阅读说明：本技术 通过抑制或调节t细胞受体信号传导来治疗t细胞耗竭的方法 (Methods of treating T cell depletion by inhibiting or modulating T cell receptor signaling ) 是由 瑞秋·琳恩 克里斯托·麦考尔 埃文·温伯 桑杰·马尔霍特拉 于 2018-03-30 设计创作，主要内容包括：本文提供了用于预防或逆转T细胞耗竭的组合物和方法。特别地,本发明涉及通过使经历T细胞耗竭的T细胞暴露于特定酪氨酸激酶抑制剂(例如达沙替尼、普纳替尼)或通过在特定酪氨酸激酶抑制剂(例如达沙替尼、普纳替尼)存在下扩增遗传工程化的T细胞来预防或逆转T细胞耗竭的方法。(Provided herein are compositions and methods for preventing or reversing T cell depletion. In particular, the present invention relates to methods of preventing or reversing T cell depletion by exposing T cells undergoing T cell depletion to specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) or by expanding genetically engineered T cells in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).)

1. A method for preventing and/or reversing T cell depletion in a subject, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor.

2. The method of claim 1, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

3. The method of claim 1, wherein the tyrosine kinase inhibitor is an Lck inhibitor.

4. The method of claim 1, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

5. The method of claim 1, wherein treatment increases IL-2 secretion by T cells in the subject.

6. The method of claim 1, wherein treatment reduces apoptosis of T cells in the subject.

7. The method of claim 1, wherein treatment reduces expression of at least one T cell depletion marker selected from the group consisting of PD-1, TIM-3, and LAG-3.

8. The method of claim 1, wherein treatment increases expression of CD62L or CCR 7.

9. The method of claim 1, wherein the subject is administered multiple cycles of treatment.

10. The method of claim 7, wherein the tyrosine kinase inhibitor is administered intermittently.

11. The method of claim 1, wherein the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least a portion of T cell function and then discontinued.

12. The method of claim 1, wherein the tyrosine kinase inhibitor is administered orally.

13. The method of claim 1, wherein the subject is a human.

14. The method of claim 1, wherein the subject has a chronic infection or cancer.

15. The method of claim 1, wherein treatment is prophylactic.

16. A method for treating an immune system related disorder or disease in a subject, the method comprising administering to the subject genetically engineered T cells and a therapeutically effective amount of a tyrosine kinase inhibitor.

17. The method of claim 16, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

18. The method of claim 16, wherein the tyrosine kinase inhibitor is an Lck inhibitor.

19. The method of claim 16, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

20. The method of claim 16, wherein the tyrosine kinase inhibitor and the genetically engineered T cell are administered simultaneously and/or at different time points.

21. The method of claim 16, wherein the immune system related disorder or disease is selected from cancer or an autoimmune disease or disorder.

22. The method of claim 16, wherein the genetically engineered T cell is selected from a CAR T cell, a genetically engineered TCR-expressing T cell, a genetically engineered T cell configured for Tumor Infiltrating Lymphocyte (TIL) therapy, a genetically engineered T cell configured for transduction T cell therapy, and/or a virus-specific T cell re-engineered with a TCR or CAR.

23. the method of claim 16, further comprising administering to the subject one or more anti-cancer agents.

24. The method of claim 23, wherein the one or more anti-cancer agents are selected from chemotherapeutic agents and radiation therapy.

25. A composition comprising a population of genetically engineered T cells, wherein the population of genetically engineered T cells is expanded in the presence of a tyrosine kinase inhibitor.

26. The composition of claim 25, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

27. The composition of claim 25, wherein the tyrosine kinase inhibitor is an Lck inhibitor.

28. The composition of claim 25, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

29. The composition of claim 25, wherein the population of genetically engineered T cells is selected from a population of CAR T cells, a population of genetically engineered TCR-expressing T cells, a population of genetically engineered T cells configured for tumor-infiltrating lymphocyte (TIL) therapy, a population of genetically engineered T cells configured for transduction T cell therapy, and/or a population of virus-specific T cells re-engineered with a TCR or a CAR.

30. A method of generating a population of genetically engineered T cells that are resistant to T cell depletion, the method comprising expanding a population of genetically engineered T cells in the presence of a tyrosine kinase inhibitor.

31. The method of claim 30, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

32. The method of claim 30, wherein the tyrosine kinase inhibitor is an Lck inhibitor.

33. The method of claim 30, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

34. The method of claim 30, wherein the population of genetically engineered T cells is selected from a population of CAR T cells, a population of genetically engineered TCR-expressing T cells, a population of genetically engineered T cells configured for tumor-infiltrating lymphocyte (TIL) therapy, a population of genetically engineered T cells configured for transduction T cell therapy, and/or a population of virus-specific T cells re-engineered with TCRs or CARs.

35. A method of treating an immune system related disorder or disease comprising administering to a subject a population of genetically engineered T cells expanded in the presence of a tyrosine kinase inhibitor.

36. The method of claim 35, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

37. The method of claim 35, wherein the tyrosine kinase inhibitor is an Lck inhibitor.

38. The method of claim 35, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

39. The method of claim 35, wherein the population of genetically engineered T cells is selected from a population of CAR T cells, a population of genetically engineered TCR-expressing T cells, a population of genetically engineered T cells configured for tumor-infiltrating lymphocyte (TIL) therapy, a population of genetically engineered T cells configured for transduction T cell therapy, and/or a population of virus-specific T cells re-engineered with TCRs or CARs.

40. The method of claim 35, wherein the subject is undergoing adoptive T cell therapy.

41. The method of claim 40, wherein the adoptive T cell therapy is CAR T cell therapy.

42. The method of claim 40, wherein the adoptive T cell therapy is a transduced T cell therapy.

43. The method of claim 40, wherein the adoptive T cell therapy is Tumor Infiltrating Lymphocyte (TIL) therapy.

44. The method of claim 35, wherein the immune system related disorder or disease is selected from cancer or an autoimmune disease or disorder.

45. The method of claim 35, further comprising administering to the subject one or more anti-cancer agents.

46. The method of claim 45, wherein the one or more anti-cancer agents are selected from chemotherapeutic agents and radiation therapy.

47. A method for preventing and/or reversing toxicity associated with genetically engineered T cells administered to a subject, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor.

48. The method of claim 47, wherein the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling.

49. The method of claim 47, wherein the tyrosine kinase inhibitor is an Lck kinase inhibitor.

50. The method of claim 47, wherein the tyrosine kinase inhibitor is dasatinib or ponatinib.

51. The method of claim 33, wherein the genetically engineered T cell is selected from a CAR T cell, a genetically engineered TCR-expressing T cell, a genetically engineered T cell configured for Tumor Infiltrating Lymphocyte (TIL) therapy, a genetically engineered T cell configured for transduction T cell therapy, and/or a virus-specific T cell re-engineered with a TCR or CAR.

