Compositions for improving CAR-T cell function and uses thereof

文档序号：1219516 发布日期：2020-09-04 浏览：7次中文

阅读说明：本技术 用于改善car-t细胞功能的组合物及其用途 (Compositions for improving CAR-T cell function and uses thereof ) 是由沙德哈克·森古普塔于 2018-11-20 设计创作，主要内容包括：本发明涉及包含CAR-T细胞和GSK3β抑制剂的组合物和试剂盒,包括此类组合物和/或试剂盒在治疗诸如癌症等的疾病中的使用。(The present invention relates to compositions and kits comprising CAR-T cells and GSK3 β inhibitors, including the use of such compositions and/or kits in the treatment of diseases such as cancer.)

1. a method for expanding a population of T cells in vitro comprising contacting the T cells with a GSK3 β inhibitor.

2. The method of claim 1, wherein the T cell is transfected with a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen prior to contacting the T cell with the GSK3 β inhibitor.

3. The method of claim 1, wherein the T cell is isolated from a subject.

4. The method of claim 1, further comprising contacting the transduced T cells with a tumor antigen.

5. The method of claim 1 or 2, wherein the T cell is contacted with the GSK3 β inhibitor and the tumor antigen simultaneously.

6. The method according to any one of the preceding claims, wherein the T cell is transduced with a nucleic acid encoding a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T).

7. The method of claim 4, wherein the nucleic acid encodes interleukin 13 variant IL13.E13K.R109K or a fragment thereof.

8. The method of claim 6, wherein the nucleic acid encodes a fragment of interleukin 13 comprising a domain that binds to an interleukin 13 receptor or an extracellular domain thereof and a fusion protein comprising an interleukin 13 receptor or an extracellular domain thereof.

9. The method of claim 6, wherein the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof.

10. The method of claim 9, wherein the tumor antigen comprises an alpha (a) chain of interleukin 13 receptor (IL13R a) or a variant thereof.

11. The method of any one of the preceding claims, wherein the GSK3 β inhibitor is:

(a) a chemical agent selected from SB216763, 1-Azakenpaulolone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or

(b) A genetic agent selected from the group consisting of micro RNA (mirna), small interfering RNA (sirna), DNA-directed RNA interference (ddRNAi) oligonucleotides, antisense oligonucleotides, or a combination thereof.

12. The method of any one of the preceding claims, wherein the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

13. The method of claim 1, wherein the expanded T cells are subsequently administered back to the patient to treat a disease.

14. The method of claim 13, wherein the disease is cancer.

15. The method of claim 14, wherein the cancer is a solid tumor.

16. The method of claim 15, wherein the tumor expresses a tumor antigen.

17. A method for expanding a population of T cells in vitro comprising:

a. isolating a sample comprising T cells from a subject;

b. transducing the population of T cells with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; and

c. the transduced T cells are contacted with a GSK3 β inhibitor.

18. A composition comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor.

19. The composition of claim 19, wherein the chimeric antigen receptor protein binds a tumor antigen.

20. The composition of claim 18 or claim 19, wherein the T cell expresses a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T).

21. The composition of claim 20, wherein the T cell expresses a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k.

22. The composition of claim 18, wherein the GSK3 β inhibitor is a small molecule or a genetic agent.

23. The composition of any one of claims 18 to 22, wherein the inhibitor of GSK3 β is a small molecule that is SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO) or a genetic agent that is a siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN).

24. The composition of any one of claims 18 to 22, wherein the inhibitor of GSK3 β is a genetic agent selected from microrna (mirna), small interfering RNA (sirna), DNA-directed RNA interference (ddRNAi) oligonucleotide, antisense oligonucleotide, or a combination thereof, and dominant negative allele of GSK3 (GSK3 DN).

25. A separately administered formulation comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor.

26. The formulation of claim 25, wherein the GSK3 β inhibitor is a small molecule or genetic agent that is SB216763, TWS-119, 1-Azakenpaullone, or 6-bromoisatin-3' -oxime (BIO); the genetic agent is an siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN).

27. A kit comprising in one or more than one package a CAR nucleic acid construct, a GSK3 β inhibitor; and optionally comprising a first agent for transducing a T cell by the CAR nucleic acid construct; and further optionally a second agent for activating T cells, wherein the CAR nucleic acid construct encodes a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T).

28. The kit of claim 27, wherein the second reagent is IL13R α 2-Fc.

29. The kit of claim 27 or 28, wherein the nucleic acid construct encodes a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k.

30. The kit of any one of claims 27 to 29, wherein the inhibitor of GSK3 β is a small molecule that is SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO) or a genetic agent that is an siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN).

31. The kit of any one of claims 27 to 29, wherein the GSK3 β inhibitor is a genetic agent comprising microrna (mirna), small interfering RNA (sirna), DNA-directed RNA interference (ddRNAi) oligonucleotide, antisense oligonucleotide, or a combination thereof, and GSK3 DN.

32. A T cell that inhibits GSK β expression or activity compared to a native or wild-type T cell.

33. The T cell of claim 32, which is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

34. The T cell of claim 32, wherein the T cell comprises a genetic inhibitor comprising a microrna (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof, wherein the genetic inhibitor inhibits the activity or expression of GSK3 β in the T cell.

