regulated in HA-28z CAR T cells include genes associated with naïve T cells and T -memory cells (IL7R, TCF7, LEF1 and KLF2). Using GSEA, demonstrated that genes upregulated in 10-day HA-28z CAR T cells compared to CD19-28z CAR T cells overlap with exhaustion-associated gene sets previously described in mouse models of chronic LCMV ¹³ (Fig. 18g). Although the degree of exhaustion in GD2-28z CAR T cells is less pronounced, analysis of differential gene expression at the single cell level of GD2-28z versus CD19-28z CAR T cells revealed a similar gene expression profile (Figure 19). Together, these data support the rationale for recommending HA-28z- and GD2-28z-expressing T cells as models for studying human T-cell exhaustion.

T cell depletion is associated with changes in chromatin accessibility in a mouse model and in human patients with chronic viral infections and cancer (refs. 12, 17; incorporated herein by reference in its entirety). Analysis of chromatin accessibility using ATAC-seq20 (Figure 20) of CD4+ and CD8+HA-28z derived from N or CM CD4+ and CD8+HA-28z compared to CD19-28z CAR T cells demonstrated significant changes in the epigenetic signature on Day 10 of culture (Figure 12g), CD8+HA-28z CAR T cells exhibited >20,000 unique accessible chromatin regions (peaks), compared to <3,000 unique peaks in CD8+CD19-28z CAR T cells (FDR<0.1 and log2FC>1). These patterns of depletion-induced changes in chromatin accessibility were similar in CD4+ T cells (Fig. 21a). Similar to transcriptomic analysis, PCA identified HA- versus CD19-CAR as the strongest driver of differential chromatin states (PC1 variance 79.6%, Fig. 12h), with weaker but significant differences observed between N and SM cells ( PC2 variance 7.4%), and between CD4 and CD8 subgroups (PC3 variance 6.5%) (Fig. 21b). Clustering of the top 5000 differentially accessible regions (peaks) revealed globally similar chromatin accessibility in HA-28z CAR T cells regardless of the subset of origin (Figure 12i). HA-28z CAR T cells showed increased chromatin accessibility at regulatory sites near exhaustion-related genes such as CTLA4 and decreased accessibility at regulatory sites near memory-related genes such as IL7R (Figure 12j).

Epigenetic and transcriptional analyzes show a strong AP-1 signature in CAR-depleted T cells

To identify transcriptional programs predicted to be unregulated by epigenetic changes induced in exhausted T cells, transcription factor (TF) motif bias was compared between the open chromatin of exhausted and healthy CAR T cells. Using ChromVAR analysis (ref. 21; incorporated herein by reference in its entirety), the 25 most distinctive motifs across all 8 samples were identified, and many of them were found to belong to the AP-1 (bZIP) family (Fig. 13a). Likewise, TF motif enrichment analysis identified AP-1/bZIP and bZIP/IRF binding motifs as among the most enriched in CAR-depleted T cells (Fig. 13b and Fig. 21c).

Clustering of differentially accessible peaks by co-enrichment of the TF motif identified 4 clusters associated with HA-28z-depleted CAR T cells (Fig. 21d, EX1-EX4). Depletion-related clusters contained peaks in close proximity to genes such as BTLA, CD39, IFNG, and CTLA4, indicating common TF regulation of depletion-related genes. All 4 wasting-associated clusters showed strong enrichment for AP-1 family and AP-1-related family TFs, indicating widespread AP-1 TF modulation of exhaustion-related gene regulation. Strong enrichment for NFκB, NFAT, and RUNX family TF motifs was also observed in some of the attrition clusters, indicating that a subset of attrition-related genes may be regulated by these transcriptional programs and recapitulate the epigenetic signatures of attrition observed in other models (refs. 12, 17, 22; incorporated herein by reference in their entirety). Clusters associated with healthy CD19-28z CAR T cells (HLT1-2) showed a profile similar to the cluster significantly associated with the original naïve subgroup. This observation is consistent with the idea that healthy CAR T cells maintain an epigenetic signature more similar to naïve-derived T cells, a subset associated with increased persistence and efficacy of adoptive T-cell therapy (ref. 23; incorporated herein in its entirety by reference), whereas chronic antigenic stimulation results in wide divergence in epigenetic reprogramming.