52. The method of claim 47, wherein the subject is undergoing adoptive T cell therapy.

53. The method of claim 52, wherein the adoptive T cell therapy is CAR T cell therapy.

54. The method of claim 52, wherein the adoptive T cell therapy is a transduced T cell therapy.

55. The method of claim 52, wherein the adoptive T cell therapy is Tumor Infiltrating Lymphocyte (TIL) therapy.

56. The method of claim 47, wherein the toxicity associated with genetically engineered T cells administered to a subject is cytokine release syndrome.

57. The method of claim 47, wherein the toxicity associated with genetically engineered T cells administered to a subject is off-target or off-target off-tumor toxicity.

Technical Field

Provided herein are compositions and methods for preventing or reversing T cell depletion. In particular, the present invention relates to methods of preventing or reversing T cell depletion by exposing T cells undergoing T cell depletion to specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) or by expanding genetically engineered T cells in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).

Background

T cells are immune cells that become activated via T Cell Receptor (TCR) signaling upon engagement with an antigen. Physiological activation through T cell receptors enables T cells to mediate potent antitumor or anti-infectious 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 infection or cancer, T cells rarely undergo pathological differentiation towards a dysfunctional state, which has been referred to as T cell depletion. T cell depletion is characterized by significant changes in metabolic function, transcriptional programming, loss of effector function (e.g., cytokine secretion, killing ability), and co-expression of multiple surface inhibitory receptors. The underlying cause of T cell depletion is sustained antigen exposure, resulting in sustained TCR signaling. Prevention or reversal of T cell depletion has long been sought as a means of enhancing T cell effectiveness in cancer or chronically infected patients.

The present invention addresses this urgent need.

Disclosure of Invention

Immune cells have a wide range of responses to the presence of foreign antigens, including secretion of preformed and newly formed mediators, phagocytosis of particles, endocytosis, cytotoxicity against target cells, and cell proliferation and/or differentiation. T cells are a subset of cells that, together with other immune cell types (e.g., polymorphonuclear, eosinophilic, basophilic, mast, B, and NK cells), constitute the cellular component of the immune system (see, e.g., U.S. patent No. 6,057,294; U.S. patent application 20050070478). Under physiological conditions, T cells play a role in immune surveillance and elimination of foreign antigens. However, in pathological conditions, there is compelling evidence that T cells play a major role in the etiology and spread of the disease. In these disorders, the breakdown of central or peripheral T cell immune tolerance is a fundamental process leading to autoimmune diseases.

It is well known that T Cell Receptor (TCR) engagement and costimulatory signaling provide key signals for regulating T cell activation, proliferation and cytolytic function. T cells react to antigen via a polypeptide complex consisting of ligand-bound T Cell Receptor (TCR) disulfide-linked alpha and beta subunits (or gamma and delta subunits in gamma delta T cells), each subunit having a single Transmembrane (TM) span and a small intracellular tail, and non-covalently bound to heterodimeric (CD3 gamma epsilon and CD3 delta epsilon) and homodimeric (zeta) signaling subunits (see, e.g., Cambier J.C. curr Opin Immunol 1992; 4: 257-64). The CD3 epsilon, delta, and gamma chains have a single Ig family extracellular domain, a single approximate alpha-helix TM span, and an inherently disordered intracellular domain of 40-60 residues, while each zeta subunit has a small extracellular region (9 residues) carrying an intersubunit disulfide bond, a single approximate alpha-helix TM span per subunit, and a large inherently disordered cytoplasmic domain of about 110 residues. Therefore, understanding the processes of TCR-mediated TM signaling and subsequent T cell activation (leading to T cell proliferation and differentiation) is crucial for both health and disease. Disorders of TCR signaling can lead to inflammation and other T cell-related disorders.

T cells expressing Chimeric Antigen Receptors (CARs) at high levels experience tonic, antigen-independent signaling due to receptor clustering. Such T cells do not function well due to T cell depletion, as evidenced by high levels of PD-1, TIM-3, LAG-3, reduced antigen-induced cytokine production, and excessive programmed cell death. Tonic signaling can be prevented by transiently lowering a CAR-associated TCR signaling protein (e.g., TCR ζ) to a level below a threshold required for tonic signaling.

Experiments conducted during the course of developing embodiments of the present invention show that treatment with specific tyrosine kinase inhibitors that inhibit T cell receptor signaling (e.g., Lck tyrosine kinase inhibitors (e.g., dasatinib)) (e.g., Src family tyrosine kinase inhibitors) reduces expression of T cell depletion markers and improves the development of T cell memory. Accordingly, the present invention relates to methods of preventing or reversing T cell depletion by transiently inhibiting T Cell Receptor (TCR) signaling to restore T cell function with specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).

Additional experiments determined that CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib exhibited reduced activation and degranulation, were unable to secrete cytokines, and demonstrated reduced killing in response to tumor antigens.

Additional experiments determined that dasatinib was effective in inhibiting phosphorylation of CAR CD3z and remote signaling proteins after CAR cross-linking.

Additional experiments determined that tonic signaling CAR T cells expanded in the presence of dasatinib exhibited a reduction in expression of classical depletion markers, retained the ability to form memory, exhibited enhanced cytokine secretion in response to tumor antigens, and exhibited enhanced cytotoxicity in a dose-dependent manner.

Additional experiments established that dasatinib treatment in vivo suppresses expression of the depletion marker, enhances memory formation and promotes cell survival/proliferation.

Accordingly, provided herein are compositions and methods for preventing or reversing T cell depletion. In particular, the present invention relates to methods of preventing or reversing T cell depletion by exposing T cells undergoing T cell depletion to specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) or by expanding genetically engineered T cells in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).

In certain embodiments, the present invention provides a method for treating a subject to reduce T cell depletion, the method comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor. Such embodiments are not limited to a particular tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling. In some embodiments, the tyrosine kinase inhibitor is an Lck kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is a Fyn inhibitor. In some embodiments, 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.

Such methods are not limited to a particular manner of treating T cell depletion in the subject. In some embodiments, the treatment increases secretion of IL-2 by T cells in the subject. In some embodiments, the treatment reduces apoptosis of T cells in the subject. In some embodiments, the treatment decreases expression of at least one T cell depletion marker selected from the group consisting of PD-1, TIM-3, and LAG-3. In some embodiments, the treatment increases expression of CD62L or CCR 7.

Such methods are not limited to a particular mode of administration. In some embodiments, the subject is administered multiple cycles of treatment. In some embodiments, the tyrosine kinase inhibitor is administered intermittently. In some embodiments, the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least a portion of T cell function, and then discontinued. In some embodiments, the tyrosine kinase inhibitor is administered orally.

Such methods are not limited to a particular type or class of subject. In some embodiments, the subject is a human. In some embodiments, the subject has a chronic infection or cancer.

In certain embodiments, the invention provides methods for treating an immune system related disorder or disease in a subject, comprising administering to the subject genetically engineered T cells and a therapeutically effective amount of a tyrosine kinase inhibitor. Such embodiments are not limited to a particular tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling. In some embodiments, the tyrosine kinase inhibitor is an Lck kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is a Fyn inhibitor. In some embodiments, 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 cell are administered simultaneously and/or at different time points.

Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Such methods are not limited to the treatment of particular immune system related disorders or diseases. In some embodiments, the immune system related disorder or disease is selected from cancer or an autoimmune disease or disorder.

In certain embodiments, the present invention provides methods for preventing and/or reversing toxicity associated with genetically engineered T cells administered to a subject, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor. Such embodiments are not limited to a particular tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling. In some embodiments, the tyrosine kinase inhibitor is an Lck kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is a Fyn inhibitor. In some embodiments, the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib.

Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Such methods are not limited to a particular type or kind of adoptive T cell therapy. In some embodiments, the adoptive T cell therapy is CAR T cell therapy. In some embodiments, the adoptive T cell therapy is a transduction T cell therapy. In some embodiments, the adoptive T cell therapy is Tumor Infiltrating Lymphocyte (TIL) therapy.

Such methods are not limited to a particular type or kind of toxicity associated with genetically engineered T cells administered to a subject. In some embodiments, the toxicity associated with genetically engineered T cells administered to a subject is cytokine release syndrome. In some embodiments, the toxicity associated with genetically engineered T cells administered to a subject is an on-target off-tumor (on-target off-tumor) toxicity or an off-target off-tumor (off-target off-tumor) toxicity.

In certain embodiments, the present invention provides compositions comprising a population of genetically engineered T cells, wherein the population of genetically engineered T cells is expanded in the presence of a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling inhibitors. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib.

In certain embodiments, the invention provides a method of producing a genetically engineered T cell population that is resistant to T cell depletion, the method comprising expanding the genetically engineered T cell population in the presence of a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting TCR signaling and/or CAR signaling inhibitors. In some embodiments, the tyrosine kinase inhibitor is dasatinib or ponatinib. Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs. Such methods are not limited to particular amplification techniques, as such techniques are well known in the art.

In certain embodiments, the invention provides a method of treating an immune system-related disorder or disease in a subject undergoing adoptive T cell therapy, the method comprising administering to the subject a population of genetically engineered T cells expanded in the presence of a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is capable of inhibiting a TCR signaling inhibitor and/or CAR signaling. In some embodiments, the tyrosine kinase inhibitor is an Lck kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is a Fyn inhibitor. In some embodiments, 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 disorder or disease is selected from cancer or an autoimmune disease or disorder.

Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Such methods are not limited to a particular type or kind of adoptive T cell therapy. In some embodiments, the adoptive T cell therapy is CAR T cell therapy. In some embodiments, the adoptive T cell therapy is a transduction T cell therapy. In some embodiments, the adoptive T cell therapy is Tumor Infiltrating Lymphocyte (TIL) therapy.

The present invention contemplates that exposing an animal (e.g., a human) having cancer (e.g., and/or a cancer-related disorder) to an adoptive T cell therapy (e.g., CAR T cell therapy, transduction T cell therapy, and tumor-infiltrating lymphocyte (TIL) therapy) using a genetically engineered population of T cells and a composition comprising a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) will completely inhibit the growth of cancer cells or supporting cells and/or render such cells as a population more susceptible to cell death-inducing activity of a cancer treatment drug or radiation therapy. In such embodiments, the methods result in improved therapeutic outcomes because such specific tyrosine kinase inhibitors are capable of 1) modulating TCR signaling (e.g., reducing expression of one or more of PD-1, TIM-3, and LAG-3; increasing the expression of a memory marker (e.g., CD62L or CCR 7); increase secretion of IL-2 and other cytokines), and 2) prevent and/or reverse T cell depletion within the genetically engineered T cell population. Accordingly, the present invention provides methods for treating cancer (e.g., and/or cancer-related disorders) in a subject using adoptive T cell therapies (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy), comprising administering to the subject (e.g., simultaneously and/or at different time points) genetically engineered T cells, specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), and additional cancer treatment drugs or radiation therapies.

The present invention contemplates that exposing an animal (e.g., a human) having a cancer (e.g., and/or a cancer-related disorder) to an adoptive T cell therapy (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) using a population of genetically engineered T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) will completely inhibit the growth of the cancer cells or supporting cells and/or render such cells as the population more susceptible to cell death-inducing activity of a cancer treatment drug or radiation therapy. In such embodiments, the methods result in improved therapeutic outcomes because such genetically engineered T cell populations are resistant to and/or less prone to T cell depletion. Accordingly, the present invention provides methods for treating cancer (e.g., and/or cancer-related disorders) in a subject using adoptive T cell therapies (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) comprising administering to the subject (e.g., simultaneously and/or at different time points) a genetically engineered population of T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and an additional cancer treatment drug or radiation therapy.

The present invention contemplates that such methods (e.g., adoptive T cell therapy using genetically engineered T cell populations and compositions comprising specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib)), e.g., adoptive T cell therapy using genetically engineered T cell populations expanded in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib), satisfy an unmet need for treating multiple cancer types when administered as monotherapy or when administered in a temporal relationship (combination therapy) with additional agents, such as other cancer treatment drugs or radiation therapies that induce cell death or disrupt the cell cycle, thereby making a greater proportion of cancer cells or supporting cells more susceptible to performing an apoptotic program than a corresponding proportion of cells in animals treated with only the cancer treatment drug or radiation therapy alone.

In certain embodiments of the invention, treatment of animals with such methods (e.g., adoptive T cell therapy using a genetically engineered T cell population and a composition comprising a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib)) in combination (e.g., adoptive T cell therapy using a genetically engineered T cell population expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) produces greater tumor responses and clinical benefits in such animals than those treated with the anticancer drug/radiation alone.

A non-limiting, exemplary list of cancers (e.g., and/or cancer-related conditions) includes, but is not limited to, pancreatic cancer (pancreatic cancer), breast cancer (breast cancer), prostate cancer (prostate cancer), lymphoma, skin cancer, colon cancer (colon cancer), melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer (bladder cancer), non-small cell lung cancer, head or neck cancer, breast cancer (breast cancer), ovarian cancer, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer (bladder cancer), pancreatic cancer (pancreatic cancer), gastric cancer, colon cancer (colon cancer), prostate cancer (prostate cancer), reproductive system cancer, urinary cancer, esophageal cancer, myeloma, multiple myeloma, and cervical cancer, Renal cell carcinoma, endometrial carcinoma, adrenocortical carcinoma, malignant pancreatic islet tumor, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, retinoblastoma, and other T and B cell mediated autoimmune diseases; inflammatory diseases; (ii) infection; hyperproliferative diseases; AIDS; degenerative diseases, vascular diseases, and the like. In some embodiments, the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are resistant to an anticancer agent.

Drawings

FIG. 1: characterization of fkbp CAR gd2.28z. T cells were transduced with lentiviruses encoding gd2.28z. fkbp CARs on day 1 post-activation and subsequently cultured with various concentrations of shield-1 in growth medium. On day 7, CAR expression was quantified via FACS.

FIG. 2: removal of S1 from the medium resulted in reversal of surface expression of the T cell depletion marker.

