US20200101108A1 - Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling - Google Patents

Methods of treating t cell exhaustion by inhibiting or modulating t cell receptor signaling Download PDF

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US20200101108A1
US20200101108A1 US16/499,762 US201816499762A US2020101108A1 US 20200101108 A1 US20200101108 A1 US 20200101108A1 US 201816499762 A US201816499762 A US 201816499762A US 2020101108 A1 US2020101108 A1 US 2020101108A1
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cells
cell
car
tyrosine kinase
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Rachel Lynn
Crystal MacKall
Evan Weber
Sanjay Malhotra
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Leland Stanford Junior University
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Leland Stanford Junior University
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Definitions

  • compositions and methods for preventing or reversing T cell exhaustion relate to methods of preventing or reversing T cell exhaustion by exposing T cells experiencing T cell exhaustion to particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), or by expanding genetically engineered T cells in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • particular tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • T cells are immune cells that become activated via T cell receptor (TCR) signaling following engagement with antigen.
  • TCR T cell receptor
  • Physiologic activation through the T cell receptor renders T cells capable of mediating potent antitumor or anti-infective effects.
  • T cells During resolution of an acute inflammatory response, a subset of activated effector T cells differentiate into long-lived memory cells.
  • T cell exhaustion T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector function (e.g., cytokine secretion, killing capacity), and co-expression of multiple surface inhibitory receptors.
  • the root cause of T cell exhaustion is persistent antigen exposure leading to continuous TCR signaling. Prevention or reversal of T cell exhaustion has been long sought as a means to enhance T cell effectiveness in patients with cancer or chronic infections.
  • the present invention addresses this urgent need.
  • T cells respond to the presence of foreign antigens with a wide range of responses, including the secretion of preformed and newly formed mediators, phagocytosis of particles, endocytosis, cytotoxicity against target cells, as well as cell proliferation and/or differentiation.
  • T cells are a subgroup of cells which together with other immune cell types (e.g., polymorphonuclear, eosinophils, basophils, mast cells, B cells, and NK cells), constitute the cellular component of the immune system (see, e.g., U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478).
  • T cells function in immune surveillance and in the elimination of foreign antigen.
  • pathological conditions there is compelling evidence that T cells play a major role in the causation and propagation of disease. In these disorders, breakdown of T cell immunological tolerance, either central or peripheral is a fundamental process in the causation of autoimmune disease.
  • T cell receptor (TCR) engagement and costimulatory signaling provide the critical signals that regulate T cell activation, proliferation and cytolytic functions.
  • T cells respond to antigen via a polypeptide complex composed of the ligand-binding T cell receptor (TCR) disulfide-linked ⁇ and ⁇ subunits (or ⁇ and ⁇ subunits in ⁇ T cells) that have single transmembrane (TM) spans per subunit and small intracellular tails and associate non-covalently with hetero- (CD3 ⁇ and CD3 ⁇ ) and homodimeric ( ⁇ ) signaling subunits (see, e.g., Cambier J. C. Curr Opin Immunol 1992; 4:257-64).
  • TCR ligand-binding T cell receptor
  • TM transmembrane
  • the CD3 ⁇ , ⁇ , and ⁇ chains have single Ig-family extracellular domains, single presumably ⁇ -helical TM spans, and intrinsically disordered intracellular domains of 40-60 residues, whereas each subunit has a small extracellular region (9 residues) carrying the intersubunit disulfide bond, a single presumably ⁇ -helical TM span per subunit, and a large, intrinsically disordered cytoplasmic domain of approximately 110 residues.
  • An understanding of the process of TCR-mediated TM signal transduction and subsequent T cell activation, leading to T cell proliferation and differentiation, is therefore pivotal to both health and disease. Disturbance in TCR signaling can lead to inflammatory and other T cell-related disorders.
  • T cells expressing chimeric antigen receptors (CARs) at high levels undergo tonic, antigen independent signaling due to receptor clustering.
  • CARs chimeric antigen receptors
  • Such T cells function poorly as a result of T cell exhaustion, as evidenced by high levels of PD-1, TIM-3, LAG-3, diminished antigen induced cytokine production, and excessive programmed cell death.
  • Tonic signaling can be prevented by transiently decreasing CAR associated TCR signaling proteins (e.g., TCR zeta) to levels below the threshold required for tonic signaling.
  • tyrosine kinase inhibitor that inhibits T cell receptor signaling
  • a particular tyrosine kinase inhibitor that inhibits T cell receptor signaling e.g., a Lck tyrosine kinase inhibitor (e.g., dasatinib)
  • a Src family tyrosine kinase inhibitor reduced expression of the T cell exhaustion markers and improved formation of T cell memory.
  • the present invention relates to methods of preventing or reversing T cell exhaustion by transiently inhibiting T cell receptor (TCR) signaling to restore T cell function with particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • TCR T cell receptor
  • CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib exhibit attenuated activation and degranulation, fail to secrete cytokine, and display attenuated killing in response to tumor antigen.
  • compositions and methods for preventing or reversing T cell exhaustion relate to methods of preventing or reversing T cell exhaustion by exposing T cells experiencing T cell exhaustion to particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), or by expanding genetically engineered T cells in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • particular tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • the present invention provides methods for treating a subject to mitigate T cell exhaustion, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor.
  • a tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.
  • the tyrosine kinase inhibitor is a Lck kinase inhibitor.
  • the tyrosine kinase inhibitor is a Fyn kinase inhibitor.
  • the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. In some embodiments, tyrosine kinase inhibitor is dasatinib or ponatinib. In some embodiments, the treatment is prophylactic.
  • the treatment increases secretion of IL-2 by T cells in the subject.
  • the treatment decreases apoptosis of T cells in the subject.
  • the treatment decreases expression of at least one T cell exhaustion marker selected from the group consisting of PD-1, TIM-3, and LAG-3.
  • the treatment increases expression of CD62L or CCR7.
  • Such methods are not limited to particular manner of administration.
  • multiple cycles of treatment are administered to the subject.
  • the tyrosine kinase inhibitor is administered intermittently.
  • the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least partial T cell function then discontinued.
  • the tyrosine kinase inhibitor is administered orally.
  • Such methods are not limited to a particular type or kind of subject.
  • the subject is a human.
  • the subject has a chronic infection or cancer.
  • the present invention provides for treating an immune system related condition or disease in a subject comprising administering to the subject genetically engineered T cells and a therapeutically effective amount of a tyrosine kinase inhibitor.
  • a tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.
  • the tyrosine kinase inhibitor is a Lck kinase inhibitor.
  • the tyrosine kinase inhibitor is a Fyn kinase inhibitor.
  • the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib. In some embodiments, the treatment is prophylactic. In some embodiments, the tyrosine kinase inhibitor and the genetically engineered T cells are administered simultaneously and/or at different time points.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • Such methods are not limited to treating a specific immune system related condition or disease.
  • the immune system related condition or disease is selected from cancer or an autoimmune disease or condition.
  • the present invention provides methods for preventing and/or reversing toxicity related to genetically engineered T cell administered to a subject, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor.