AP-1-associated TFs are coordinated to form a diverse set of homo- and heterodimers through interactions in a common bZIP domain and can dimerize with IRF transcription factors (refs. 14, 24; incorporated herein by reference in their entirety). AP-1 factor complexes compete for binding to DNA elements containing the TGA-G/C-TCA nuclear consensus motifs. Activating complexes, such as those containing the classically described heterodimer of c-Fos and c-Jun AP-1, drive IL-2 transcription. Conversely, other AP-1 and IRF family members can directly antagonize c-Jun activity and/or drive the expression of immunoregulatory genes in T cells (refs. 14, 24-29; incorporated herein by reference in their entirety). To assess whether changes in the accessibility of AP-1 binding chromatin were associated with increased availability of the activating and inhibitory TFs bZIP and IRF, during the development of embodiments of the present invention, experiments were conducted to compare transcript levels of members of the bZIP and IRF families using RNA sequencing in depleted HA-28z versus healthy CD19-28z CAR T cells. Pairwise RNA-seq analysis from 3 different donors revealed a consistent pattern of overexpression of members of the bZIP and IRF families, which was most significant for JUNB, FOSL1, BATF, BATF3, ATF3, ATF4 and IRF4 (Fig. 13c and Fig. 22a). Western blot analysis confirmed consistent protein overexpression of JunB, IRF4 and BATF3 in HA cells compared to CD 19 CAR T cells (Fig. 13d and Fig. 22b), with the immunoregulatory TFs BATF/IRF showing higher expression levels over compared to c-Jun. The biological significance of elevated levels of inhibitory bZIP/IRF family members was further demonstrated by the demonstration that Western blot analysis of Jun immunoprecipitates (IP) revealed that several inhibitory family members were in direct complex with c-Jun and JunB in HA-28z-depleted CAR T cells (Fig. 22c). Comparison of TF profiles by single-cell RNA-seq analysis of CD8+ T cells expressing CD19-28z versus GD2-28z CAR confirmed that bZIP family members: JUN, JUNB, JUND, and ATF4 were among the most differentiated expressed and widely associated in networks of depleted GD2-28z CAR T cells (Fig. 13e and Fig. 19).

Overexpression (OE) of c-Jun reduces functional exhaustion in CAR T cells

Based on the finding that exhausted CAR T cells exhibit very low production of IL-2 (Fig. 12d) and predominantly overexpression of the transcription factors bZIP and IRF, which drive immunoregulatory and exhaustion-associated programs, it can be hypothesized that T-cell dysfunction cells in starved cells may be due to a relative deficiency of c-Jun/c-Fos heterodimers required to drive IL-2 transcription. HA-28z and CD19-28z CAR T cells were co-transduced with a bicistronic lentiviral vector to overexpress c-Jun and c-Fos. HA-28z CAR T cells engineered to overexpress AP-1 showed increased IL-2 production upon antigen stimulation (FIG. 23a-c). However, when using separate expression vectors, enhanced functionality was observed only with c-Jun OE in HA-28z CAR T cells, while transduction with a separate c-Fos expression vector did not consistently improve function (Fig. 23d-e).

To further examine the ability of c-Jun to enhance the function of exhausted CAR T cells and to promote constitutive expression of c-Jun in all CAR-expressing T cells, bicistronic vectors were generated that coexpress c-Jun and CAR transgenes separated by the viral skipping peptide P2A (JUN -CAR, Fig. 14a). These expression vectors increased c-Jun expression in both CD19 and HA-28z CAR T cells (Figure 14b), although c-Jun was predominantly activated (phosphorylated) in JUN-HA CAR T cells (HA-28z ), which is consistent with N-terminal phosphorylation of c-Jun (JNP) by JNK proteins activated downstream of the tonic TCR signaling cascade propagated through HA-28z CAR30. After stimulation with GD2+ tumor cell lines JUN-HA-28z CAR T cells showed a marked increase in IL-2 and IFNg production compared to control HA-28z CAR T cells (Fig. 14d-e). The fold increase in cytokine production under c-Jun OE conditions was substantially greater for JUN-HA CAR compared to JUN-CD19 CAR T cells (Fig. 24a-b). Similarly, JUN-HA CAR T cells showed an increased frequency of SCM/CM subsets as opposed to E/EM compared to HA-28z CAR T cells (Fig. 14f), while between CD19 and JUN-CD19 CAR T -cells there were no significant differences in the composition of subgroups on the 10th day of cultivation. Taken together, the data are consistent with a model in which the c-Jun OE is functionally more significant in exhausted T cells that overexpress the inhibitory TFs bZIP and IRF.