FIG. 3: removal of S1 from the medium allowed for maintenance of CD62L expression and prevention of apoptosis.

FIG. 4: removal of S1 from the medium resulted in reversal of functional T cell depletion.

FIG. 5: removal of surface CAR makes T cell depletion more effective to prevent than PD-1/PDL-1 blocking.

FIG. 6: after only 4 days, removal of the surface CAR rescued the depletion of PD-1/TIM-3/LAG-3 triple positive CAR T cells.

FIG. 7: dasatinib inhibits cytokine secretion by CAR T cells in response to tumor antigens.

FIG. 8: dasatinib reverses depletion marker expression and co-expression.

FIG. 9: dasatinib treatment allowed CD62L expression to be maintained.

FIG. 10: dasatinib treatment produces enhanced IL-2 and IFN γ secretion in response to tumor antigens.

FIG. 11: CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib showed reduced activation and degranulation. As shown, cd19.28z CAR T cells were cultured for at least 48 hours in the presence or absence of various concentrations of dasatinib or ponatinib. CAR T cells were then co-cultured with Nalm6 tumor cells bearing CD19 for 6 hours. CD69 and CD107a surface expression were subsequently assessed via FACS. The figure shows cells gated on the CD8+ CAR + population. Such results demonstrate that 80% of cd19.28z CAR T cells become activated (surface CD69 is the activated surrogate) and degranulated (surface CD107a is the degranulated surrogate) in response to tumors. However, dasatinib and ponatinib dose-dependently inhibited the ability of CAR T cells to respond to tumors in this manner.

FIG. 12: CAR T cells co-cultured with tumor cells in the presence of dasatinib or ponatinib are unable to secrete cytokines. As shown, high affinity gd2.28z (HA-gd2.28z) CAR T cells were co-cultured with nalm6 overexpressing GD2 in the presence or absence of various concentrations of dasatinib or ponatinib for 24 hours. Supernatants were then collected and analyzed for IL-2 and IFNy via ELISA. These results indicate that inhibition of CAR T cells using HA-gd2.28z CAR, dasatinib, and ponatinib responds to tumor secretion of IL-2 and IFNy.

FIG. 13: CAR T cells cultured in the presence of dasatinib exhibited attenuated killing in response to tumor antigens. An incucyte assay was performed in which cd19.bbz CAR T cells were co-cultured with nalm6 tumor cells expressing the GFP reporter gene in the presence of 1uM dasatinib or vehicle (DMSO) for 72 hours. Tumor GFP fluorescence was measured over time. GFP values were normalized to the fluorescence intensity at the first time point. These results demonstrate that dasatinib attenuated the ability of cd19.28z CARs to kill tumor cells. Figures 11, 12 and 13 demonstrate that dasatinib or ponatinib can act as a rapid and reversible safety "off switch for CAR T cells with deleterious effects in a given patient.

FIG. 14: dasatinib effectively inhibits phosphorylation of CAR CD3z and remote signaling proteins after CAR cross-linking. On day 10 post-activation, 2E6 HA-gd2.28z CAR T cells cultured in 1uM dasatinib or vehicle were removed from the culture. The idiotype primary antibody and cross-linked secondary antibody were then added to the cells at 5ug/mL to initiate signaling through the CAR. As shown herein, dasatinib effectively inhibited cross-linking induced phosphorylation of the CD3z domain on CARs, as well as phosphorylation of the remote signaling kinases Akt and ERK 1/2. This is a representative blot of n-3 independent experiments.

FIG. 15: tonic signaling CAR T cells expanded in the presence of dasatinib showed a reduction in expression of classical depletion markers in a dose-dependent manner. HA-gd2.28z CAR T cells were expanded in the presence of various concentrations of dasatinib or vehicle (DMSO). At day 14 post-activation, cells were removed from culture, stained, and their depletion phenotype was analyzed via FACS. Representative blots from 3 independent experiments. FIG. 15A: CAR + T cell classical depletion marker expression. FIG. 15B: CAR + CD4+ (left) or CAR + CD8+ (right) marker for depletion co-expression. These results demonstrate that HA-gd2.28z CAR signals catatonic in the absence of antigen, which ultimately induces T cell depletion as defined by expression of multiple inhibitory receptors, lack of memory formation, and reduced effector function. Figure 14 demonstrates that expansion of HA-gd2.28z CAR T cells in the presence of dasatinib dose-dependently attenuated depletion marker single expression (a) or co-expression (b).

FIG. 16: the tonic signaling CAR T cells expanded in the presence of dasatinib retained the ability to form memory. CD19.28z or HA-GD2.28z was amplified in the presence or absence of 1uM dasatinib or vehicle (DMSO). At day 14 post-activation, cells were removed from the culture for FACS analysis. This representative figure shows CAR + T cells. The red box highlights the low CD45RA, CCR7 high population, which corresponds to central memory-like T cells. These results demonstrate that expansion of tonic signaling HA-gd2.28z CAR T cells in dasatinib also enhances memory formation, here demonstrated by a significant increase in the CD45RA low, CCR7 high population (which corresponds to the central memory-like phenotype).

FIG. 17: the tonic signaling CAR T cells expanded in the presence of dasatinib displayed enhanced cytokine secretion in response to tumor antigens. HA-gd2.28z CAR T cells were expanded in the presence or absence of various concentrations of dasatinib or ponatinib. Drugs were removed from T cells 24 hours prior to co-culture with nalm6 tumor cells that overexpress GD2, to restore the T cells ability to respond to tumor signaling. After 24 hours, supernatants were collected and evaluated for IL-2 and IFNy secretion via ELISA. Figures 11, 12, 13, 15 and 16 demonstrate that dasatinib and ponatinib can inhibit CAR T cell signaling and function. Figure 17 shows that expansion of tonic signaling HA-gd2.28z CAR T cells in the presence of these drugs, followed by removal of the drug prior to co-culture with tumor cells resulted in an increase in IL-2 and IFNy.

FIG. 18: the tonic signaling CAR T cells expanded in the presence of dasatinib displayed enhanced cytotoxicity. HA-gd2.28z CAR T cells were expanded for 96 hours in the presence or absence of dasatinib or vehicle (DMSO). Dasatinib was removed from T cells 24 hours prior to the incucyte assay at day 14 post-activation, in which T cells were co-cultured with nalm6 tumors overexpressing GD2 at an E: T ratio of 1: 8. Tumor GFP fluorescence was measured over time. GFP values were normalized to the fluorescence intensity at the first time point. These results demonstrate that inhibition of the tonic signaling of HA-gd2.28z CAR T cells rescues the ability of these CAR T cells to kill tumors by including dasatinib in the culture medium during expansion, followed by removal of dasatinib prior to co-culture.