  • a tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.
  • the tyrosine kinase inhibitor is a Lck kinase inhibitor.
  • the tyrosine kinase inhibitor is a Fyn kinase inhibitor.
  • the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • the adoptive T cell therapy is a CAR T-cell therapy.
  • the adoptive T cell therapy is a transduced T-cell therapy.
  • the adoptive T cell therapy is a tumor infiltrating lymphocyte (TIL) therapy.
  • Such methods are not limited to a particular type or kind of toxicity related to genetically engineered T cell administered to a subject.
  • the toxicity related to genetically engineered T cell administered to a subject is cytokine release syndrome.
  • the toxicity related to genetically engineered T cell administered to a subject is on-target off tumor toxicity or off-target off-tumor toxicity.
  • the present invention provides compositions comprising a genetically engineered T cell population, wherein the genetically engineered T cell population was expanded in the presence of tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling inhibitor.
  • the tyrosine kinase inhibitor dasatinib or ponatinib.
  • the present invention provides methods of generating a population of genetically engineered T cells resistant to T cell exhaustion, comprising expanding a population of genetically engineered T cells in the presence of a tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling inhibitor.
  • the tyrosine kinase inhibitor is dasatinib or ponatinib. Such methods are not limited to a specific type or kind of genetically engineered T cells.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • the present invention provides methods of treating an immune system related condition or disease in a subject undergoing an adoptive T cell therapy, comprising administering to the subject a genetically engineered T cell population that were expanded in the presence of a tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor is capable of inhibiting TCR signaling inhibitor and/or CAR signaling.
  • the tyrosine kinase inhibitor is a Lck kinase inhibitor.
  • the tyrosine kinase inhibitor is a Fyn kinase inhibitor.
  • the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib. In some embodiments, the immune system related condition or disease is selected from cancer or an autoimmune disease or condition.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • the adoptive T cell therapy is a CAR T-cell therapy.
  • the adoptive T cell therapy is a transduced T-cell therapy.
  • the adoptive T cell therapy is a tumor infiltrating lymphocyte (TIL) therapy.
  • the present invention contemplates that exposure of animals (e.g., humans) suffering from cancer (e.g., and/or cancer related disorders) to adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) with genetically engineered T cell populations and compositions comprising particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will inhibit the growth of cancer cells or supporting cells outright and/or render such cells as a population more susceptible to the cell death-inducing activity of cancer therapeutic drugs or radiation therapies.
  • T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • the methods result in improved therapy outcome as such particular tyrosine kinase inhibitors are capable of 1) modulating TCR signaling within the genetically engineered T cell population (e.g., decreasing expression of one or more of PD-1, TIM-3, and LAG-3; increasing expression of memory markers (e.g., CD62L or CCR7); increasing secretion of IL-2 and other cytokines), and 2) preventing and/or reversing T cell exhaustion within the genetically engineered T cell population.
  • TCR signaling e.g., decreasing expression of one or more of PD-1, TIM-3, and LAG-3
  • memory markers e.g., CD62L or CCR7
  • IL-2 and other cytokines e.g., CD62L or CCR7
  • the present invention provides methods for treating cancer (e.g., and/or cancer related disorders) with adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) in a subject comprising administering to the subject (e.g., simultaneously and/or at different time points) genetically engineered T cells, particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), and additional cancer therapeutic drugs or radiation therapies.
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • adoptive T cell therapies e.g., a CAR T
  • the present invention contemplates that exposure of animals (e.g., humans) suffering from cancer (e.g., and/or cancer related disorders) to adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) with genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will inhibit the growth of cancer cells or supporting cells outright and/or render such cells as a population more susceptible to the cell death-inducing activity of cancer therapeutic drugs or radiation therapies.
  • T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • the methods result in improved therapy outcome as such genetically engineered T cell populations are resistant and/or less prone to T cell exhaustion.
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • the present invention contemplates that such methods (e.g., adoptive T cell therapies with genetically engineered T cell populations and compositions comprising particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib)) (e.g., adoptive T cell therapies with genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib)) satisfy an unmet need for the treatment of multiple cancer types, either when administered as monotherapy or when administered in a temporal relationship with additional agent(s), such as other cell death-inducing or cell cycle disrupting cancer therapeutic drugs or radiation therapies (combination therapies), so as to render a greater proportion of the cancer cells or supportive cells susceptible to executing the apoptosis program compared to the corresponding proportion of cells in an animal treated only with the cancer therapeutic drug or radiation therapy alone.
  • additional agent(s) such as other cell death-inducing or cell
  • combination treatment of animals with such methods e.g., adoptive T cell therapies with genetically engineered T cell populations and compositions comprising particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib))
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • adoptive T cell therapies with genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • the present invention contemplates the various combinations of them with such methods.
  • a non-limiting exemplary list of cancer includes, but is not limited to, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia
  • FIG. 1 Characterization of the GD2.28z.FKBP CAR.
  • T cells were transduced with lentivirus encoding the GD2.28z.FKBP CAR on day 1 after activation and subsequently cultured with various concentrations of shield-1 in the growth medium. On day 7, CAR expression was quantified via FACS.
  • FIG. 2 Removal of S1 from culture medium results in reversal of T cell exhaustion marker surface expression.
  • FIG. 3 Removal of S1 from culture medium results in maintenance of CD62L expression and prevention of apoptosis.
  • FIG. 4 Removal of S1 from culture medium results in reversal of function T cell exhaustion.
  • FIG. 5 Removal of surface CAR results in more effective prevention of T cell exhaustion compared PD-1/PDL-1 blockade.
  • FIG. 6 Removal of surface CAR rescues exhaustion in PD-1/TIM-3/LAG-3 triple positive CAR T cells after only 4 days.
  • FIG. 7 Dasatinib inhibits cytokine secretion of CAR T cells in response to tumor antigen.
  • FIG. 8 Dasatinib reverses exhaustion marker expression and co-expression.
  • FIG. 9 Dasatinib treatment results in maintenance of CD62L expression.
  • FIG. 10 Dasatinib Treatment results in augmented IL-2 and IFN ⁇ secretion in response to tumor antigen.
  • FIG. 11 CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib exhibit attenuated activation and degranulation.
  • CD19.28z CAR T cells were cultured in the presence or absence of various concentrations of dasatinib or ponatinib for at least 48 hours.
  • CAR T cells were then co-cultured with CD19-bearing Nalm6 tumor cells for 6 hours.
  • CD69 and CD107a surface expression was subsequently assessed via FACS.
  • Plots display cells gated on the CD8+ CAR+ population.
  • FIG. 12 CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib fail to secrete cytokine.
  • high affinity GD2.28z (HA-GD2.28z) CAR T cells were co-cultured with GD2-overexpressing nalm6 for 24 hours in the presence of absence of various concentrations of dasatinib or ponatinib. Supernatant was then collected and analyzed for IL-2 and IFN ⁇ via ELISA.
  • FIG. 13 CAR T cells cultured in the presence of dasatinib display attenuated killing in response to tumor antigen.