To evaluate whether c-Jun OE increases long-term proliferative capacity; which is associated with antitumor effects in solid tumors (ref. 31; incorporated herein by reference in its entirety), and test whether c-Jun OE can enhance function in CAR T cells without tonic signaling (CD19-28z, CD19-BBz) or in cells with lower levels of tonic signaling (GD2-BBz), in vitro expansion of JUN-CAR T cells was measured in 3 different healthy donors over a long period (Fig. 24c). In the presence of OE c-Jun, a pattern of increased long-term proliferative potential was observed that remained IL-2 dependent, as these cells immediately ceased expansion in the absence of IL-2 (Fig. 14g). Consistent with the ability of c-Jun to confer resistance to exhaustion, late-expanding CD8+ JUN- CD19-28z CAR T cells exhibited reduced expression of exhaustion markers and maintained a robust subset of cells exhibiting a stem cell memory (SCM) phenotype (CD45RA+CD62L+) by compared to control CD19-28z CAR T cells tested at the same time point (Fig. 14h-j). Homeostatic expansion of JUN-CAR T cells adopted into tumor-free NSG mice was assessed. The number of peripheral blood T cells was increased in mice treated with both JUN-CD19-28z and JUN-CD19-BBz CAR T cells compared to the control group at 25 days post-infusion (Figure 14k). which resulted in accelerated GVHD in mice treated with JUN-CD19-BBz CAR T cells. Taken together, the data show that c-Jun OE reduces T cell exhaustion in multiple CARs tested, including those that include CD28 or 4-1BB co-stimulatory domains, and regardless of whether the exhaustion is caused by forced long-term expansion or tonic transmission of signals.

Example 12

Molecular Requirements for c-Jun-Mediated Restoration of CAR T Cell Depletion

Experiments were conducted during the development of embodiments herein to determine whether c-Jun OE could restore exhausted T cells by two different mechanisms that are not mutually exclusive. OE c-Jun may directly enhance AP-1-mediated gene transcription by increasing the availability of Fos/Jun or Jun/Jun dimers or may work indirectly by disrupting AP-1/IRF inhibitory transcription complexes (AP-1-i) ^14,24 , which control the expression of genes associated with exhaustion (Fig. 15a). To better understand the mechanisms by which c-Jun OE mitigates T cell exhaustion, a destabilization domain (DD) derived from E. coli dihydrofolate reductase (DHFR) was fused to the N terminus of c-Jun to transiently regulate c-Jun expression. Jun (JUN-DD, fig. 15b). DD is stabilized in the presence of cell-permeable low-molecular-weight trimethoprim (TMP) and leads to stable expression of c-Jun, but in the absence of TMP, DD is destabilized, causing proteasomal degradation of the entire fusion protein (Fig. 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), whereas JUN-DD quickly became undetectable in the absence of TMP (t1/2 of 1. 84 hours) (Fig. 15d and Fig. 25a-b). JUN-DD-CAR T cells mediated increased production of IL-2 and IFNg only in the presence of TMP, confirming the critical role of c-Jun contents in modulating CAR T cell functionality (Fig. 15e). It was hypothesized that a direct effect of c-Jun on transcription could be accomplished rapidly, and therefore it was tested whether functional restoration would occur 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 were required to compete with AP-1 inhibitory complexes (AP-1i) during exhaustion induction, longer exposure during T cell expansion (ON-OFF) may be required (Fig. 15f). Compared to HA-28z CAR T cells that never experienced c-Jun OE (OFF-OFF), both conditions mediated partial restoration, however full restoration of IL-2 function required c-Jun OE as during T cell expansion , and upon stimulation with antigen (ON-ON) (Fig. 15g). This finding is consistent with a model in which c-Jun OE directly enhances gene transcription during acute stimulation following antigen encounter and also indirectly modulates molecular reprogramming during the development of wasting. In addition, there was a decrease in the expression of both protein and mRNA of JUNB and BATF/BATF3 family members upon overexpression of c-Jun (Fig. 25c-d), as well as a decrease in JunB/BATF complexes by IP (Fig. 25e).

The indirect model of c-Jun-mediated destruction of AP-1 inhibitory complexes does not depend on direct transcriptional activation of c-Jun. To test whether direct c-Jun-mediated gene activation is required for functional recovery of exhausted T cells, we generated JNP-deficient c-Jun with Ser63 and Ser73 alanine substitutions in the c-Jun transactivation domain to prevent phosphorylation at these sites (c-JunAA ), which have been shown to be important for c-Jun-mediated gene transcription (refs. 33, 34; incorporated herein by reference in their entirety) (Fig. 15h-i). JUNAA-HA-28z CAR T cells showed equivalent increases in IL-2 and IFNg production compared to wild-type JUN-CAR T cells (Figure 25j). Together, these data are consistent with a model in which c-Jun-mediated restoration of depletion does not require direct gene activation.