FIG. 19: nalm6 overexpressing GD2 in the presence and absence of dasatinib. 0.5E 6143B tumor cells were implanted intramuscularly in the legs of mice. On day 3 post-implantation, 10E6 gd2.bbz CAR T cells expanded in the presence of dasatinib or vehicle (DMSO) were infused intravenously into mice. The left panel shows the mean leg area +/-SEM (n ═ 5 mice). Fig. 19 and 20 summarize the findings from fig. 14, 15, 16 and 17 in an in vivo environment. Different types of CARs (gd2.bbz, HA-gd2.28z) were cultured in dasatinib and then infused in vivo to enhance their anti-tumor function.

FIG. 20A: 0.5E 6143B tumor cells were implanted intramuscularly in the legs of mice. On day 3 post-implantation, 10E6 HA-gd2.28z CAR T cells expanded in the presence of dasatinib or vehicle (DMSO) were infused intravenously into mice. The upper panel shows the mean leg area +/-SEM (n ═ 5 mice). Fig. 19 and 20 summarize the findings from fig. 14, 15, 16 and 17 in an in vivo environment. Different types of CARs (gd2.bbz, HA-gd2.28z) were cultured in dasatinib and then infused in vivo to enhance their anti-tumor function.

FIG. 20B: nalm6 tumor cells overexpressing 1E6 GD2 were implanted intravenously into mice. On day 3 post-implantation, 2E6 CAR + HA-gd2.28z CAR T cells expanded in the presence of dasatinib or vehicle (DMSO) were infused intravenously into mice. The upper panel shows mean tumor fluorescence +/-SEM (n ═ 5 mice). Fig. 19 and 20 summarize the findings from fig. 14, 15, 16 and 17 in an in vivo environment. Different types of CARs (gd2.bbz, HA-gd2.28z) were cultured in dasatinib and then infused in vivo to enhance their anti-tumor function.

FIG. 21: nalm6 tumor cells overexpressing 1E6 GD2 were implanted intravenously into mice. On day 3 post-implantation, 2E6 HA-gd2.28z CAR T cells expanded in the presence of dasatinib or vehicle (DMSO) were infused intravenously into mice. On day 17 post-implantation, blood samples were taken from each mouse and mixed with counting beads. FACS analysis was performed and the number of CD4+ and CD8+ cells per mouse was counted. This figure shows the mean CD4+ or CD8+ cells +/-SEM per mouse (n-5 mice). Fig. 21 shows one of the mechanisms by which dasatinib enhances function. After infusion of dasatinib-treated CAR T cells into mice, blood samples were taken and analyzed for the number of circulating CAR T cells, which is a typical reading of CAR T cell proliferation in response to tumors in vivo. The vehicle HA-gd2.28z CAR T cells did not exhibit significantly more in vivo proliferation than mock T cells, as these cells are likely to be depleted when they are initially infused into mice. However, CAR T cells expanded in dasatinib retain their anti-tumor function and therefore proliferate robustly in vivo.

Fig. 22A, 22B, 22C, 22D, 22E: in vivo dasatinib treatment suppressed expression of the depletion marker, enhanced memory formation and promoted cell survival/proliferation. Mice were implanted with nalm6 tumor cells overexpressing 1E6 GD2 via intravenous injection. On day 4 post-implantation, 2E6 HA-gd2.28z CAR T cells were infused intravenously into mice. Mice were administered 50mg/kg dasatinib via intraperitoneal injection on days 21-23 after tumor implantation. At 5 hours after dasatinib administration on day 23, 1 mouse receiving vehicle and 1 mouse receiving dasatinib were sacrificed and spleen/blood was harvested, surface stained and phenotyped via FACS. A and C) CAR + T cells accounted for a higher percentage of total circulating cells (a) or total splenocytes (C) in mice treated with dasatinib compared to vehicle control. B and D) in contrast to mice treated with vehicle (red), circulating or splenic CD8+ CAR + T cells exhibited a phenotype consistent with non-activated or resting T cells in dasatinib-treated mice (blue), indicating that dasatinib suppressed CAR T cell activation and induced memory formation (i.e., higher CD62L expression) in vivo. E) On days 27-29 post tumor implantation, 1 mouse received 50mg/kg dasatinib daily, and different mice received vehicle controls. On days 30-32, mice were untreated. On day 32, tumor luminescence was assessed. Dasatinib administration for 3 days was sufficient to induce robust recovery of the anti-tumor response (blue). These data indicate that repeated administration of dasatinib can restore depleted T cells in vivo.

FIG. 23: nucleic acid and amino acid sequences of CD19.28z (FMC63 scFv).

FIG. 24: nucleic acid and amino acid sequences of bbz (FMC63 scFv).

FIG. 25: nucleic acid and amino acid sequences of bbz (14G2a scFv).

FIG. 26: nucleic acid and amino acid sequences of HA-GD2.28z (high affinity 14G2a scFv).

Definition of

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a T cell" includes two or more T cells and the like.

The term "about" with respect to a given amount is intended to encompass deviations of plus or minus 5%.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor engineered to be expressed on immune effector cells and specifically bind an antigen. The CARs are useful as therapies using adoptive cell transfer. T cells are removed from a patient and modified so that they express receptors specific for a particular form of antigen. For example, in some embodiments, the CAR has been expressed with specificity for a tumor associated antigen. The CAR can also comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. The specificity of CAR design may be derived from the ligand of the receptor (e.g., peptide). In some embodiments, the CAR can target the cancer by redirecting the specificity of T cells expressing the CAR specific for the tumor-associated antigen.

By "pharmaceutically acceptable excipient or carrier" is meant an excipient that may optionally be included in the compositions of the present invention and that does not cause significant detrimental toxicological effects to the patient.

"pharmaceutically acceptable salts" include, but are not limited to, amino acid salts; salts prepared with inorganic acids such as chloride, sulfate, phosphate, diphosphate, bromide and nitrate; or salts prepared from the corresponding mineral acid forms of any of the foregoing, e.g., hydrochloride salts and the like; or salts prepared with organic acids such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, p-toluenesulfonate, pamoate (palmoate), salicylate and stearate, and etoate (estolates), gluconate and lactobionate. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

The term "T cell" refers to a T lymphocyte as defined in the art and is intended to include a thymocyte, an immature T lymphocyte, a mature T lymphocyte, a resting T lymphocyte, or an activated T lymphocyte. The T cell may be CD4⁺T cell, CD8⁺T cell, CD4⁺CD8⁺T cells or CD4^-CD8^-A cell. The T cell may also be a T helper cell, such as a T helper 1(TH1) cell or a T helper 2(TH2) cell or a TH17 cell, as well as a cytotoxic T cell, a regulatory T cell, a natural killer T cell, a naive T cell, a memory T cell or a γ δ T cell.

The T cells may be a purified population of T cells, or alternatively the T cells may be in a population with different types of cells such as B cells andAnd/or other peripheral blood cell populations. The T cells may be a subset of T cells (e.g., CD 4)⁺T cells), or they may be a population of T cells comprising different T cell subsets. In another embodiment of the invention, the T cells are T cell clones that have been maintained in culture for an extended period of time. T cell clones can be transformed to varying degrees. In a specific embodiment, the T cells are T cell clones that proliferate indefinitely in culture.