  • An incucyte assay was conducted in which CD19.BBz CAR T cells were co-cultured with nalm6 tumor cells expressing a GFP reporter for 72 hours in the presence of 1 uM dasatinib or vehicle (DMSO). Tumor GFP fluorescence was measured over time. GFP values were normalized to the fluorescence intensity at the first time point.
  • FIGS. 11, 12 and 13 demonstrate that dasatinib or ponatinib could serve as a rapid and reversible safety “OFF” switch for CAR T cells that are having deleterious effects in a given patient.
  • FIG. 14 Dasatinib potently inhibits the phosphorylation of CAR CD3z as well as distal signaling proteins after CAR crosslinking.
  • 2E6 HA-GD2.28z CAR T cells cultured in 1 uM dasatinib or vehicle were removed from culture on day 10 post-activation. Idiotype primary antibody and a crosslinking secondary antibody were then added to the cells at 5 ug/mL to initiate signaling through the CAR.
  • FIG. 15 Tonically signaling CART cells expanded in the presence of dasatinib exhibit a reduction in canonical exhaustion marker expression in a dose-dependent manner.
  • HA-GD2.28z CART cells were expanded in the presence of various concentrations of dasatinib or vehicle (DMSO).
  • DMSO vehicle
  • FIG. 15A CAR+ T cell canonical exhaustion marker expression.
  • FIG. 15B CAR+ CD4+ (left) or CAR+ CD8+ (right) exhaustion marker co-expression.
  • FIG. 16 Tonically signaling CAR T cells expanded in the presence of dasatinib retain the capacity to form memory.
  • CD19.28z or HA-GD2.28z were expanded in the presence or absence of 1 uM dasatinib or vehicle (DMSO).
  • DMSO vehicle
  • FIG. 16 Tonically signaling CAR T cells expanded in the presence of dasatinib retain the capacity to form memory.
  • CD19.28z or HA-GD2.28z were expanded in the presence or absence of 1 uM dasatinib or vehicle (DMSO).
  • DMSO dasatinib
  • FIG. 16 Tonically signaling CAR T cells expanded in the presence of dasatinib retain the capacity to form memory.
  • CD19.28z or HA-GD2.28z were expanded in the presence or absence of 1 uM dasatinib or vehicle (DMSO).
  • DMSO dasatinib
  • FIG. 16 Tonically signaling CAR T cells expanded in the presence of dasatinib retain the capacity
  • FIG. 17 Tonically signaling CAR T cells expanded in the presence of dasatinib display augmented cytokine secretion in response to tumor antigen.
  • HA-GD2.28z CAR T cells were expanded in the presence or absence of various concentrations of dasatinib or ponatinib.
  • Drug was removed from the T cells 24 hours prior to co-culture with GD2-overexpressing nalm6 tumor cells in order to allow the T cells to regain the ability to signal in response to tumor. After 24 hours, supernatants were collected and IL-2 and IFN ⁇ secretion was assessed via ELISA.
  • FIGS. 11, 12, 13, 15 and 16 demonstrate that dasatinib and ponatinib can inhibit CART cell signaling and function.
  • FIG. 17 shows that expansion of tonically signaling HA-GD2.28z CART cells in the presence of these drugs followed by remove of the drugs prior to co-culturing with tumor cells results in augmentation of IL-2 and IFN ⁇ .
  • FIG. 18 Tonically signaling CART cells expanded in the presence of dasatinib display augmented cytotoxicity.
  • HA-GD2.28z CAR T cells were expanded in the presence or absence of dasatinib or vehicle (DMSO) for 96 hours.
  • dasatinib was removed from the T cells 24 hours prior to an incucyte assay in which T cells were co-cultured at a 1:8 E:T ratio with GD2-overexpressing nalm6 tumor.
  • Tumor GFP fluorescence was measured over time. GFP values were normalized to the fluorescence intensity at the first time point.
  • FIG. 19 GD2-overexpressing Nalm6 in the presence and absence of dasatinib.
  • 0.5E6 143B tumor cells were engrafted intramuscularly in the legs of mice.
  • 10E6 GD2.BBz CART cells expanded in the presence of dasatinib or vehicle (DMSO) were infused into mice intravenously.
  • FIGS. 19 and 20 recapitulate the findings from FIGS. 14, 15, 16 and 17 in an in vivo setting. Culturing different types of CARs (GD2.BBz, HA-GD2.28z) in dasatinib and then infusing them in vivo augments their anti-tumor function.
  • FIG. 20A 0.5E6 143B tumor cells were engrafted intramuscularly in the legs of mice.
  • 10E6 HA-GD2.28z CAR T cells expanded in the presence of dasatinib or vehicle (DMSO) were infused into mice intravenously.
  • FIGS. 19 and 20 recapitulate the findings from FIGS. 14, 15, 16 and 17 in an in vivo setting. Culturing different types of CARs (GD2.BBz, HA-GD2.28z) in dasatinib and then infusing them in vivo augments their anti-tumor function.
  • FIGS. 19 and 20 recapitulate the findings from FIGS. 14, 15, 16 and 17 in an in vivo setting. Culturing different types of CARs (GD2.BBz, HA-GD2.28z) in dasatinib and then infusing them in vivo augments their anti-tumor function.
  • FIG. 21 demonstrates one of the mechanisms by which dasatinib augments function.
  • CAR T cells After infusing dasatinib-treated CAR T cells into mice, blood samples were taken and the number of circulating CAR T cells analyzed, a typical readout for in vivo CAR T cell proliferation in response to tumor.
  • the vehicle HA-GD2.28z CAR T cells did not exhibit significantly more in vivo proliferation than mock T cells, as these cells were likely exhausted when they were initially infused into the mice.
  • CAR T cells that were expanded in dasatinib retained their anti-tumor function and thus proliferated robustly in vivo.
  • FIG. 22A ,B,C,D,E In vivo dasatinib treatment suppresses exhaustion marker expression, augments memory formation, and facilitates cell survival/proliferation.
  • Mice were engrafted with 1E6 GD2-overexpressing nalm6 tumor cells via intravenous injection. On day 4 post-engraftment, 2E6 HA-GD2.28z CAR T cells were infused into mice intravenously. Mice were dosed with 50 mg/kg dasatinib via intraperitoneal injection on days 21-23 post-tumor engraftment.
  • C CAR+ T cells constituted a higher percentage of total circulating cells (A) or total splenic cells (C) in the mouse treated with dasatinib versus the vehicle control.
  • FIG. 23 The nucleic acid and amino acid sequence for CD19.28z (FMC63 scFv).
  • FIG. 24 The nucleic acid and amino acid sequence for CD19.BBz (FMC63 scFv).
  • FIG. 25 The nucleic acid and amino acid sequence for GD2.BBz (14G2a scFv).
  • FIG. 26 The nucleic acid and amino acid sequence for HA-GD2.28z (High affinity 14G2a scFv).
  • chimeric antigen receptor refers to an artificial T cell receptor that is engineered to be expressed on an immune effector cell and specifically bind an antigen.