35. A method for expanding T cells in vitro comprising: isolating a sample comprising T cells from a subject; contacting a T cell with a GSK3 β inhibitor; transducing the T cell with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; contacting the transduced T cells with the tumor antigen to expand the transduced T cells.

36. A method for expanding T cells in vitro comprising: isolating a sample comprising T cells from a subject; contacting a T cell with a GSK3 β inhibitor; transducing the T cell with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; contacting the transduced T cells with the tumor antigen to activate and/or expand the transduced T cells.

37. The method of claim 35 or 36, wherein the T cell is transduced with a nucleic acid encoding a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T).

38. The method of claim 37, wherein the nucleic acid encodes interleukin 13 variant il13.e13k.r109k or a fragment thereof.

39. The method of claim 38, wherein the nucleic acid encodes a fragment of interleukin 13 comprising a domain that binds to an interleukin 13 receptor or extracellular domain thereof, or a fusion protein comprising an interleukin 13 receptor or extracellular domain thereof.

40. The method of claim 39, wherein the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof.

41. The method of claim 40, wherein said tumor antigen comprises the alpha (α) chain of interleukin 13 receptor (IL13R α) or a variant thereof.

42. The method of claim 35 or 36, wherein the GSK3 β inhibitor is

(a) A chemical agent selected from SB216763, 1-Azakenpaulolone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or

(b) A genetic agent selected from the group consisting of a microrna (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof.

43. The method of claim 35 or 36, wherein the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

44. A method for treating a disease treatable by adoptive transfer of T cells in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a plurality of activated and expanded T cells, wherein the activation comprises contacting CAR-T with an antigen and the expansion comprises contacting the activated CAR-T cells with a GSK3 β inhibitor.

45. The method of claim 44, wherein said GSK3 β inhibitor is

(a) A chemical agent selected from SB216763, TWS-119, 1-Azakenpullone, or 6-bromoisatin-3' -oxime (BIO); and/or

46. The method of claim 44, wherein the disease is a neoplastic disease, a pathogenic disease selected from bacterial disease, viral disease and protozoal disease, or an autoimmune disease.

47. A composition comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor.

48. A method for treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a plurality of activated and expanded T cells expressing a chimeric antigen receptor protein (CAR-T) comprising a molecule that binds to a tumor antigen, wherein the activation comprises contacting the CAR-T with the tumor antigen and the expansion comprises contacting the activated CAR-T cells with a GSK3 β inhibitor, wherein the activated CAR-T cells express the chimeric antigen receptor protein, wherein the chimeric antigen receptor protein binds to the tumor antigen.

49. The method of claim 48, wherein said T cells are autologous T cells.

50. The method of claim 48, wherein said tumor antigen is interleukin 13 receptor (IL13R) or a ligand binding domain thereof.

51. The method of claim 48, wherein said chimeric antigen receptor protein comprises Il13 or a variant or fragment thereof.

52. The method of claim 48, wherein said chimeric antigen receptor protein comprises the IL13 variant IL13. E13K.R109K.

53. The method of claim 48, wherein the GSK3 β inhibitor is:

(a) a chemical agent selected from SB216763, 1-Azakenpaulolone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or

54. The method of claim 48, wherein said T cells are activated and expanded simultaneously or sequentially.

55. The method of claim 48, wherein the tumor is IL13R positive.

56. The method of claim 48, wherein the tumor is an IL13R positive glioma.

57. A method for generating tumor-specific memory T cells, comprising: transducing a T cell isolated from a biological sample of a subject (CAR-T) with a nucleic acid encoding a chimeric antigen receptor comprising a molecule that binds to a tumor antigen; contacting a CAR-T cell with the tumor antigen and a GSK3 β inhibitor; detecting a first marker specific for memory cells and a second marker specific for a tumor antigen, thereby generating tumor-specific memory T cells.

58. The method of claim 57, wherein the CAR-T cells are transduced with a nucleic acid encoding IL13 or a fragment or variant thereof.

59. The method of claim 58, wherein the CAR-T cells are transduced with a nucleic acid encoding an IL13 variant IL13.E13K.R109K.

60. The method of claim 59, wherein the tumor antigen is the IL13 receptor or its ligand binding domain.

61. The method of claim 57, wherein said GSK3 β inhibitor is

(a) A chemical agent selected from SB216763, 1-Azakenpaulolone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or

62. The method of claim 57, wherein the memory cell specific marker is selected from the group consisting of CD45RO + and CD45RA + and the tumor antigen specific marker comprises expression of a protein that binds to the tumor antigen.

63. The method of claim 57, wherein the CAR-T cells have specificity for IL 13R-positive tumor cells, the specificity comprising binding to IL 13R-positive cells and optionally disrupting IL 13R-positive cells as determined by a functional assay.

64. The method of claim 57, wherein the memory T cells are CD8+ T cells.

65. The method of claim 57, further comprising detecting a third marker of memory CAR-T cell homeostasis.

66. The method of claim 57, wherein the third marker is IL13R expression, T-beta expression, and/or PD-1 expression.

67. The method of claim 66, wherein increased T-beta expression and/or decreased PD-1 expression is indicative of improved CAR-T cell homeostasis.