Example 13

JUN-CAR T cells mediate enhanced antitumor activity in vivo

Experiments were conducted during the development of embodiments herein to determine whether JUN-CAR T cells would exhibit enhanced activity in vivo. Nalm6-GD2+ leukemia cells were inoculated into 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 ultimately failed as all mice succumbed to the disease (median survival of d59). In contrast, JUN-HA-28z CAR T cells mediated complete tumor regression by day 24 and provided long-term tumor-free survival (Fig. 16a-c). To determine whether c-Jun OE could enhance the functionality of CARs targeting solid tumors, the effect of JUN-CAR was assessed using Her2- and GD2-targeted CARs incorporating the 4-1 BB co-stimulatory domain, which has emerged as the preferred signaling domain for imparting long-term persistence (refs. 35-37; incorporated herein by reference in their entirety). In a long-term ex vivo killing assay of osteosarcoma 143b JUN-Her2-BBz CAR T cells demonstrated significantly greater killing activity at an effector:target ratio of 1:8 (E:T), consistent with increased activity per cell (Fig. .16d-e). Likewise, JUN-Her2-BBz CAR T cells prevented tumor growth in vivo and resulted in a significant improvement in long-term survival, which was 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 osteosarcoma 143b (Fig. 26), confirming the superiority of c-Jun OE in CAR T cells responding to solid tumors and in CAR T cells , including signaling domains 4-1 BB.

Example 14

Overexpression of c-Jun lowers the activation threshold of CAR T cells and allows recognition of tumor cells with lower antigen density

Elevated levels of inhibitory API family members in exhausted CAR T cells raised the possibility that dysfunction in exhausted CAR T cells is due, at least in part, to a higher activation threshold that can be normalized by restoring the balance of activation relative to inhibitory family members. API. To test this, cytokine production by HA-CAR versus JUN-HA-CAR T cells was compared in response to serial dilutions of plate-bound 1A7, an anti-idiotypic antibody that binds scFv 14g2a, allowing control of stimulus strength. OE c-Jun significantly increased the maximum production of IL-2 and IFNg, and also significantly reduced the amount of 1A7 required to induce IL-2 secretion, consistent with the reduced activation threshold in JUN-HA-CAR T cells (Fig. 17a-b ).

Limitation of target antigen expression levels on tumor cells is increasingly recognized as a factor limiting CAR functionality (refs. 5, 6, 38; incorporated herein by reference). CD22dim relapses have recently been reported in patients with leukemia after initial responses to CD22 CAR therapy. Because c-Jun OE lowers the activation threshold in tonically signaling HA-28z CAR T cells, it was assessed whether JUN-CAR would recognize and kill tumor cells with lower antigen density that may evade recognition by control CAR T cells. When JUN-CD22-BBz CAR (Fig. 17) T cells were sensitized with CD22low leukemia cells (Fig. 17d), JUN-CAR T cells showed increased cytokine production in vitro (Fig. 17e) and dramatically increased antitumor activity in vivo . (Fig. 17f-i). Control CD22-BBz CAR T cells showed initial activity when administered at a dose of 3x10 ⁶ CAR T cells, but this treatment ultimately failed to control tumor growth (median survival d45). In contrast, JUN-CD22-BBz CAR T cells mediated significant antitumor effects and were completely curative. In summary, c-Jun OE demonstrates significantly improved antitumor control in 4 tumor models and is associated with improved expansion, resistance to depletion, and improved ability to recognize low-antigen targets.

Example 15

Truncated JUN proteins

Experiments were conducted during the development of embodiments herein to identify the required JUN domains that are responsible for mediating the recovery of dysfunctional T cells. A series of JUN mutants with various domains deleted were created (Fig. 27a). JUN mutations with N-terminal deletions and truncations (JUN-AA, JUN-Dd, and JUN-DTAD) retain the ability to restore function of HA-28z-depleted CAR T cells, whereas C-terminal mutations in the epsilon and bZIP domains do not restore functionality. Constructs JUN-AA, JUN-Dd and JUN-DTAD retain equivalent increases in cytokine production compared to JUN-WT (Figure 27b,d) and improve long-term killing at low E:T ratios (Figure 27c) compared to JUN mutations -De, JUN-Dbasic, JUN-DLeu and JUN-DbZIP. Consistent with the improved in vitro functional activity of HA-28z CAR T cells expressing JUN-WT and JUN-DTAD exhibited increased proliferation in vivo compared with JUN-DLeu and JUN-DbZIP when administered to N6-GD2 leukemia mice.

Example 16

IRF4 knockdown dramatically increases the functional activity of HA-28z-depleted CAR T cells

Experiments were performed that optimized CRISPR gRNA for 9 different bZIP/IRF family components and tested the ability of knockdown to restore functional activity (cytokine secretion) of HA-28z-depleted CAR T cells. Summary data showing the results of 3 to 6 independent experiments with healthy donors are presented in FIG. 28. As shown in FIG. 28, while knockout of JunB and BATF3 improved functional activity in some donors, knockout of IRF4 significantly improved IL-2 secretion in HA-28z stimulated CAR T cells (left and middle) in all donors tested. IRF4-KO CAR T cells even showed improved basal IL-2 secretion during tonic signaling.