  • CARs may be used as a therapy with adoptive cell transfer. T cells are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some embodiments, the CARs have been expressed with specificity to a tumor associated antigen, for example. CARs may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising a tumor associated antigen binding region.
  • the specificity of CAR designs may be derived from ligands of receptors (e.g., peptides).
  • a CAR can target cancers by redirecting the specificity of a T cell expressing the CAR specific for tumor associated antigens.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • T cell refers to T lymphocytes as defined in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • the T cells can be CD4 + T cells, CD8 + T cells, CD4 + CD8 + T cells, or CD4 ⁇ CD8 ⁇ cells.
  • the T cells can also be T helper cells, such as T helper 1 (TH1), or T helper 2 (TH2) cells, or TH17 cells, as well as cytotoxic T cells, regulatory T cells, natural killer T cells, na ⁇ ve T cells, memory T cells, or gamma delta T cells.
  • the T cells can be a purified population of T cells, or alternatively the T cells can be in a population with cells of a different type, such as B cells and/or other peripheral blood cells.
  • the T cells can be a purified population of a subset of T cells, such as CD4 + T cells, or they can be a population of T cells comprising different subsets of T cells.
  • the T cells are T cell clones that have been maintained in culture for extended periods of time. T cell clones can be transformed to different degrees.
  • the T cells are a T cell clone that proliferates indefinitely in culture.
  • the T cells are primary T cells.
  • the term “primary T cells” is intended to include T cells obtained from an individual, as opposed to T cells that have been maintained in culture for extended periods of time.
  • primary T cells are particularly peripheral blood T cells obtained from a subject.
  • a population of primary T cells can be composed of mostly one subset of T cells.
  • the population of primary T cells can be composed of different subsets of T cells.
  • the T cells can be from previously stored blood samples, from a healthy individual, or alternatively from an individual affected with a condition.
  • the condition can be an infectious disease, such as a condition resulting from a viral infection, a bacterial infection or an infection by any other microorganism, or a hyperproliferative disease, such as cancer like melanoma.
  • the T cells are from a subject suffering from or susceptible to an autoimmune disease or T-cell pathologies.
  • the T cells can be of human origin, murine origin or any other mammalian species.
  • T cell exhaustion refers to loss of T cell function, which may occur as a result of an infection or a disease. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, apoptosis, and reduced cytokine secretion.
  • terapéuticaally effective dose or amount of an inhibitor of TCR signaling is intended an amount that, when administered as described herein, brings about a positive therapeutic response in treatment of T cell exhaustion, such as restored T cell function.
  • Improved T cell function may include decreased expression of PD-1, TIM-3, and LAG-3, maintenance of memory markers (e.g., CD62L or CCR7), prevention of apoptosis, and increased secretion of IL-2 and other cytokines.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • subject refers to any vertebrate subject, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • the invention is based on the discovery that transient inhibition or modulation of TCR signaling and/or CAR signaling in human T cells can prevent or reverse T cell exhaustion and restore T cell function.
  • the inventors have shown that GD2-CAR expressing T cells develop functional exhaustion, exhibited by expression of PD-1, TIM-3, and LAG-3 exhaustion markers. Cessation of tonic signaling restores the ability of T cells to secrete IL-2 in response to tumor antigen.
  • the inventors further showed that treatment with dasatinib, a Lck tyrosine kinase inhibitor that inhibits T cell receptor signaling, reduced expression of the T cell exhaustion markers and improved preservation of T cell memory.
  • Protein tyrosine kinases are a family of enzymes catalysing the transfer of the terminal phosphate of adenosine triphosphate to tyrosine residues in protein substrates. Phosphorylation of tyrosine residues on protein substrates leads to transduction of intracellular signals which regulate a wide variety of intracellular processes such as growth and activation of cells of the immune system, e.g. T-cells. As T-cell activation is implicated in a number of inflammatory conditions and other disorders of the immune system (e.g. autoimmune diseases), modulation of the activity of protein tyrosine kinases appears to be an attractive route to the management of inflammatory diseases. A large number of protein tyrosine kinases have been identified which may be receptor protein tyrosine kinases, e.g. the insulin receptor, or non-receptor protein tyrosine kinases.
  • receptor protein tyrosine kinases e.g. the insulin receptor,
  • Protein tyrosine kinases of the Src family have been found to be particularly important for intracellular signal transduction related to inflammatory responses (see, e.g., D. Okutani et al., Am. J. Physiol. Lung Cell Mol. Physiol. 291, 2006, pp. L129-L141; CA. Lowell, Mol. Immunol. 41, 2004, pp. 631-643). While some of Src family protein tyrosine kinases, e.g. Src, Yes and Fyn, are expressed in a variety of cell types and tissues, the expression of others is restricted to specific cell types, e.g. hematopoietic cells.
  • the protein tyrosine kinase Lck is expressed almost exclusively in T-cells as the first signalling molecule to be activated downstream of the T-cell receptor, and its activity is essential for T-cell signal transduction.
  • Expression of Hck, Lyn and Fgr is increased by inflammatory stimuli such as LPS in mature monocytes and macrophages.
  • gene expression of the main B-cell Src family kinases, namely Lyn, Fyn and BIk is disrupted, immature B-cells are prevented from developing into mature B-cells.
  • Src family kinases have also been identified as essential for the recruitment and activation of monocytes, macrophages and neutrophils as well as being involved in the inflammatory response of tissue cells.
  • Lck p56 lck or lymphocyte specific kinase
  • NK natural killer
  • the invention is further based on the discovery that the Lck kinase inhibitor dasatinib and the receptor tyrosine kinase inhibitor ponatinib have the potential to address several important challenges currently facing the field of adoptive T cell therapies (e.g., CART cell therapies).
  • these drugs were shown to potently inhibit CAR signaling, which provides a method to regulate CAR activity and thus mitigate CAR T cell toxicity while preserving the option to continue therapy once the toxicity has resolved, as the inhibitory effect of dasatinib and ponatinib on CAR T cell function is reversible.
  • compositions and methods for preventing or reversing T cell exhaustion relate to methods of preventing or reversing T cell exhaustion by exposing T cells experiencing T cell exhaustion to particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), or by expanding genetically engineered T cells in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • particular tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • the present invention contemplates that exposure of animals (e.g., humans) undergoing adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) with genetically engineered T cell populations to compositions comprising particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will result in improved therapy outcome as such particular tyrosine kinase inhibitors are capable of 1) modulating TCR signaling within the genetically engineered T cell population (e.g., decreasing expression of one or more of PD-1, TIM-3, and LAG-3; increasing expression of memory markers (e.g., CD62L or CCR7); increasing secretion of IL-2 and other cytokines), and 2) preventing and/or reversing T cell exhaustion within the genetically engineered T cell population.
  • adoptive T cell therapies e.g., a CAR
  • the present invention contemplates that the use of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors) within adoptive T cell therapies satisfies an unmet need as the effectiveness of such therapies are frequently compromised by such T cell populations experiencing T cell exhaustion.