68. The method of claim 67, wherein T cell homeostasis comprises reduced T cell failure, sustained cytokine expression, T cell clonal maintenance, and/or promotion of CAR-T memory development.

69. The method of any one of claims 57 to 68, wherein CAR-T cells produced by activation of said tumor antigen and expansion in the presence of a GSK3 β inhibitor exhibit increased specificity and memory for tumor cells expressing said tumor antigen.

Technical Field

The disclosure herein relates to compositions and methods for improving the function of genetically modified or chimeric antigen receptor T cells (e.g., CAR-T) expressing receptor proteins, which can be used for a variety of therapeutic applications, such as the treatment of tumors.

Background

The use of engineered T cells expressing chimeric antigen receptors (CAR-T) as immunotherapeutic strategies against malignancies has become a hallmark of successful treatment of peripheral liquid tumors. However, CAR-T therapy shows a mixed response in solid tumor treatment. The success of adoptive T cell therapy depends on the ready availability of antigen sources and co-stimulatory signals by therapeutic T cells, which can lead to a strong activation pattern and strong cytotoxic effects, such as CAR-T cells exposed to a large number of malignant B cells in lymph nodes in hematological tumors or during treatment of highly immunogenic tumors (e.g., melanoma). In contrast, during CAR-T treatment of solid tumors, poor activation of T cells due to limited exposure to tumor antigens leads to unstable immune responses, anemia of clonal expansion and early contraction of clones.

Various approaches have been used in the art to overcome the problems of clonal shrinkage and weak T cell activation, with some degree of success. For example, CD28 signaling molecules were attached to the intracellular portion of CAR constructs to design so-called second generation CAR-T cells to overcome clonal shrinkage, promote rapid proliferation, and overcome cytokine deficiency. Over time, it was observed that this modification did not overcome all of the obstacles to the use of CAR-T for solid tumors. CAR-T cells have been further modified to create "third generation" CARs with the addition of co-stimulatory molecules such as 41BB and/or OX 40. In addition, patients are often treated with IL2 to maintain survival and function of the transferred T cells, which results in uncontrolled cytokine production by the therapeutic T cells.

Although these approaches have been able to enhance T cell activation to some extent in the treatment of solid tumors, there is still a need for further innovations to completely overcome clonal shrinkage and promote rapid T cell proliferation and activation. Such methods are provided herein.

Disclosure of Invention

The disclosure herein relates to compositions and methods for improving CAR-T therapy. It has been recognized in the art that one of the major obstacles impeding the success of CAR-T cell immunotherapy in solid tumors is weak antigen exposure, resulting in insufficient CAR-T cell activation, with concomitant generation of weak anti-tumor immune responses, and the present invention provides compositions and methods to overcome existing obstacles in CAR-T therapy. In particular, the compositions and methods described herein overcome many of the limitations of CD28 and other costimulatory signaling moieties in second generation CARs, as well as the cytotoxicity associated with supplemental IL2 therapy.

In various embodiments, provided herein is a method for expanding a T cell population in vitro, comprising contacting a T cell population with a GSK3 β inhibitor. In various embodiments, T cells are first transduced with a nucleic acid encoding a chimeric antigen T cell receptor. In various embodiments, the T cell is derived from a mammal. In various embodiments, the mammal is a human.

In various embodiments, the method further comprises contacting the transduced cell with a tumor antigen.

In various embodiments, provided herein is a method for expanding a population of T cells in vitro, comprising: isolating a sample comprising said T cells from the subject; transducing the population of T cells with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; and, contacting the transduced T cells with a GSK3 β inhibitor.

In various embodiments, the method further comprises contacting the transduced T cells with a tumor antigen. In various embodiments, the T cell is contacted with both the GSK3 β inhibitor and the tumor antigen.

In various embodiments, a T cell (IL13CAR-T) is transduced with a nucleic acid encoding a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof. In various embodiments, the nucleic acid encodes an interleukin 13 variant il13.e13k.r109k or a fragment thereof.

In various embodiments, the nucleic acid encodes a fragment of interleukin 13 comprising a domain that binds to an interleukin 13 receptor or extracellular domain thereof, or a fusion protein comprising a fusion protein of an interleukin 13 receptor or extracellular domain thereof. In various embodiments, the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof.

In various embodiments, the tumor antigen comprises the alpha (α) chain of interleukin 13 receptor (IL13R α) or a variant thereof.

In various embodiments, the GSK3 β inhibitor is (a) a chemical agent selected from SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or (b) a genetic agent selected from the group consisting of a micro RNA (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof.

In various embodiments, the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

In various embodiments, the expanded T cells are subsequently administered back to the patient to treat the disease. In various embodiments, the disease is cancer. In various embodiments, the cancer is a solid tumor. In various embodiments, the tumor expresses a tumor antigen.

In various embodiments, provided herein is a composition comprising the chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor. In various embodiments, the chimeric antigen receptor binds to a tumor antigen.

In various embodiments, the T cell expresses a chimeric antigen receptor (IL13CAR-T) comprising interleukin 13 or a variant or fragment thereof. In various embodiments, the T cell expresses a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k.