Example 17

Transcription mutant (JUN-AA) also restores functional activity and proliferative capacity in CD19 CAR T cells

Experiments were carried out involving the creation of JUN-AA mutations in CD19 CAR T cells. JUN-AA maintained increased reactivity against low-density antigen (Fig. 29A) and increased long-term proliferation in culture (Fig. 29B). In vivo, c-Jun demonstrated improved survival in Nalm6 leukemia mice treated with a low dose CD19 CAR T cell “stress test” dose (Figure 29C).

Example 18

Enhanced in vivo function of c-Jun-modified HA-28z CAR T cells cannot be reproduced by ex vivo provision of IL-2

Although IL-2 production is an excellent biomarker for exhaustion-resistant cells, enhanced JUN-CAR T cell function cannot be reproduced by IL-2 alone (Figure 30). On days 7, 9, 11, 13, and 15 after tumor engraftment, IP was given at 250,000 IU/mouse. 1x10 ⁶ CAR+ T cells were administered intravenously on day 7. A representative result from 3 independent experiments with similar results is shown in FIG. thirty

Example 19

c-Jun enhances Her2-BBz CAR T cell activity in a suppressive solid tumor microenvironment

JUN-CAR T cells exhibit reduced exhaustion and increased functional activity ex vivo. Osteosarcoma 143B xenografts were implanted via intramuscular injection into NSG mice. After solid tumor mass was measured (on day 14 after tumor inoculation), 1 x 10 ⁷ control Her2-BBz (blue) CAR T cells or JUN-Her2-BBz (red) CAR T cells were injected intravenously. In fig. 31A shows that JUN-Her2-BBz CAR T cells mediated regression of large, established solid tumors 143B, while Her2-BBz CAR T cells showed no change compared to control non-transduced mock T cells. Two weeks after T cell injection (until complete tumor clearance in JUN mice), mice were sacrificed and solid tumor tissue was removed and mechanically dissociated (n = 6–8 mice per group). In fig. 31B showed that digests of solid tumors from JUN-treated mice contained a significantly higher percentage of CD8+ T cells (frequency of total viable cells) and maintained a higher frequency of CAR+ CD8+ T cells. In fig. 31C shows that tumor-localized CD8+ T cells showed reduced expression (and coexpression) of the depletion-associated inhibitory receptors PD-1 and CD39 in JUN-Her2-BBz (n = 6 mice per group, left) (representative flow cytometry plots on right).

Experiments were then performed to evaluate the functional capacity of tumor-localized CAR T cells by ex vivo restimulation with Nalm6-Her2+ target cells (FIG. 31D and E). As shown in FIG. 31D, 5x10 ⁴ FACS sorted CD45+ T cells were restimulated with 5x10 ⁴ target cells and IL-2 secretion was measured after 24 hours by ELISA. As shown in FIG. 31E, 3x10 ⁵ digests of single tumor cells were restimulated with 3x10 ⁵ isolated Nalm6-Her2+ T cells for 6 hours in the presence of monensin and anti-CD107a antibody. After 6 hours, single-cell production of CD107a, IL-2, IFNγ, and TNFα was assessed by intracellular cytokine staining. Ex vivo stimulated JUN-Her2-BBz CAR T cells exhibited significantly higher frequencies of CD107a, IL-2, IFNγ, TNFα, and dual cytokine-producing cells compared to Her2-BBz controls.

Example 20

Overexpression of c-Jun increases resistance to TGFβ-mediated suppression of exhausted HA-28z CAR T cells

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 5 nM TGFβ. As shown in FIG. 32 left, IL-2 secretion measured by ELISA, one representative donor shown. As shown in FIG. 32 right, fold reduction in IL-2 secretion in TGFβ+ conditions compared to non-TGFβ conditions (n = 3 independent donors from 3 independent experiments).

Example 21

Transcriptional changes in c-Jun-modified cells are consistent with reduced exhaustion and increased memory formation

In fig. 33A shows a heat map of genes differentially regulated in HA-28z depleted CAR T cells upon addition of c-Jun overexpression (193 genes down-regulated by c-Jun, 176 genes up-regulated by c-Jun p(adjusted) < 0.05. n = 3 independent donors). Genes of interest are highlighted on the right. Genes downregulated in Jun cells include inhibitory receptors (LAG3, ENTPD1) and other potentially inhibitory AP-1 family members (BATF3, JUNB), as well as other genes associated with exhaustion (IL13, GZMB, LTA, TNFRSF9 ). Genes positively regulated by c-Jun are associated with naïve and memory T cells (IL7R, SELL (CD62L), CD44, and transcription factors LEF1 and foxp1).