In some embodiments, the T cell is a primary T cell. The term "primary T cell" is intended to include T cells obtained from an individual, as opposed to T cells that have been maintained in culture for an extended period of time. Thus, primary T cells are in particular peripheral blood T cells obtained from a subject. The population of primary T cells may consist essentially of a subset of T cells. Alternatively, the population of primary T cells may consist of different subpopulations of T cells.

T cells may be from a previously stored blood sample, from a healthy individual, or alternatively from an individual with a disorder. The condition may be an infectious disease, such as a condition caused by a viral infection, a bacterial infection, or an infection by any other microorganism; or hyperproliferative diseases, such as cancer, like melanoma. In another embodiment of the invention, the T cell is from a subject suffering from or susceptible to an autoimmune disease or T cell pathology. The T cells may be of human origin, murine origin, or any other mammalian species.

"T cell depletion" refers to loss of T cell function, which may occur as a result of infection or disease. T cell depletion is associated with increased expression of PD-1, TIM-3 and LAG-3, apoptosis and decreased cytokine secretion.

By "therapeutically effective dose or amount" of a TCR signaling inhibitor (e.g., dasatinib) is meant an amount that, when administered as described herein, produces a positive therapeutic response (e.g., restored T cell function) in the treatment of T cell depletion. 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 precise 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, the mode of administration and the like. An appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

The terms "subject," "individual," and "patient" are used interchangeably herein and refer to any vertebrate subject, including but not limited to 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 indicate a particular age. Thus, it is expected that adult as well as neonatal individuals will be covered.

Detailed Description

The present invention is based on the following findings: transient inhibition or modulation of TCR signaling and/or CAR signaling in human T cells can prevent or reverse T cell depletion and restore T cell function. The inventors have shown that GD2-CAR expressing T cells develop functional depletion, which is manifested by the expression of PD-1, TIM-3 and LAG-3 depletion markers. Cessation of tonic signaling restores the ability of T cells to secrete IL-2 in response to tumor antigens. The inventors further showed that treatment with dasatinib, an Lck tyrosine kinase inhibitor that inhibits T cell receptor signaling, reduced the expression of T cell depletion markers and improved the preservation of T cell memory.

Protein tyrosine kinases are a family of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate to tyrosine residues in protein substrates. Phosphorylation of tyrosine residues on protein substrates results in the transduction of intracellular signals that regulate a variety of intracellular processes, such as the growth and activation of cells of the immune system (e.g., T cells). Since T cell activation is implicated in a variety of inflammatory disorders of the immune system and other disorders (e.g., autoimmune diseases), modulation of protein tyrosine kinase activity appears to be an attractive approach to control inflammatory diseases. A number of protein tyrosine kinases have been identified which may be receptor protein tyrosine kinases such as insulin receptors, or non-receptor protein tyrosine kinases.

Protein tyrosine kinases of the Src family have been found to be particularly important for intracellular signal transduction associated with inflammatory responses (see, e.g., d.okutani et al, am.j.physiol.lung Cell moi.physiol.291,2006, pages L129-L141; ca.lowell, moi.immunol.41,2004, pages 631-643). While some of the Src family protein tyrosine kinases (e.g., Src, Yes, and Fyn) are expressed in a variety of cell types and tissues, others are restricted to specific cell types, such as hematopoietic cells. Therefore, the protein tyrosine kinase Lck is almost completely expressed in T cells as the first signaling molecule to be activated downstream of the T cell receptor, and its activity is crucial for T cell signaling. In mature monocytes and macrophages, inflammatory stimuli such as LPS increase expression of Hck, Lyn, and Fgr. Likewise, if gene expression of the major B cell Src family kinases (i.e., Lyn, Fyn, and BIk) is disrupted, the immature B cells are prevented from developing into mature B cells. Src family kinases have also been identified as critical to the recruitment and activation of monocytes, macrophages and neutrophils and are involved in the inflammatory response of tissue cells.

As noted, receptor tyrosine kinases are important components of signal transduction pathways that mediate cell-to-cell communication, and they serve as relay points for signal transduction pathways. They play a key role in controlling cell proliferation and differentiation, regulating cell growth and cell metabolism, and promoting cell survival and apoptosis in a multitude of processes. Lck (p 56)^lckOr lymphocyte specific kinases) are cytoplasmic tyrosine kinases of the Src family that are expressed in T cells and Natural Killer (NK) cells. Genetic evidence from knockout mouse and human mutations suggests that Lck kinase activity is critical for T Cell Receptor (TCR) -mediated signaling, leading to normal T cell development and activation. Thus, selective inhibition of Lck is useful in the treatment of T cell mediated autoimmune and inflammatory disordersAnd/or organ transplant rejection.

The present 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 therapy (e.g., CAR T cell therapy). First, these drugs were shown to effectively inhibit CAR signaling, which provides a means for modulating CAR activity and thus mitigating CAR T cell toxicity, while retaining the option to continue treatment once toxicity subsides, as the inhibitory effects of dasatinib and ponatinib on CAR T cell function are reversible. Second, expansion of CART cells in the presence of dasatinib or ponatinib was shown to prevent CAR tonic signaling and thereby enhance the functional capacity of CAR T cells. Finally, providing short-term CAR T cell "rest" in vivo via repeated drug administration has been shown to be a method of preventing or reversing CAR T cell depletion and/or inducible memory.

Accordingly, provided herein are compositions and methods for preventing or reversing T cell depletion. In particular, the present invention relates to methods of preventing or reversing T cell depletion by exposing T cells undergoing T cell depletion to specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) or by expanding genetically engineered T cells in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib).

As such, the present invention contemplates that exposing an animal (e.g., a human) undergoing adoptive T cell therapy (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) using a genetically engineered T cell population to a composition comprising a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) will yield improved therapeutic outcomes in that such particular tyrosine kinase inhibitor is capable of 1) modulating TCR signaling (e.g., reducing expression of one or more of PD-1, TIM-3, and LAG-3 within the genetically engineered T cell population; increasing the expression of a memory marker (e.g., CD62L or CCR 7); increase secretion of IL-2 and other cytokines), and 2) prevent and/or reverse T cell depletion within the genetically engineered T cell population. Indeed, the present invention contemplates that the use of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors) in adoptive T cell therapies satisfies an unmet need, as the effectiveness of such therapies is often compromised by such T cell populations undergoing T cell depletion. Accordingly, the present invention provides methods for treating an immune system-related disorder or disease (e.g., cancer) in a subject, the methods comprising administering to the subject (e.g., simultaneously and/or at different time points) genetically engineered T cells and a specific tyrosine kinase inhibitor (e.g., dasatinib, ponatinib). Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Such tyrosine kinase inhibitors may be administered by any suitable mode of administration, but are typically administered orally. Multiple cycles of treatment may be administered to the subject. In certain embodiments, the tyrosine kinase inhibitor is administered according to a daily dosing regimen or intermittently. In another embodiment, the tyrosine kinase inhibitor is administered for a period of time sufficient to restore at least a portion of T cell function and then stopped.