In various embodiments, the GSK3 β inhibitor is a small molecule or a genetic agent. In various embodiments, the GSK3 β inhibitor is a small molecule or genetic agent that is SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); the genetic agent is an siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN). In various embodiments, the inhibitor of GSK3 β is a genetic agent selected from microrna (mirna), small interfering RNA (sirna), DNA-directed RNA interference (ddRNAi) oligonucleotides, antisense oligonucleotides, or a combination thereof, and a dominant negative allele of GSK3 (GSK3 DN).

In various embodiments, provided herein is a separately administered formulation comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor.

In various embodiments, the GSK3 β inhibitor is a small molecule that is SB216763, TWS-119, 1-Azakenpaullone, or 6-bromoisatin-3' -oxime (BIO), or a genetic agent; the genetic agent is an siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN).

In various embodiments, provided herein is a kit, wherein the kit comprises, in one or more than one package, a CAR nucleic acid construct encoding a chimeric antigen receptor protein (IL13CAR-T) comprising interleukin 13 or a variant or fragment thereof; GSK3 β inhibitors; and optionally comprising a first agent for transducing a T cell by the CAR nucleic acid construct; and further optionally a second agent for activating T cells.

In various embodiments, the second agent is IL13R α 2-Fc. In various embodiments, the nucleic acid construct encodes a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k. In various embodiments, the GSK3 β inhibitor is a small molecule that is SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO), or a genetic agent; the genetic agent is an siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN). In various embodiments, the GSK3 β inhibitor is a genetic agent comprising a microrna (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof, and GSK3 DN.

In various embodiments, provided herein are T cells that inhibit GSK β expression or activity as compared to native or wild-type T cells. In various embodiments, the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

In various embodiments, provided herein is a method for expanding T cells in vitro, comprising: isolating a sample comprising T cells from a subject; contacting a T cell with a GSK3 β inhibitor; transducing T cells with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; the transduced T cells are contacted with a tumor antigen to expand the transduced T cells.

In various embodiments, the T cell is transduced by a nucleic acid encoding a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T). In various embodiments, the nucleic acid encodes an interleukin 13 variant il13.e13k.r109k or a fragment thereof. In various embodiments, the nucleic acid encodes a fragment of interleukin 13 comprising a domain that binds to an interleukin 13 receptor or extracellular domain thereof, or a fusion protein comprising an interleukin 13 receptor or extracellular domain thereof. In various embodiments, the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof. In various embodiments, the tumor antigen comprises the alpha (α) chain of interleukin 13 receptor (IL13R α) or a variant thereof.

In various embodiments, the GSK3 β inhibitor is (a) a chemical agent selected from SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO); and/or (b) a genetic agent selected from the group consisting of a microrna (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof.

In various embodiments, the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, or a gamma T cell.

In various embodiments, provided herein is a method of treating a disease treatable by adoptive transfer T cells in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a plurality of activated and expanded T cells, wherein the activating comprises contacting CAR-T with an antigen and the expanding comprises contacting the activated CAR-T cells with a GSK3 β inhibitor.

In various embodiments, the GSK3 β inhibitor is (a) a chemical agent selected from SB216763, TWS-119, 1-Azakenpaullone, or 6-bromoisatin-3' -oxime (BIO); and/or (b) a genetic agent selected from the group consisting of a microrna (mirna), a small interfering RNA (sirna), a DNA-directed RNA interference (ddRNAi) oligonucleotide, an antisense oligonucleotide, or a combination thereof.

In various embodiments, the disease is a neoplastic disease, a pathogenic disease selected from bacterial disease, viral disease, and protozoal disease, or an autoimmune disease.

In various embodiments, provided herein is a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a plurality of activated and expanded T cells (CAR-T) expressing a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen, wherein the activating comprises contacting the CAR-T with the tumor antigen and the expanding comprises contacting the activated CAR-T cells with a GSK3 β inhibitor, wherein the activated CAR-T cells express the chimeric antigen receptor protein, wherein the chimeric antigen receptor protein binds to the tumor antigen.

In various embodiments, the T cell is an autologous T cell.

In various embodiments, T cells are activated and expanded simultaneously or sequentially. In various embodiments, the tumor is positive for IL 13R. In various embodiments, the tumor is an IL13R positive glioma.

In various embodiments, provided herein is a method for generating tumor-specific memory T cells, the method comprising: transducing a T cell (CAR-T) isolated from a biological sample of a subject with a nucleic acid encoding a chimeric antigen receptor comprising a molecule that binds to a tumor antigen; contacting a CAR-T cell with the tumor antigen and a GSK3 β inhibitor; detecting a first marker specific for memory cells and a second marker specific for a tumor antigen, thereby generating tumor-specific memory T cells.

In various embodiments, the CAR-T cell is transduced with a nucleic acid encoding IL13 or a fragment or variant thereof. In various embodiments, the CAR-T cell is transduced with a nucleic acid encoding an IL13 variant il13.e13k.r109k. In various embodiments, the tumor antigen is an IL13 receptor or a ligand binding domain thereof.

In various embodiments, T cells are activated and expanded simultaneously or sequentially. In various embodiments, the marker specific for memory cells is selected from the group consisting of CD45RO + and CD45RA +, and the marker specific for a tumor antigen includes expression of a protein that binds to the tumor antigen. In various embodiments, the CAR-T cells have specificity for IL 13R-positive tumor cells, as determined by a functional assay, comprising binding to IL 13R-positive cells and optionally disrupting IL 13R-positive cells. In various embodiments, the memory T cell is a CD8+ T cell.