In fig. 33B shows Venn diagrams showing the overlap of genes down-regulated in c-Jun-HA-28z CAR T cells and genes promoted by exhaustion in PC1, while FIG. 33C shows that genes up-regulated in c-Jun show overlap with genes associated with healthy (CD 19) CAR T cells (below). Approximately 25% of the top performing genes that differentiate between healthy and exhausted T cells can be modulated by c-Jun overexpression, suggesting that the AP-1 family may account for ~25% of the exhausted phenotype in this model.

In fig. 33D is a heat map showing gene expression of 50 genes in the overlap regions from b-c in all 12 samples (n=3 donors per condition).

Table 2 provides a complete list of genes altered by c-Jun

Example 22

IRF4 Inhibition Reduces T Cell Exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF4 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF4 inhibitors compared to control T cells.

Example 23

IRF8 Inhibition Reduces T Cell Exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF8 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF8 inhibitors compared to control T cells.

Example 24

BATF Inhibition Reduces T Cell Exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the BATF inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with BATF inhibitors compared to control T cells.

Example 25

BATF3 Inhibition Reduces T Cell Exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the BATF3 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with BATF3 inhibitors compared to control T cells.

Example 26

JUNB inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the JUNB inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with JUNB inhibitors compared to control T cells.

Example 27

IRF1 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF1 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF1 inhibitors compared to control T cells.

Example 28

IRF2 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF2 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF2 inhibitors compared to control T cells.

Example 29

IRF3 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF3 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF3 inhibitors compared to control T cells.

Example 30

IRF5 Inhibition Reduces T Cell Exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF5 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF5 inhibitors compared to control T cells.

Example 31

IRF6 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF6 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF6 inhibitors compared to control T cells.

Example 32

IRF7 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF7 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF7 inhibitors compared to control T cells.

Example 33

IRF9 inhibition reduces T cell exhaustion

Control and exhausted T cells are contacted with effective amounts (0.1-1000 μM) of the IRF9 inhibitors shown in Table 1. Samples are collected and processed for cytokine and interferon production assays. Increased production of the cytokine IL2 and/or interferon IFNγ is observed in exhausted T cells treated with IRF9 inhibitors compared to control T cells.

All publications and patents mentioned in the above description are incorporated herein by reference. Various modifications and variations of the described method and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described methods of carrying out the present invention, which are obvious to those skilled in the relevant fields, are intended to be within the scope of the following claims.

Links

The following references, some of which are cited above by number (eg, reference X), are incorporated herein by reference in their entirety.

1. Maude, S. L. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 378, 439-448, doi: 10.1056/NEJMoal709866 (2018).

2. Neelapu, S. S. 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).

3. Fraietta, J. A. et al. Determinants of response and resistance to CD 19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 24, 563-571, doi:10.1038/s41591-018-0010-l (2018).

4. June, S. H., O'Connor, R. S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. Science 359, 1361-1365 (2018).

5. Walker, A. J. et al. Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. Mol Ther 25, 2189-2201, doi:10.1016/j.ymthe.2017.06.008(2017).

6. Fry, T. J. et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD 19-targeted CAR immunotherapy. Nat Med 24, 20-28, (2018).

7. Hegde, M., Moll, A. J., Byrd, T. T., Louis, C. U. & Ahmed, N. Cellular immunotherapy for pediatric solid tumors. Cytotherapy 17, 3-17, (2015).

8. Long, A.H. et al. Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy

Efficacy for Sarcomas. Cancer Immunol Res 4, 869-880, doi:10.1158/2326-6066.CIR-15-0230 (2016).

9. Long, A. H. et al. 4-IBB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21, 581-590, doi:10.1038/nm.3838 (2015).

10. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumor rejection. Nature 543, 113-117, doi: 10.1038/nature21405 (2017).

11. Fraietta, J. A. et al. Disruption of TET2 promotes the therapeutic efficacy of CD 19-targeted T cells. Nature 558, 307-312, doi:10.1038/s41586-018-0178-z (2018).

12. Sen, D.R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165-1169, doi:10.1126/science.aae0491 (2016).

13. Wherry, E.J. et al. Molecular signature of CD8+T cell exhaustion during chronic viral infection. Immunity 27, 670-684, doi:10.1016/j.immuni.2007.09.006 (2007).

14. Man, K. et al. Transcription Factor IRF4 Promotes CD8(+) T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection. Immunity 47, 1129-1141 ell25, doi:10.1016/j.immuni.2017.11.021 (2017).

15. Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15, 486-499, doi: 10.1038/nri3862 (2015).

16. Bengsch, B. et al. Epigenomic-Guided Mass Cytometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells. Immunity 48, 1029-1045 el 025, doi:10.1016/j.immuni.2018.04.026 (2018).