The present invention contemplates that ex vivo expansion of a T cell population with a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) will result in a T cell population that is resistant to and/or less prone to T cell depletion. Accordingly, the present invention provides compositions comprising a population of T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) (e.g., a Src family kinase inhibitor) (e.g., an Lck inhibitor). Accordingly, the present invention provides methods of expanding T cell populations by expanding such T cells in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) to produce T cell populations that are resistant to and/or less prone to T cell depletion. Accordingly, the invention provides kits comprising a population of T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and an additional agent (e.g., additional agents that can be used to expand T cells) (e.g., additional agents that can be used in adoptive T cell therapies (e.g., CAR T cell therapy, transduction T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy). 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

The present invention contemplates ex vivo expansion of a population of genetically engineered T cells (e.g., genetically engineered to be resistant to and/or less prone to T cell depletion) with a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) (e.g., a Src family kinase inhibitor) (e.g., an Lck inhibitor) will result in genetically engineered T cells that are resistant to and/or less prone to T cell depletion in adoptive T cell therapies (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) Methods for genetically engineered T cell populations that are sexually and/or less prone to T cell depletion. Accordingly, the present invention provides kits comprising a population of genetically engineered T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib). Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

The present invention contemplates that exposure of animals (e.g., humans) undergoing adoptive T cell therapy (e.g., CAR T cell therapy, transduced T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) using genetically engineered T cell populations expanded in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) will yield improved therapeutic outcomes because such genetically engineered T cell populations are resistant to T cell depletion and/or less prone to T cell depletion. Accordingly, the present invention provides methods of treating an immune system related disorder or disease (e.g., cancer) in a subject comprising administering a population of genetically engineered T cells expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lck inhibitors). Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Such embodiments are not limited to a particular type or kind of immune system related disorder or disease.

For example, in some embodiments, the immune system-related disorder or disease is an autoimmune disease or disorder (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, chylomicronemia-like sprue, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, acroscleroderma, crohn's disease, Degos' disease, dermatomyositis juvenile, discoid, primary mixed cryoglobulinemia, annuomyelitis, or a disease, Fibromyalgia-fibromyositis, Graves ' disease, Guillain-Barre syndrome (Guillain-Barre syndrome), hashimoto's thyroiditis, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (stills's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon (Raynaud's paranema), reis syndrome (Reiter ' syndrome), Rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as Systemic Sclerosis (SS)), Sjogren's syndrome, stiff person syndrome, systemic lupus erythematosus, takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo, wegener's granulomatosis, and any combination thereof).

For example, in some embodiments, the immune system related disorder 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 cancer).

The present invention contemplates that the use of genetically engineered T cell populations expanded in the presence of specific tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) in adoptive T cell therapies (e.g., CAR T cell therapy, transduced T-cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy) satisfies an unmet need as such therapies are often compromised by such T cell populations undergoing T cell depletion. Such methods are not limited to a particular type or kind of genetically engineered T cell. In some embodiments, 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 transduction T cell therapy, and/or virus-specific T cells re-engineered with TCRs or CARs.

Embodiments of the present invention are not limited to a particular type of tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is an Lck tyrosine kinase inhibitor. In some embodiments, 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). In some embodiments, the tyrosine kinase inhibitor is dasatinib

Or a pharmaceutically acceptable salt, solvate or prodrug thereof. In some embodiments, the tyrosine kinase inhibitor is ponatinibOr a pharmaceutically acceptable salt, solvate or prodrug thereof.

Some embodiments of the invention provide for the administration of such methods (e.g., adoptive T cell therapy using a genetically engineered T cell population and a composition comprising a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib)), e.g., adoptive T cell therapy using a genetically engineered T cell population expanded in the presence of a particular tyrosine kinase inhibitor (e.g., dasatinib, ponatinib), in combination with an effective amount of at least one additional therapeutic agent (including, but not limited to, chemotherapeutic anti-neoplastic agents, apoptosis modulators, antimicrobial agents, antiviral agents, antifungal agents, and anti-inflammatory agents) and/or therapeutic technique (e.g., surgical intervention and/or radiation therapy). In particular embodiments, the additional therapeutic agent is an anti-cancer agent.

Tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) may be formulated into pharmaceutical compositions, optionally comprising one or more pharmaceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for use in injectable compositions include water, alcohols, polyols, glycerol, vegetable oils, phospholipids and surfactants. Carbohydrates, such as sugars, derivatised sugars (such as sugar alcohols, aldonic acids, esterified sugars and/or sugar polymers) may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, and the like. Excipients may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof.

The surfactant may 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, New Jersey); sorbitan esters; lipids, such as phospholipids, e.g., lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposome form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; as well as zinc and other such suitable cations.

The acid or base may be present in the pharmaceutical composition as an excipient. Non-limiting examples of acids that can be used include those 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. Examples of suitable bases include, but are not limited to, 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 fumarate, and combinations thereof.

The amount of tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) in the pharmaceutical composition (e.g., when included in a drug delivery system) will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is in unit dosage form or in a 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 the clinically desirable endpoint.

The amount of any individual excipient in a pharmaceutical composition will vary depending on the nature and function of the excipient and the particular needs of the composition. Typically, the optimum amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of excipient (ranging from low to high), examining stability and other parameters, and then determining the range at which optimum performance is obtained without significant adverse effects. Generally, however, the excipient will be present in the composition in an amount of from 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 a concentration of less than 30% by weight being most preferred. These aforementioned pharmaceutical excipients are described, along with other excipients, in "Remington: The Science & Practice of Pharmacy", 19 th edition, Williams & Williams, (1995); "Physician's Desk Reference", 52 th edition, Medical Economics, Montvale, NJ (1998) and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3 rd edition, American Pharmaceutical Association, Washington, D.C., 2000.

Pharmaceutical compositions encompass all types of formulations, and especially those suitable for injection, such as powders or lyophilized powders that can be reconstituted with a solvent and prepared for injection as a solution or suspension prior to use, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates that are diluted prior to administration. Examples of suitable diluents for reconstituting the solid composition prior to injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are contemplated. Other preferred compositions include those for oral, ocular or topical delivery.

The pharmaceutical formulations herein may also be contained in a syringe, an implant device, or the like, depending on the intended mode of delivery and use. Preferably, the pharmaceutical composition comprising one or more tyrosine kinase inhibitors (e.g. dasatinib, ponatinib) as described herein is in unit dosage form, meaning an amount of the conjugate or composition of the invention suitable for a single dose in a pre-measured or pre-packaged form.

The pharmaceutical compositions herein may optionally comprise, or may be combined with, one or more additional agents, such as other drugs for treating T cell depletion (e.g., anti-PD-1 checkpoint inhibitors, such as nivolumab) or other drugs for treating infections or diseases associated with T cell depletion in a subject (e.g., antiviral, antibiotic or anti-cancer drugs and therapies, including adoptive T cell therapies). Co-formulations comprising at least one tyrosine kinase inhibitor (e.g. dasatinib, ponatinib) and one or more other agents (such as other drugs for the treatment of T cell depletion or infections or diseases associated with T cell depletion) may be used. Alternatively, such agents may be included in a composition different from the composition comprising the tyrosine kinase inhibitor (e.g., dasatinib, ponatinib), and may be co-administered simultaneously, prior to, or after the composition comprising the tyrosine kinase inhibitor (e.g., dasatinib, ponatinib).