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • Src family kinase inhibitors e.g., Lck inhibitors
  • the present invention provides methods for treating an immune system related condition or disease (e.g., cancer) in a subject comprising administering to the subject (e.g., simultaneously and/or at different time points) genetically engineered T cells and particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • an immune system related condition or disease e.g., cancer
  • administering e.g., simultaneously and/or at different time points
  • genetically engineered T cells and particular tyrosine kinase inhibitors e.g., dasatinib, ponatinib.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • T cells configured for transduced T-cell therapy
  • viral specific T cells reengineered with a TCR or CAR include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • Such tyrosine kinase inhibitors may be administered by any suitable mode of administration, but is typically administered orally. Multiple cycles of treatment may be administered to a subject. In certain embodiments, the tyrosine kinase inhibitors are administered according to a daily dosing regimen or intermittently. In another embodiment, the tyrosine kinase inhibitors are administered for a period of time sufficient to restore at least partial T cell function, then discontinued.
  • the present invention contemplates that ex vivo expansion of a population of T cells with particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will result in a population T cells that are resistant and/or less prone to T cell exhaustion.
  • the present invention provides compositions comprising a population of T cells that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors).
  • the present invention provides methods of expanding a population of T cells to generate T cell populations that are resistant and/or less prone to T cell exhaustion through expanding such T cells in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib.
  • kits comprising T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) and additional agents (e.g., additional agents useful in expanding T cells) (e.g., additional agents useful in adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy).
  • additional agents e.g., additional agents useful in expanding T cells
  • additional agents useful in adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy.
  • TIL tumor infiltrating lymphocyte
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • T cells configured for transduced T-cell therapy
  • viral specific T cells reengineered with a TCR or CAR include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • the present invention contemplates that ex vivo expansion of a population of genetically engineered T cells (e.g., genetically engineered for use within adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy)) with particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors) will result in genetically engineered T cells that are resistant and/or less prone to T cell exhaustion.
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • tyrosine kinase inhibitors e.g., dasatinib,
  • compositions comprising a population of genetically engineered T cells that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib.
  • the present invention provides methods of expanding a population of genetically engineered T cells to generate genetically engineered T cell populations that are resistant and/or less prone to T cell exhaustion through expanding such T cells in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • kits comprising genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • Such methods are not limited to a specific type or kind of genetically engineered T cells.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • the present invention contemplates that exposure of animals (e.g., humans) undergoing adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) with genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will result in improved therapy outcome as such genetically engineered T cell populations are resistant and/or less prone to T cell exhaustion.
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • the present invention provides methods of treating an immune system related condition or disease (e.g., cancer) in a subject comprising administering a population of genetically engineered T cells expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors).
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • Src family kinase inhibitors e.g., Lck inhibitors
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • T cells configured for transduced T-cell therapy
  • viral specific T cells reengineered with a TCR or CAR include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • Such embodiments are not limited to a particular type or kind of an immune system related condition or disease.
  • the immune system related condition or disease is an autoimmune disease or condition (e.g., Acquired Immunodeficiency Syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-
  • AIDS Acquired
  • the immune system related condition or disease is cancer (e.g., breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid carcinoma).
  • cancer e.g., breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid carcinoma.
  • the present invention contemplates that the use of genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) within adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy) satisfies an unmet need as such therapies are frequently compromised by such T cell populations experiencing T cell exhaustion.
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • adoptive T cell therapies e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy
  • TIL tumor infiltrating lymphocyte
  • Such methods are not limited to a specific type or kind of genetically engineered T cells.
  • the genetically engineered T cells include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • CAR T cells genetically engineered TCR expressing T cells
  • TIL tumor infiltrating lymphocyte
  • T cells configured for transduced T-cell therapy
  • viral specific T cells reengineered with a TCR or CAR include, but are not limited to, CAR T cells, genetically engineered TCR expressing T cells, genetically engineered T cells configured for tumor infiltrating lymphocyte (TIL) therapy, genetically engineered T cells configured for transduced T-cell therapy, and/or viral specific T cells reengineered with a TCR or CAR.
  • TIL tumor infiltrating lymphocyte
  • the embodiments of the present invention are not limited to specific types of tyrosine kinase inhibitors.
  • the tyrosine kinase inhibitors are a Lck tyrosine kinase inhibitors.
  • the tyrosine kinase inhibitor is a Src family kinase inhibitor (e.g., Src kinase inhibitor, Yes kinase inhibitor, Fyn kinase inhibitor, Fgr kinase inhibitor, Lck kinase inhibitor, Hck kinase inhibitor, Blk kinase inhibitor, Lyn kinase inhibitor).
  • the tyrosine kinase inhibitor is dasatinib
  • the tyrosine kinase inhibitor is ponatinib
  • Some embodiments of the present invention provide for administering such methods (e.g., adoptive T cell therapies with genetically engineered T cell populations and compositions comprising particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib)) (e.g., adoptive T cell therapies with genetically engineered T cell populations that were expanded in the presence of particular tyrosine kinase inhibitors (e.g., dasatinib, ponatinib)) in combination with an effective amount of at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g., surgical intervention, and/or radiotherapies).
  • the additional therapeutic agent(s) is an anticancer agent.
  • Tyrosine kinase inhibitors can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients.
  • excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphat
  • a surfactant can be present as an excipient.
  • exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the pharmaceutical composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • the amount of the tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) (e.g., when contained in a drug delivery system) in the pharmaceutical composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial).
  • a therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the pharmaceutical composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • the pharmaceutical compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned. Additional preferred compositions include those for oral, ocular, or localized delivery.
  • compositions herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.
  • pharmaceutical compositions comprising one or more tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) described herein are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre-packaged form.
  • compositions herein may optionally include one or more additional agents, or may be combined with one or more additional agents, such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitor, such as nivolumab), or other medications used to treat a subject for an infection or disease associated with T cell exhaustion (e.g., antiviral, antibiotic, or anti-cancer drugs and therapies, including adoptive T cell therapies).
  • additional agents such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitor, such as nivolumab), or other medications used to treat a subject for an infection or disease associated with T cell exhaustion (e.g., antiviral, antibiotic, or anti-cancer drugs and therapies, including adoptive T cell therapies).
  • Compounded preparations may be used including at least one tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and one or more other agents, such as other drugs for treating T
  • such agents can be contained in a separate composition from the composition comprising a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and co-administered concurrently, before, or after the composition comprising a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib).
  • a tyrosine kinase inhibitor e.g., dasatinib, ponatinib
  • a tyrosine kinase inhibitor e.g., dasatinib, ponatinib
  • At least one therapeutically effective cycle of treatment with a tyrosine kinase inhibitor will be administered to a subject for treatment of T cell exhaustion.
  • a tyrosine kinase inhibitor e.g., a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib)
  • therapeutically effective cycle of treatment is intended a cycle of treatment that when administered, brings about a positive therapeutic response with respect to treatment of an individual for T cell exhaustion.
  • a cycle of treatment with a tyrosine kinase inhibitor e.g., dasatinib, ponatinib
  • restores T cell function when administered transiently as described herein, restores T cell function.