In various embodiments, the methods described herein further comprise detecting a third marker of memory CAR-T cell homeostasis. In various embodiments, the third marker is expression, T-beta expression, and/or PD-1 expression. In various embodiments, wherein an increase in T-beta expression and/or a decrease in PD-1 expression indicates an improvement in CAR-T cell homeostasis. In various embodiments, T cell homeostasis includes reduced T cell failure, sustained cytokine expression, T cell clonal maintenance, and/or promotion of CAR-T memory development. In various embodiments, CAR-T cells produced by activation of the tumor antigen and expansion in the presence of a GSK3 β inhibitor exhibit increased specificity and memory for tumor cells expressing the tumor antigen.

Drawings

The details of one or more embodiments disclosed herein are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings/tables and detailed description, and from the claims.

Figure 1 shows that GSK3 β inhibition protects activated CAR-T cells from ATCD in the absence of IL2 supplementation in vitro. Figure 1A shows that survival of IL13R α 2-Fc activated IL13CAR-T steadily decreased in the absence of SB21763 (open squares, solid lines; upper panel; p ═ 0.2), and survival of IL13R α 2-Fc activated IL13CAR-T was rescued to the survival level of IL2 supplemented IL13CAR-T (closed squares, dashed lines) (lower panel; p <0.05) after inhibition of GSK3 β using SB 216763. Results represent 1 out of 2 experiments, with n-3 wells per sample at each time point. Error bars represent SD. FIG. 1B shows the results of flow cytometry of the frequency of FasL-expressing IL13CAR-T cells after activation with IL13R α 2-Fc only (upper panel) and after activation with GSK3 β inhibition (lower panel). The results are representative of n-3 independent experiments. Figure 1C shows representative FACS properties of CFSE dilutions, showing IL13CAR-T cell proliferation without any treatment (top curve), IL13CAR-T cell proliferation treated with SB216763 only (second curve), IL13CAR-T cell proliferation activated with IL13R α 2-Fc only (third curve) and IL13CAR-T cell proliferation activated with IL13R α 2-Fc + SB216763 (bottom curve).

FIG. 1-supplementation shows the results of IL13R α 2 specificity of IL13CAR-T cells of the invention FIG. 1A-supplementation is shown at different effector to target cell (E: T) ratios (left) to IL13R α 2⁺U251MG tumor cells were co-cultured, flow cytometry results enriched for IL13CAR-T after activation with 1. mu.g/ml and 10. mu.g/ml of IL13R α 2-Fc (middle) and IL13R α 1-Fc (right), untransduced T cells are indicated by open lines and IL13CAR-T by closed lines FIG. 1B-supplement shows flow cytometry results of CFSE dilutions showing IL13R α 2-specific proliferation of IL 13-T cells after activation with 0. mu.g/ml (black), 1. mu.g/ml (grey) and 10. mu.g/ml (open) of IL13R α 2-Fc (middle panel) and IL13R α 1-Fc (lower panel) CAR in the presence of U251MG 125 cells (upper panel) (E: T ratios 1: 0 (black), 1:1 (grey) and 1: 2 (open), respectively).

FIG. 2 shows GSK3 β inhibition results in T-beta upregulation and PD-1 expression reduction in activated CAR-T cells FIG. 2A shows flow cytometry results for intranuclear T-beta expression in IL13CAR-T cells (left panel), and frequency of PD-1+ IL13CAR-T cells after activation with IL13R α 2-Fc in the absence or presence of SB216763 (right panel). results are representative n ═ 3 independent experiments FIG. 2B shows relative expression (qPCR) of TBX21 gene (T-beta; left panel) and PDCD1 gene (PD-1; right panel) in IL13CAR-T cells activated with IL13R α 2-Fc (right panel). data are normalized using 2 PDH after GAPDH normalization^-ΔΔC _TThe method is used for analysis. Error bars represent from 3 independent N ═ sSEM of the experiment.

Figure 2-supplement shows transduction efficiency of IL13 CAR. OKT-3 and IL2 were extracted from PBMCs of three blind donors to enrich for T cells and transduced 3 times with retroviral supernatants expressing IL13CAR to maximize Transduction Efficiency (TE). TE was measured by observing the expression of human IL13 on CD3+ T cells using flow cytometry 48 hours after final transduction. All experiments in this study were normalized to TE of IL13CAR to eliminate donor-dependent changes.

Figure 3 shows that GSK3 β inhibition results in increased expression of β -catenin in the nucleus of antigen-specific CAR-T cells. Representative histograms of nuclear β -catenin expression in unstimulated IL13CAR-T cells (upper panel); representative histograms of nuclear β -catenin expression in IL13CAR-T cells activated by IL13R α 2-Fc (middle panel); and SB 216763-treated IL13R α 2-Fc activated IL13CAR-T cells (lower panel). Treated or untreated IL13CAR-T cells were stained with a rat anti-human IL13 primary anti/APC anti-rat IgG1 secondary antibody, and a rabbit anti- β -catenin MAb/FITC anti-rabbit IgG secondary antibody. Specific antibodies were used as controls to eliminate background staining. The results are representative n 2 experiments.