At least one therapeutically effective treatment cycle using a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib)) is administered to the subject to treat T cell depletion. By "therapeutically effective treatment cycle" is meant a treatment cycle that, when administered, results in a positive therapeutic response with respect to the depletion of T cells in the treated individual. Of particular interest are treatment cycles with tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) that restore T cell function when administered transiently as described herein. For example, a therapeutically effective dose or amount of a tyrosine kinase inhibitor may reduce the expression of PD-1, TIM-3, and LAG-3, improve the maintenance of memory markers (e.g., CD62L or CCR7), prevent apoptosis, and increase the secretion of IL-2 and other cytokines.

In certain embodiments, multiple therapeutically effective doses of a pharmaceutical composition 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 depletion (e.g., anti-PD-1 checkpoint inhibitors, such as nivolumab) or other drugs for treating infections or diseases associated with T cell depletion in a subject (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, but not necessarily, administered orally, via injection (subcutaneous, intravenous or intramuscular), by infusion or topically. Additional modes of administration are also contemplated, such as topical, intralesional, intracerebral, intracerebroventricular, intraparenchymal, pulmonary, rectal, transdermal, transmucosal, intrathecal, pericardial, intraarterial, intraocular, intraperitoneal, and the like.

The pharmaceutical preparation may be in the form of a liquid solution or suspension immediately before administration, but may also take another form, such as syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, and the like. Pharmaceutical compositions comprising one or more tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) and other agents may be administered according to any medically acceptable method known in the art, using the same or different routes of administration.

In another embodiment, a pharmaceutical composition comprising one or more tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) and/or other agents is administered prophylactically, e.g., to prevent T cell depletion. Such prophylactic use would be of particular value for subjects with chronic infection or cancer at risk of developing T cell depletion.

In another embodiment of the invention, the pharmaceutical composition comprising one or more tyrosine kinase inhibitors (e.g. dasatinib, ponatinib) and/or other agents is in the form of a sustained release formulation, or a formulation administered using a sustained release device. Such devices are well known in the art and include, for example, transdermal patches and micro-implantable pumps that can provide drug delivery in a continuous, steady-state manner over time at various doses to achieve a sustained release effect in the case of non-sustained release pharmaceutical compositions.

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 having a disorder responsive to treatment with a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) contained in the conjugate or composition. The method comprises administering a therapeutically effective amount of the conjugate or drug delivery system by any means described herein, preferably provided as part of a pharmaceutical composition. The methods of administration can be used to treat any condition responsive to treatment with a tyrosine kinase inhibitor (e.g., dasatinib, ponatinib). More specifically, the pharmaceutical compositions herein are effective in treating T cell depletion.

One of ordinary skill in the art will appreciate which disorders tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) can effectively treat. The actual dose to be administered will vary depending on the age, weight and general condition of the subject, as well as the severity of the condition being treated, the judgment of the health professional, and the conjugate being administered. A therapeutically effective amount can be determined by one skilled in the art and will be adjusted to the specific requirements of each particular situation.

Typically, a therapeutically effective amount will range from about 0.50mg to 5 grams of tyrosine kinase inhibitor per day, more preferably from about 5mg to 2 grams per day, and even more preferably from about 7mg to 1.5 grams per day. Preferably, such doses are in the range of 10-600mg four times per day (QID), 200-500mg QID, 25-600mg three times per day (TID), 25-50mg TID, 50-100mg TID, 50-200mg TID, 300-600mg TID, 200-400mg TID, 200-600mg TID, 100-700 mg twice per day (BID), 100-600mg BID, 200-500mg BID or 200-300mg BID. The amount of compound administered will depend on the potency of the tyrosine kinase inhibitor and the degree or effect desired and the route of administration.

The purified tyrosine kinase inhibitor (again, preferably provided as part of a pharmaceutical formulation) may be administered alone or in combination with: one or more other therapeutic agents, such as other drugs for treating T cell depletion (e.g., anti-PD-1 checkpoint inhibitors, such as nivolumab) or other drugs for treating infection or disease associated with T cell depletion in a subject (e.g., antiviral, antibiotic, or anticancer drugs); or adoptive T cell therapy (e.g., CAR T cell therapy, transduction T cell therapy, and Tumor Infiltrating Lymphocyte (TIL) therapy); or other drugs used to treat a particular condition or disease according to various dosing regimens, depending on the judgment of the clinician, the needs of the patient, and the like. Specific dosing regimens will be known to those of ordinary skill in the art or may be determined experimentally using routine methods. Exemplary dosing regimens include, but are not limited to: administration is five times daily, four times daily, three times daily, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly and any combination thereof. Preferred compositions are those that require administration no more than once per day.

The tyrosine kinase inhibitor may be administered prior to, concurrently with, or subsequent to the other agent or therapy. If provided concurrently with other agents or therapies, one or more tyrosine kinase inhibitors may be provided in the same or different compositions. Thus, one or more tyrosine kinase inhibitors and other agents may be provided to an individual by way of concurrent therapy. By "concurrent therapy" is meant administration to a subject such that a therapeutic effect of the combination of substances is elicited in the subject undergoing treatment. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising a tyrosine kinase inhibitor in combination with a dose of a pharmaceutical composition comprising at least one other agent (such as another drug for treating T cell depletion), the pharmaceutical composition comprising a therapeutically effective dose according to a particular dosing regimen. Similarly, one or more tyrosine kinase inhibitors and one or more other therapeutic agents may be administered in at least one therapeutic dose. Administration of separate pharmaceutical compositions or therapies may be simultaneous or at different times (i.e., sequentially, in any order, on the same day, or on different days), so long as the therapeutic effect of the combination of these substances is produced in the subject undergoing therapy.

The invention also provides kits comprising one or more containers holding a composition comprising at least one tyrosine kinase inhibitor (e.g., dasatinib, ponatinib) and optionally one or more other agents for treating T cell depletion. The composition may be in liquid form or may be lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, including glass or plastic. The container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle).

The kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution, or dextrose solution. It may also include other materials useful to the end user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles and syringes or other delivery devices. The delivery device may be pre-filled with the composition.

The kit may further comprise a package insert containing written instructions for a method of treating T cell depletion in a subject using a composition comprising at least one tyrosine kinase inhibitor (e.g., dasatinib, ponatinib). The package insert may be a draft package insert that is not approved or may be a package insert approved by the Food and Drug Administration (FDA) or other regulatory agency.

Those skilled in the art will readily appreciate that the foregoing represents only a detailed description of certain preferred embodiments of the invention. Various modifications and alterations of the above-described compositions and methods can be readily made using the expertise available in the art and are within the scope of the present invention.