17. Pauken, K. E. et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165, doi:10.1126/science.aaf2807 (2016).

18. Heczey, A. et al. CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther 25, 2214-2224, doi:10.1016/j.ymthe.2017.05.012(2017).

19. Horwacik, I. et al. Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody. Mol Cell Proteomics 14, 2577-2590, doi: 10.1074/mcp.Ml 15.052720 (2015).

20. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of the open

chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10, 1213-1218, doi:10.1038/nmeth.2688 (2013).

21. Schep, A. N., Wu, W., Buenrostro, J. D. & Greenleaf, W. J. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nat Methods 14, 975-978, doi: 10.1038/nmeth.4401 (2017).

22. Philip, M. et al. Chromatin states define tumor-specific T cell dysfunction and reprogramming. Nature 545, 452-456, doi: 10.1038/nature22367 (2017).

23. Singh, N., Perazzelli, J., Grupp, S. A. & Barrett, D. M. Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies. Sci Transl Med 8, 320ra323, doi: 10.1126/scitranslmed. aad5222 (2016).

24. Murphy, T. L., Tussiwand, R. & Murphy, K. M. Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks. Nat Rev Immunol 13, 499-509, doi:10.1038/nri3470 (2013).

25. Meixner, A., Karreth, F., Kenner, L. & Wagner, E. F. JunD regulates lymphocyte proliferation and T helper cell cytokine expression. EMBO J 23, 1325-1335, doi:10.1038/sj.emboj.7600133 (2004).

26. Chiu, R., Angel, P. & Karin, M. Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun. Cell 59, 979-986 (1989).

27. Echlin, D. R., Tae, H. J., Mitin, N. & Taparowsky, E. J. B-ATF functions as a negative regulator of AP-1 mediated transcription and blocks cellular transformation by Ras and Fos. Oncogene 19, 1752-1763, doi: 10.1038/sj.one. 1203491 (2000).

28. Li, P. et al. BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543-546, doi:10.1038/naturell530 (2012).

29. Quigley, M. et al. Transcriptional analysis of HIV-specific CD8+T cells shows that PD-1 inhibits T cell function by upregulating BATF. Nat Med 16, 1147-1151, doi: 10.1038/nm.2232 (2010).

30. Derijard, B. et al. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037 (1994).

31. S, P. D. A. et al. Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-l(c259)T Cells in Synovial Sarcoma. Cancer Discov, doi: 10.1158/2159-8290.CD-17-1417 (2018).

32. Iwamoto, M., Bjorklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem Biol 17, 981-988, doi:10.1016/j.chembiol.2010.07.009 (2010).

33. Bannister, A. J., Oehler, T., Wilhelm, D., Angel, P. & Kouzarides, T. Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73 are required for CBP induced stimulation in vivo and CBP binding in vitro. Oncogene 11, 2509-2514 (1995).

34. Weiss, C. et al. JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO J 22, 3686-3695, doi: 10.1093/emboj/cdg364 (2003).

35. Milone, M. C. et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 17, 1453-1464, doi:10.1038/mt.2009.83 (2009).

36. Porter, D. L. et al. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 7, 303ra139, doi:10.1126/scitranslmed.aac5415 (2015).

37. Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6, 224ra225, doi: 10.1126/scitranslmed.3008226 (2014).

38. Majzner, R. G. & Mackall, C. L. Tumor Antigen Escape from CAR T-cell Therapy. Cancer Discov, doi:10.1158/2159-8290.CD-18-0442 (2018).

39. Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of activated

CD8(+) T cells. Immunity 42, 265-278, doi:10.1016/j.immuni.2015.01.006 (2015).

40. Roychoudhuri, R. et al. BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers. Nat Immunol 17, 851-860, doi:10.1038/ni.3441 (2016).

41. Bohmann, D. et al. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science 238, 1386-1392 (1987).

42. Mariani, O. et al. JUN oncogene amplification and overexpression block adipocytic differentiation in highly aggressive sarcomas. Cancer Cell 11, 361-374, doi:10.1016/j.ccr.2007.02.007 (2007).

43. Shaulian, E. AP-l~The Jun proteins: Oncogenes or tumor suppressors in disguise? Cell Signal 22, 894-899, doi:10.1016/j.cellsig.2009.12.008 (2010).

44. Behrens, A., Jochum, W., Sibilia, M. & Wagner, E. F. Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal phosphorylation. Oncogene 19, 2657-2663, doi: 10.1038/sj.onc. 1203603 (2000).

45. Di Stasi, A. et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365, 1673-1683, doi: 10.1056/NEJMoal 106152 (2011).

46. Hudecek, M. et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 3, 125-135, doi: 10.1158/2326-6066.CIR-14-0127 (2015).

47. Jena, B. et al. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials. PLoS One 8, e57838, doi:10.1371/journal.pone.0057838 (2013).

48. Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14, 959-962, doi: 10.1038/nmeth.4396 (2017).

49. Wickham, H. Ggplot2: elegant graphics for data analysis. (Springer, 2009).

50. Mootha, V. K. et al. PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267-273, doi:10.1038/ng1180 (2003).

51. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102, 15545-

15550, doi:10.1073/pnas.0506580102 (2005).

52. Zheng, G. X. et al. Massively parallel digital transcriptional profiling of single cells. NatCommun 8, 14049, doi: 10.1038/ncomms 14049 (2017).

Claims (31)

1. A composition for treating a disease or condition in a subject amenable to treatment by administration of T cells, wherein the composition comprises isolated T cells and wherein the isolated T cells contain an administered nucleic acid to overexpress c-Jun. 2. The composition according to claim 1, wherein the isolated T cells express the recombinant receptor. 3. The composition according to claim 2, wherein the recombinant receptor is a T-cell receptor (TCR). 4. The composition according to claim 2, wherein the recombinant receptor is a chimeric antigen receptor (CAR). 5. Composition according to any one of paragraphs. 2-4, in which the recombinant receptor is specific for a tumor antigen. 6. The composition of claim 5, wherein the tumor antigen is selected from the group consisting of the following: 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. 7. Composition according to any one of paragraphs. 1-6, in which the isolated T cells are native, naturally occurring T cells. 8. The composition of claim 7, wherein the native, naturally occurring T cells are tumor infiltrating lymphocytes (TILs). 9. The composition of claim 7, wherein native, naturally occurring T cells are obtained by leukapheresis of a blood sample. 10. The composition according to claim 9, wherein the expansion of T cells is carried out ex vivo. 11. Composition according to any one of paragraphs. 1-10, wherein the T cells are selected from the group consisting of the following: CD3+ T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, gamma delta T cells, a combination of CD4+ and CD8+ T cells, memory T cells, cytokine-induced killer cells, and combinations thereof. 12. Composition according to any one of paragraphs. 1-11, in which c-Jun is a mutated/truncated c-Jun. 13. The composition of claim 12, wherein the mutated/truncated c-Jun contains an N-terminal deletion. 14. The composition of claim 12, wherein the mutated/truncated c-Jun (i) does not contain a transactivation domain or (ii) contains an inactive transactivation domain. 15. Composition according to any one of paragraphs. 1-11, in which c-Jun represents wild-type c-Jun. 16. Composition according to any one of paragraphs. 2-15, wherein the T cells are modified by introducing a nucleic acid encoding c-Jun, and wherein c-Jun and the recombinant receptor are expressed from separate expression vector constructs. 17. Composition according to any one of paragraphs. 2-15, wherein the T cells are modified by introducing a nucleic acid encoding c-Jun, and wherein c-Jun and the recombinant receptor are coexpressed from a single expression vector construct. 18. Composition according to any one of paragraphs. 1-17, wherein the isolated T cells are T cells with tumor specificity and activity. 19. The composition of claim 18, wherein the T cells are peripheral blood T cells genetically modified to express a receptor that recognizes and responds to a tumor. 20. Composition according to any one of paragraphs. 1-19, wherein the T cells are modified to reduce and/or eliminate the expression and/or activity of one or more members of the AP-1 inhibitory complex. 21. The composition of claim 20, wherein the AP-1 inhibitory complex member is selected from the group consisting of JunB, a BATF family member, IRF4, an ATF family member, or a combination thereof. 22. The composition of claim 20, wherein the AP-1 inhibitory complex member is JunB and/or BATF3. 23. A method of treating a disease or pathological condition in a subject, comprising administering to the subject, characterized by the presence of the disease or pathological condition, an effective amount of the composition according to any one of paragraphs. 1-22, wherein the disease or condition is treatable by administration of T cells. 24. The method of claim 23, wherein the T cells in the composition are less susceptible to T cell exhaustion. 25. The method of claim 23 or 24, wherein the T cells in the composition are characterized by increased reactivity to a low density antigen on the T cell target cells. 26. Method according to any one of paragraphs. 23-25, in which the T cells in the composition are less susceptible to reduced memory formation. 27. Method according to any one of paragraphs. 23-26, in which the T cells in the composition are less susceptible to a decrease in proliferative potential. 28. Method according to any one of paragraphs. 23-27, in which the disease or condition is a tumor or cancer. 29. The method of claim 28, further comprising administering to the patient one or more anticancer agents and/or one or more chemotherapeutic agents. 30. The method according to claim 29, wherein the administration occurs before, simultaneously and/or after the patient receives radiation therapy. 31. Method according to any one of paragraphs. 23-27, in which the disease or condition is a viral, bacterial and/or parasitic infection.