  • a therapeutically effective dose or amount of a tyrosine kinase inhibitor may decrease expression of PD-1, TIM-3, and LAG-3, improve maintenance of memory markers (e.g., CD62L or CCR7), prevent apoptosis, and increase secretion of IL-2 and other cytokines.
  • memory markers e.g., CD62L or CCR7
  • multiple therapeutically effective doses of pharmaceutical compositions comprising one or more tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), and/or one or more other therapeutic agents, such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitor, such as nivolumab), or other medications used to treat a subject for an infection or disease associated with T cell exhaustion (e.g., antiviral, antibiotic, or anti-cancer drugs and therapies, including adoptive T cell therapies) will be administered.
  • the pharmaceutical compositions of the present invention are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, or intramuscularly), by infusion, or locally.
  • Additional modes of administration are also contemplated, such as topical, intralesion, intracerebral, intracerebroventricular, intraparenchymatous, pulmonary, rectal, transdermal, transmucosal, intrathecal, pericardial, intra-arterial, intraocular, intraperitoneal, and so forth.
  • the pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like.
  • the pharmaceutical compositions comprising one or more tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) and other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.
  • compositions comprising one or more tyrosine kinase inhibitors are administered prophylactically, e.g., to prevent T cell exhaustion.
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • other agents are administered prophylactically, e.g., to prevent T cell exhaustion.
  • prophylactic uses will be of particular value for subjects with a chronic infection or cancer, who are at risk of developing T cell exhaustion.
  • the pharmaceutical compositions comprising one or more tyrosine kinase inhibitors are in a sustained-release formulation, or a formulation that is administered using a sustained-release device.
  • tyrosine kinase inhibitors e.g., dasatinib, ponatinib
  • other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device.
  • sustained-release devices include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.
  • the invention also provides a method for administering a conjugate comprising a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) as provided herein to a patient suffering from a condition that is responsive to treatment with a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) contained in the conjugate or composition.
  • the method comprises administering, via any of the herein described modes, a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition.
  • the method of administering may be used to treat any condition that is responsive to treatment with a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib). More specifically, the pharmaceutical compositions herein are effective in treating T cell exhaustion.
  • a tyrosine kinase inhibitor e.g., dasatinib, ponatinib.
  • tyrosine kinase inhibitor e.g., dasatinib, ponatinib
  • the actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered.
  • Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.
  • a therapeutically effective amount will range from about 0.50 mg to 5 grams of a tyrosine kinase inhibitor daily, more preferably from about 5 mg to 2 grams daily, even more preferably from about 7 mg to 1.5 grams daily.
  • such doses are in the range of 10-600 mg four times a day (QID), 200-500 mg QID, 25-600 mg three times a day (TID), 25-50 mg TID, 50-100 mg TID, 50-200 mg TID, 300-600 mg TID, 200-400 mg TID, 200-600 mg TID, 100 to 700 mg twice daily (BID), 100-600 mg BID, 200-500 mg BID, or 200-300 mg BID.
  • the amount of compound administered will depend on the potency of the tyrosine kinase inhibitor and the magnitude or effect desired and the route of administration.
  • a purified tyrosine kinase inhibitor (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents, such as other drugs for treating T cell exhaustion (e.g., anti-PD-1 checkpoint inhibitor, such as nivolumab), or other medications used to treat a subject for an infection or disease associated with T cell exhaustion (e.g., antiviral, antibiotic, or anti-cancer drugs); or adoptive T cell therapies (e.g., a CAR T-cell therapy, a transduced T-cell therapy, and a tumor infiltrating lymphocyte (TIL) therapy); or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • other drugs for treating T cell exhaustion e.g., anti-PD-1 checkpoint inhibitor, such as nivolumab
  • dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof.
  • Preferred compositions are those requiring dosing no more than once a day.
  • a tyrosine kinase inhibitor can be administered prior to, concurrent with, or subsequent to other agents or therapies. If provided at the same time as other agents or therapies, one or tyrosine kinase inhibitors can be provided in the same or in a different composition. Thus, one or more tyrosine kinase inhibitors and other agents can be presented to the individual by way of concurrent therapy.
  • concurrent therapy is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy.
  • concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising a tyrosine kinase inhibitor and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating T cell exhaustion, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen.
  • a pharmaceutical composition comprising a tyrosine kinase inhibitor and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating T cell exhaustion, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen.
  • one or more tyrosine kinase inhibitors and one or more other therapeutic agents can be administered in at least one therapeutic dose.
  • Administration of the separate pharmaceutical compositions or therapies can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.
  • kits comprising one or more containers holding compositions comprising at least one tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and optionally one or more other agents for treating T cell exhaustion.
  • Compositions can be in liquid form or can be lyophilized.
  • Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
  • Containers can be formed from a variety of materials, including glass or plastic.
  • a container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the delivery device may be pre-filled with the compositions.
  • the kit can also comprise a package insert containing written instructions for methods of using the compositions comprising at least one tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) for treating a subject for T cell exhaustion.
  • the package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
  • Example I A Method of Preventing or Reversing T Cell Exhaustion by Inhibiting or Modulating TCR Signaling
  • CAR T cells were washed, resuspended in media containing S1, and mixed at a 1:1 ratio with Nalm6 leukemic cells stably expressing surface GD2. Culture supernatants were harvested approximately 24 hours later and cytokine levels were evaluated via ELISA. Similar to GD2.28z CAR that lacks a destabilization domain and therefore have persistent high levels of CAR signaling, cells expressing the GD2.28z.FKBP CAR that experienced continuous drug treatment ( FIG.
  • nivolumab (Nivo). CART cells were either treated with continuous S1 (and thus exhibit continuous tonic signaling), continuous S1+nivolumab, or no S1 until the time of the co-culture assay. Interestingly, nivolumab treatment resulted in only modest augmentation of IL-2 secretion at day 10, which was sustained until day 14, suggesting that nivolumab only partially prevented the onset of T cell exhaustion in this system ( FIG. 5 ).
  • HA-GD2.28z.FKBP CAR T cells that had experienced continuous S1 treatment were sorted in order to isolate a pure PD-1/TIM-3/LAG-3 exhausted population. “Triple positive” exhausted cells were then re-cultured either with or without S1 to test whether removal of tonic signaling could restore their function. FACS and co-culture assays were conducted 4 days later.
  • kinases in the TCR signaling pathway that are also integral to CAR signaling.
  • Lck acts to phosphorylate CD3 zeta in response to TCR or CAR ligation.
  • Dasatinib a potent receptor tyrosine kinase inhibitor and BCR/ABL antagonist, has also been shown to inhibit T cell activation, proliferation, and cytokine secretion by binding to and inhibiting Lck at low concentrations (Schade et. al, Blood, 2008 and Lee et. al, Leukemia, 2010).
  • dasatinib potently inhibits CD19.28z CART cell cytokine secretion in response to tumor antigen on day 14 post-activation ( FIG. 7 ), proving that dasatinib disrupts CAR signaling.
  • dasatinib treatment resulted in preservation of T cell memory via maintenance of CD62L expression in a dose-dependent manner ( FIG. 9 .).