FIG. 3-supplement shows experimental results of CD8 enrichment of IL13CAR-T cells FIG. 3A-supplement shows flow cytometric results of the ratio CD8: CD4 in IL13CAR-T cells activated with IL13R α 2-Fc + SB216763 Each figure represents FACS characteristics from each of 3 donors door control drawn against respective antibody controls FIG. 3B-supplement shows relative expression of IFNG (interferon- γ) gene in IL13CAR-T cells activated with IL13R α 2-Fc, data normalized with GAPDH using 2^-ΔΔC _TFigure 3C-supplement shows interferon gamma levels from IL13CAR-T cell culture supernatants measured by ELISA, treated with SB216763 alone or with SB216763 in combination with IL13R α 2-Fc activation.

Figure 4 shows the antigen specific CAR-T cell memory phenotype following GSK3 β inhibition. FIG. 4A shows representative FACS characteristics of IL13CAR-T cell frequency activated with IL13R alpha 2-Fc in the presence (right panel) or absence (left panel) of SB 216763. Figure 4B shows a line graph representation of the IL13CAR-T cell memory phenotype. Error bars represent SEM from 3 independent experiments.

FIG. 5 shows the tissue distribution of CAR-T and expression of T effector memory phenotype in tumor-bearing mice treated with IL13 CAR-T. FIG. 5A (left) shows local expression of tissue-specific IL13CAR-T distribution in tumor-draining lymph nodes (top panel), spleen (middle panel) and tumor-infiltrating lymphocytes (bottom) of tumor-bearing animals. FIG. 5B (right) shows CD45RO in tumor-draining lymph nodes (top panel), spleen (middle panel), and tumor-infiltrating lymphocytes (bottom panel) from tumor-bearing animals⁺CD127⁺Tumors were observed in all surviving xenograft animals treated with non-activated IL13CAR-T cells (100% recurrence; white circles), 67% of surviving animals treated with IL13R α 2-Fc activated IL13CAR-T cells (black circles), no tumors were detected in surviving animals treated with IL13R α 2-Fc activated IL13CAR-T cells treated with SB216763 (0% recurrence; gray circles).

Detailed Description

Exemplary embodiments and applications of the present invention are described in this specification. However, the invention is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications are operated or described herein. Other embodiments, features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. Further, where a list of elements (e.g., elements a, b, c) is recited, such reference is intended to include any one of the recited elements, less than any combination of all of the recited elements, and/or combinations of all of the recited elements. The division of the sections in this specification is for ease of reading only and does not limit any combination of the elements discussed.

As used herein, the terms "comprises," "comprising," "contains," "containing," "has," "includes," and variations thereof, are not intended to be limiting, but rather inclusive or open-ended, and do not exclude additional, unrecited additives, ingredients, integers, elements, or process steps. For example, a process, method, system, composition, kit, or apparatus that comprises a list of features is not limited to only those features but may include other features not expressly listed or inherent to such process, method, system, composition, kit, or apparatus.

Unless defined otherwise, scientific and technical terms used in connection with the teachings set forth herein shall have the meanings that are commonly understood by those of ordinary skill in the art.

The present invention relates to compositions and methods for improving CAR-T therapy. It has been recognized in the art that one of the major obstacles impeding the success of CAR-T cell immunotherapy in solid tumors is the insufficient activation of CAR-T cells resulting from weak antigen exposure, with the concomitant generation of weak anti-tumor immune responses, and the present invention provides compositions and methods to overcome the existing obstacles in CAR-T therapy. In particular, the compositions and methods described herein overcome many of the limitations of CD28 and other costimulatory signaling moieties in second generation CARs, as well as the cytotoxicity associated with supplemental IL2 therapy.

In various embodiments, the compositions and methods of the invention relate to the use of adjuvants to improve the survival and/or efficacy of CAR-T cells. In particular, the invention demonstrates that GSK3 β inhibitors can be used to increase proliferation of antigen-specific CAR-T cells, rapidly expand antigen-specific CAR-T cells and improve survival of antigen-specific CAR-T cells. As demonstrated in detail in the examples section of the invention, pharmacological inhibition of GSK3 β promotes antigen-specific CAR-T cell proliferation and long-term survival of these T cells. By alleviating PD-1 expression, inhibition of GSK3 β protects activated CAR-T cells from T cell depletion and further promotes the development of specific effector CAR-T memory phenotypes that can be modulated as a function of antigen exposure. Antigen-specific CAR-T cell treatment with GSK3 β inhibition can eliminate tumors 100% and increase the accumulation of memory CAR-T cells in the spleen and draining lymph nodes. Tumor restimulation experiments in animal models, when treated with antigen-treated (antigen-induced) CAR-T cells inhibited by GSK3 β, allowed 100% elimination of tumors and achieved progression-free survival. Taken together, these results demonstrate that this kind of adjuvant inhibition of activated CAR-T cells by GSK3 β provides an effective approach to CAR-T immunotherapy against solid tumors.