  • dasatinib treatment reinvigorated exhausted T cells in a functionally significant manner, as dasatinib-treated CAR T cells secreted more IL-2 (and to a lesser extent, IFN ⁇ ) in response to tumor antigen compared to those that never received dasatinib ( FIG. 10 ).
  • Chimeric antigen receptors are synthetic receptors that combine an extracellular tumor-targeting domain with intracellular domains that mimic endogenous TCR signaling (e.g., 1-2 costimulatory domains, like CD28 or 4-1BB, and a CD3 zeta domain) (see, e.g., Lim & June. Cell 168, 724-740 (2017)).
  • CAR T cells When CAR-expressing T cells encounter antigen-expressing tumor cells, CAR T cells form an immune synapse and initiate downstream signaling through the CAR, resulting in potent T cell activation, degranulation of cytotoxic soluble factors, cytokine release, and proliferation.
  • CAR T cell therapy has experienced unprecedented clinical success in many patients with hematological malignancies, there are several key challenges that must be addressed before this therapy can be expanded to other tumor types or offered as first-line therapy.
  • CAR toxicity typically manifests in the form of cytokine release syndrome (CRS) or on-target off-tumor activity, both of which have been observed in clinical trials and, in some instances, resulted in patient death (see, e.g., Gust et al. Cancer discovery (2017). doi:10.1158/2159-8290.cd-17-0698; Xu & Tang. Cancer Letters 343, 172-178 (2014); D'Aloia, et al. Cell Death & Disease 9, 282 (2018)).
  • CRS cytokine release syndrome
  • a second key challenge to improving the efficacy of CAR T cell therapy is the prevention of CAR T cell exhaustion.
  • T cell exhaustion results from continuous antigen exposure in the context of chronic viral infection or cancer and is characterized by a hierarchical loss of effector function, sustained co-expression of multiple inhibitory receptors (ex., PD-1, TIM-3, LAG-3), attenuated proliferative capacity, and increase apoptosis (see, e.g., Wherry & Kurachi. Nature Reviews Immunology 15, nri3862 (2015)).
  • PD-1 PD-1, TIM-3, LAG-3
  • LAG-3 multiple inhibitory receptors
  • There is strong evidence for T cell exhaustion in CART cell therapy Nearly all CD19.28z CART cells administered disappear by day 60 post-infusion (see, e.g., Lee et al.
  • CD19.BBz CART cells which are thought to be more resistant to T cell exhaustion, also exhibit features of exhaustion and are undetectable in approximately 30% of patients who receive this therapy, consequently increasing the risk of CD19 positive relapse (see, e.g., Turtle et al. Journal of Clinical Investigation 126, 2123-2138 (2016); Maude et al. The New England Journal of Medicine 371, 1507-1517 (2014)).
  • T cells expressing the CD19-targeting CAR were incubated with various concentrations of dasatinib and ponatinib for at least 24 hours.
  • CAR T cells were next co-cultured with antigen-bearing tumor cells for 6 hours in the presence or absence of dasatinib/ponatinib, and subsequently assessed CAR T cell activation and degranulation via CD69 and CD107a co-expression. Nearly 80% of control CART cells were CD69+/CD107a+ upon co-culture with tumor ( FIG. 11 a ).
  • Control HA-GD2.28z CART cells exhibited robust single marker expression and co-expression of multiple canonical exhaustion markers ( FIG. 15 ). Conversely, expansion in dasatinib reduced both the frequency of exhaustion-marker co-expressing cells as well as the extent to which these exhaustion markers were expressed in a dose-dependent manner ( FIG. 15 ).
  • CART cell expansion in the presence of dasatinib also augmented T cell memory formation, as a nearly 6-fold increase in the frequency of central-memory-like T cells (CD45RA low, CCR7 high) and a greater-than 2-fold reduction in the frequency of effector-memory-like T cells (CD45RA low, CCR7 low) was observed compared to exhausted CAR T cells cultured in the absence of dasatinib.
  • Tonically signaling CAR T cells cultured in the absence of dasatinib or ponatinib secreted low levels of cytokine in response to tumor ( FIG. 17 ) and exhibited impaired cytotoxicity ( FIG. 18 ), indicating that these cells were functionally exhausted.
  • expansion of CAR T cells in the presence of dasatinib or ponatinib dose-dependently augmented CAR T cell cytokine secretion ( FIG. 17 ) and also allowed for a more potent cytotoxic response ( FIG. 18 ), confirming that the mitigation of tonic signaling during CAR T cell expansion with these drugs confers profound functional benefits.
  • CAR T cells were expanded with or without 1 uM dasatinib and subsequently infused into NSG mice engrafted with antigen-bearing tumor.
  • both GD2.BBz and HA-GD2.28z CAR T cells grown in the absence of dasatinib failed to control tumor growth ( FIGS. 19 and 20 , respectively).
  • CAR T cells expanded in dasatinib allowed for a near complete and lasting eradication of the tumor ( FIGS. 19 and 20 ).
  • the same effect was observed in a GD2-overexpressing NALM6 leukemia model in which the tumor burden was more established at the time of CART cell infusion ( FIG. 21 ).
  • CAR T cells were infused into mice that had been engrafted with antigen-bearing tumor.
  • blood samples were taken from the mice and the number of circulating CAR T cells was assessed via FACS counting beads.
  • Tonically signaling HA-GD2.28z CAR T cells expanded in the absence of dasatinib did not expand and/or persist at levels greater than mice infused with mock untransduced T cells ( FIG. 22 ).
  • CAR T cells were infused into mice that were engrafted with antigen-bearing tumor and subsequently dosed with dasatinib for 3 consecutive days. The mice were then sacrificed and CAR T cell frequency and phenotype assessed in both the blood and the spleen.
  • the dasatinib-treated mouse exhibited a higher frequency of CAR T cells in both tissues ( FIG. 22 a, c ) compared to the vehicle-treated mouse, indicating that in vivo dasatinib treatment induced in situ proliferation or persistence.
  • CAR T cells that were recovered from the dasatinib-treated mouse exhibited reduced expression of exhaustion markers PD-1 and LAG-3, reduced expression of CD69 (i.e. lower activation state), and higher expression of the memory marker CD62L ( FIG. 22 b, d ) compared to the vehicle-treated mouse, all of which are consistent with the phenotypic changes observed upon in vitro treatment with dasatinib ( FIG. 15, 16 ).
  • dasatinib mitigates CAR T cell exhaustion phenotype and improves memory formation, and indicates that in vivo dasatinib dosing provides a functional benefit in vivo.
  • CAR T cells were infused into mice that exhibited high tumor burden.
  • the mice were dosed with dasatinib or vehicle for 3 consecutive days, then were not treated for a period of 3 additional days.
  • tumor burden was assessed and a profound decrease in tumor size in the dasatinib-treated mouse was observed ( FIG. 22 e ).
  • the tumor burden in vehicle-treated mouse continued to increase, indicating that the augmentation in anti-tumor response was specific to dasatinib treatment.
  • dasatinib and ponatinib have the potential to address several important challenges currently facing the field of adoptive T cell therapies (e.g., CART cell therapies).