The data in the examples of the invention further demonstrate that GSK3 β inhibition plays an important role in successfully modulating CAR-T cell function. Surprisingly, it was found that activity was limited to antigen-specific CAR-T cells or those activated by antigen or ligand. GSK3 β inhibition not only plays a role in the proliferation of activated CAR-T, but also promotes the generation of CD8+ CAR-T Effector Memory (TEM). The results demonstrate that GSK3 β inhibition takes advantage of the combined effect of increasing cell division and increasing survival of antigen-specific CAR-T; however, GSK3 β inhibition had no proliferative effect on non-activated CAR-T cells; GSK3B inhibition also had no effect on untransduced T cells lacking IL13CAR expression. These observations confirm that the proliferative effects of GSK3 β inhibition are specific for activated CAR-T.

In various embodiments of the invention, GSK3 β inhibition results in enhanced tumor protection for a longer duration. In various embodiments of the invention, GSK3 β inhibition results in an increase in immune memory and the production of expanded and/or proliferating CAR-T cells. Furthermore, studies of antigen-specific CAR-T inhibited by GSK3 β in experimental xenograft animals showed that CAR-T cells treated with GSK3B inhibitors had longer tumor protection time, indicating that expanded and/or proliferated CAR-T cells had immunological memory. These studies point to a hitherto undiscovered method of selectively expanding antigen-specific CAR-T cell subsets.

Accordingly, the present invention is directed to the following non-limiting embodiments:

in various embodiments, the present invention relates to a method for modulating a T cell comprising contacting the T cell with a GSK3 β inhibitor. In some embodiments, the GSK3 β inhibitor is a small molecule chemical, such as SB216763(3- (2, 4-dichlorophenyl) -4- (1-methyl-1H-indol-3 yl) -1H-pyrrole-2, 5-dione), 1-azakenpaullone, TWS-119 or 6-bromoisatin-3' -oxime (BIO), and TWS-119. In some embodiments, the GSK3 β inhibitor is a genetic agent, such as RNA interference (RNAi) by using, for example, a microrna (mirna), a small interfering RNA molecule (siRNA), a DNA-directed RNA interference (ddRNAi) oligonucleotide, or an antisense oligonucleotide specific for GSK3 β, and a dominant negative allele of GSK3 β (GSK3 DN). Preferably, the inhibitor inhibits human GSK3 β, such as human GSK3 β variant 1 (mRNA sequence in GENBANK: NM-002093; protein sequence: NP-002084), human GSK3 β variant 2 (mRNA sequence in GENBANK: NM-001146156; protein sequence: NP-001139628) or human GSK3 β variant 3 (mRNA sequence in GENBANK: NM-001354596; protein sequence: NP-001341525). In some embodiments, GSK3 β inhibition comprises deletion or disruption of GSK3 β, e.g., by targeted knock-out. In some embodiments, modulation increases T cell expansion, proliferation, survival and/or reduces depletion of activated T cells.

Any type of T cell can be modulated by the foregoing methods, including but not limited to T helper cells, cytotoxic T cells, memory T cells (e.g., central memory T cells, stem-like memory T cells (or stem-like memory T cells), or effector memory T cells (e.g., TEM cells and TEMRA cells)), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, gamma T cells, tumor infiltrating T cells (TILs), and CAR-T cells. Preferably, the T cell is a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell or a gamma T cell. In particular, the T cell is a CAR-T cell. In a particularly preferred embodiment, the T cell is an activated CAR-T cell. As known in the art, CAR-T cells are typically activated using antigenic stimulation, and CAR-T cells obtained from such processes are antigen-specific, e.g., specific for a tumor antigen, e.g., interleukin 13 receptor (IL13R) or a variant thereof.

In various embodiments, the T cell is not a memory T cell (e.g., a central memory T cell, a stem-like memory T cell (or stem-like memory T cells)), or an effector memory T cell (e.g., TEM cells and TEMRA cells)).

The invention further relates to T cells that have been modulated by the aforementioned methods, wherein the expression or activity of GSK3 β is inhibited, for example, by using a chemical agent or genetic inhibitor as described above. Preferably, the T cell has inhibited GSK3 β expression or activity compared to a wild type or normal T cell. Particularly preferably, the T cell exhibits reduced GSK3 β activity compared to a wild type or normal T cell. In particular, the T cells exhibit reduced GSK3 β activity compared to wild-type or normal T cells due to RNA interference by using siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN).

In some embodiments, the invention relates to the use of T cells that have been modulated or modified according to the methods of the invention. In this context, the modulated T cells may be used to treat any disease or disease for which adoptive transfer of T cells is considered beneficial, including, for example, treatment of cancer, treatment of pathogenic infections (e.g., viral diseases such as HIV, bacterial infections, protozoal infections), treatment of inflammatory diseases (e.g., rheumatoid arthritis or crohn's disease), and may also be used to augment the immune system.

In various embodiments, the methods disclosed herein can be used to treat cancer. The term "cancer" as used herein encompasses any cancer, including but not limited to: melanoma, sarcoma, lymphoma, cancers such as brain, breast, liver, stomach, and colon cancers, and leukemia. In various embodiments, the methods disclosed herein can be used to treat a tumor. In various embodiments, the tumor is a solid tumor. In various embodiments, the solid is a glioblastoma.