  • CART cell therapies e.g., CART cell therapies.
  • these drugs were shown potently inhibit CAR signaling, which provides a method to regulate CAR activity and thus mitigate CAR T cell toxicity while preserving the option to continue therapy once the toxicity has resolved, as the inhibitory effect of dasatinib and ponatinib on CAR T cell function is reversible.
  • Second, expansion of CAR T cells in the presence of dasatinib or ponatinib was shown to prevent CAR tonic signaling and in turn enhance the functional capacity of CAR T cells.
  • providing short periods of CAR T cell “rest” in vivo via iterative drug dosing was shown to be one method by which CAR T cell exhaustion could be prevented or reversed and/or memory could be induced.
  • Example II describes the materials and methods for Example II.
  • NALM6-GL acute lymphoblastic leukemia line, stably transfected with GFP and luciferase
  • NALM6-GL-GD2 stably transfected to overexpress GD2 synthetase cell lines
  • RPMI-1640 293T and 143B cell lines were cultured in DMEM (Life Technologies).
  • T cells Primary human T cells were obtained from healthy donor buffy coats using a Pan T cell negative selection kit (Miltenyi Biotec). Donor T cells were then aliquoted and stored in Cryostor (StemCell Technologies) in liquid nitrogen. T cells were cultured in AimV (Gibco, Life Technologies) supplemented with 5% heat-inactivated FBS, 10 mM HEPES, 1% glutamax (Gibco, Life Technologies), and 100 u/uL recombinant human IL-2 (Peprotech). Dasatinib (Sigma Aldrich and Adooq Biosciences) or ponatinib (SelleckChem) were cultured at 1 uM unless otherwise specified.
  • T cells Upon thawing, T cells were activated at a 3:1 bead:cell ratio using anti-CD3/anti-CD28-coated magnetic beads (Dynabeads, Thermo Fisher) at a concentration of 1 ⁇ 10 6 cells/mL.
  • T cells On days 2 and 3 post-activation, T cells were transduced with retrovirus encoding the CAR. Briefly, retrovirus was first spun onto retronectin-coated plates at 3000 rpm for 2 hours, after which T cells were transferred to the plates. On day 4 post-activation, magnetic beads were removed from culture, and T cells were cultured at 0.5 ⁇ 10 6 cells/mL every day thereafter. Media supplemented with IL-2 and drug was changed every two days. Transduction efficiencies were routinely 70-90% for all CARs.
  • T cell phenotype was evaluated via: CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), PD-1 (eBioJ105, eBioscience), TIM-3 (F38-2E2, Biolegend), LAG-3 (3DS223H, eBioscience), CD45RA (L48, BD Biosciences), CCR7 (150503, BD Biosciences), CD62L (DREG-56, BD Biosciences), CD69 (FN50, Biolegend), and CD107a (H4A3, eBioscience).
  • CD107a For co-culture assays in which CD107a was assessed, tumor cells and CAR T cells were co-cultured in the presence of 1:1000 monensin (eBioscience) and anti-CD107a for at least 6 hours. All FACS plots displaying CART cell phenotype data were pre-gated on CAR+ cells.
  • whole T cell populations were used for analysis.
  • 50,000 NALM6-GL-GD2 tumor cells were co-cultured with T cells at a 1:1 E:T ratio in 200 uL of complete AimV medium without IL-2 supplementation in each well of a 96-well plate. After 24 hours, supernatants were removed and stored at ⁇ 20 C. IL-2 and IFN ⁇ secretion was assessed via ELISA (Biolegend).
  • CAR′ cells were removed from culture, pelleted, and resuspended in 100 uL of RITA lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with phosphatase and protease inhibitors (Thermo Fisher), After incubating for 30 minutes at 4 C, supernatants were cleared by centrifugation at 14,000 RPM for 20 minutes at 4 C. Protein concentration in the cleared lysates was measured by a colorimetric reaction (BioRad).
  • RITA lysis buffer 10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl
  • phosphatase and protease inhibitors Thermo Fisher
  • CD3-zeta Cell signaling
  • pY142-CD3-zeta Cell Signaling
  • p44/42 MAPK Erk1/2, Cell Signaling
  • p-p44/42 MAPK p-ERK1/2, Cell Signaling
  • pSer473-Akt D9E, Cell Signaling
  • pan Akt 40D4, Cell Signaling
  • LI-COR The Odyssey (LI-COR) imaging system. LI-COR buffers, and LI-COR secondary antibodies (Goat Anti-Mouse IgG Antibody-800CW-Conjugated and Goat Anti-Rabbit IgG Antibody-680LT-Conjugated) were used for protein detection.
  • CAR T cells were incubated in 5 ug/mL anti-idiotype (clone 1A7) plus 5 ug/mL goat anti-mouse Fab secondary (Jackson Immunoresearch) or secondary alone for 5 minutes at 37 C. Cells were then quenched in ice cold PBS, pelleted at 4 C for 5 minutes, then lysed for western blot analysis.
  • mice were engrafted with 1 ⁇ 10 6 NALM6-GL-GD2 leukemia cells via intravenous injection. At day 4 post-engraftment, 2 ⁇ 10 6 HA-GD2.28z CAR+ T cells were infused intravenously.
  • NALM6-GL-GD2 tumor burden was evaluated using the Xenogen IVIS Lumina (Caliper Life Sciences). Mice were first injected intraperitoneally with 3 mg D-luciferin (Caliper Life Sciences) and then imaged 4 minutes later with an exposure time of 30 seconds, or, in cases where 30 seconds resulted in signal saturation, “auto” exposure was selected. Luminescence images were analyzed using Living Image software (Caliper Life Sciences).
  • mice 6-8 week old NSG mice were engrafted with 0.5 ⁇ 10 6 143 B osteosarcoma cells intramuscularly. On day 3 post-engraftment, 10 ⁇ 10 6 GD2.BBz or HA-GD2.28z were infused intravenously. Osteosarcoma burden was quantified via two-dimensional leg area measurements.
  • mice treated with dasatinib (Adooq Biosciences) were injected intraperitoneally at a concentration of 50 mg/kg in water+10% Kolliphor HS 15 (Sigma Aldrich). Mice treated with vehicle were injected with an equivalent volume of water+10% Kolliphor HS 15.
  • Each CAR includes a signal peptide, single chain variable fragment (scFv), extracellular hinge region, transmembrane domain, intracellular co-stimulatory domain, and intracellular CD3 zeta domain.
  • scFv single chain variable fragment
  • the nucleic acid and amino acid sequence for CD19.28z (FMC63 scFv) is provided at FIG. 23 .
  • CD19.BBz FMC63 scFv
  • FIG. 24 The nucleic acid and amino acid sequence for CD19.BBz (FMC63 scFv) is provided at FIG. 24 .
  • the nucleic acid and amino acid sequence for GD2.BBz (14G2a scFv) is provided at FIG. 25 .
  • the nucleic acid and amino acid sequence for HA-GD2.28z (High affinity 14G2a scFv) is provided at FIG. 26 .

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