In various embodiments, the tumor expresses a tumor-associated antigen. Examples of such antigens include oncofetal antigens (e.g., alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA)), surface glycoproteins (e.g., CA-125 and mesothelin), oncogenes (e.g., Her2), melanoma-associated antigens (e.g., DOPAchrome tautomerase (DCT), GP100 and MART1), cancer test antigens (e.g., MAGE protein and NY-ESO1), viral oncogenes (e.g., HPV E6 and E7), proteins ectopically expressed in tumors (which are typically restricted to embryonic or extraembryonic tissues, such as PLAC1, ECM fibrin 3 expressed by GBM tumor cells but not present in brain and Epidermal Growth Factor Receptor (EGFR)). As will be appreciated by those skilled in the art, since one or more antigens may be particularly suitable for use in the treatment of certain cancers, the antigen(s) may be selected based on the type of cancer to be treated using the methods of the present invention. For example, for the treatment of melanoma, an antigen associated with melanoma, such as DCT, may be used.

In various embodiments, the chimeric antigen receptor protein comprises interleukin 13 or a variant or fragment thereof (IL13 CAR-T). In various embodiments, the nucleic acid encodes an interleukin 13 variant il13.e13k.r109k or a fragment thereof. In various embodiments, the nucleic acid encodes a fragment of interleukin 13 comprising a domain that binds to an interleukin 13 receptor or extracellular domain thereof, or a fusion protein comprising an interleukin 13 receptor or extracellular domain thereof. In various embodiments, the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof. In various embodiments, the tumor antigen comprises an alpha (α) chain of interleukin 13 receptor (IL13R α) or a variant thereof. In various embodiments, the chimeric antigen receptor protein comprises an extracellular domain capable of targeting fibrin 3.

In various embodiments disclosed herein, the Chimeric Antigen Receptor (CAR) is directed against a tumor associated antigen. In various embodiments, the tumor-associated antigen targeted by the CAR is selected based on the type of tumor antigen expressed by the patient to be treated by the methods disclosed herein.

In preferred embodiments, the present invention relates to methods and compositions for modulating T cells that have been primed by a tumor (e.g., tumor infiltrating lymphocytes or TILs), which T cells, when modulated, can be advantageously used to kill tumor cells. Preferably, the regulated T cells are autologous transferred to the host to facilitate destruction of the tumor cells.

In particularly preferred embodiments, the present invention relates to methods and compositions for generating memory T cells, which may be used to perform one or more of the above-described therapeutic applications.

In a related embodiment, the invention relates to a method for expanding T cells in vitro, comprising: isolating a sample comprising T cells from a subject; transducing T cells with a nucleic acid encoding a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen; the transduced T cells are contacted with a GSK3 β inhibitor and a tumor antigen to expand the transduced T cells. Preferably, the T cell is transduced with a nucleic acid encoding a chimeric antigen receptor protein comprising interleukin 13(IL13CAR-T) or a variant or fragment thereof. In particular, the nucleic acid encodes a CAR comprising an interleukin 13 variant il13.e13k.r109k or fragment thereof.

In a related embodiment, the invention relates to a method for expanding T cells in vitro, comprising: isolating a sample comprising T cells from a subject; transducing a T cell with a nucleic acid encoding a fragment of interleukin 13, the fragment comprising a domain that binds to an interleukin 13 receptor or an extracellular domain thereof, or a fusion protein comprising an interleukin 13 receptor or an extracellular domain thereof; the transduced T cells are contacted with a GSK3 β inhibitor and a tumor antigen to expand the transduced T cells. Preferably, the tumor antigen comprises interleukin 13 receptor (IL13R) or a variant thereof. In particular, the tumor antigen comprises the alpha (α) chain of interleukin 13 receptor (IL13R α) or a variant thereof. GSK3 β inhibitors may be small molecule inhibitors or genetic inhibitors of GSK3 β, including siRNA, miRNA, antisense oligonucleotides, ddRNAi, or dominant negative inhibitors of GSK3 (GSK3 DN). Preferably, the GSK3 β inhibitor is a small molecule GSK3 β inhibitor, such as SB216763, TWS-119, 1-Azakenpaulolone or 6-bromoisatin-3' -oxime (BIO). In various embodiments, T cells may be activated and expanded, e.g., expanded upon activation or activated upon expansion, simultaneously or sequentially.

In various embodiments, the present invention relates to a method for treating a tumor in a subject in need thereof, comprising: administering to a subject an effective amount of a composition comprising a plurality of activated and/or expanded T cells (CAR-T) expressing a chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen, wherein said activating comprises contacting the CAR-T cells with the tumor antigen and said expanding comprises contacting the activated CAR-T cells with a GSK3 β inhibitor. For example, in some embodiments, the activated CAR-T cells preferably express a chimeric antigen receptor protein, and the chimeric antigen receptor protein binds to a tumor antigen. In various embodiments, the T cell is an autologous T cell. In particular, the tumor antigen is interleukin 13 receptor (IL13R) or a ligand binding domain thereof, and the chimeric antigen receptor protein comprises, for example, IL13 or a variant or fragment thereof that binds to tumor antigen IL13R (α 1 or α 2). In various embodiments, the inhibitor of GSK3 β may be a small molecule inhibitor or genetic inhibitor of GSK3 β, including siRNA, miRNA, antisense oligonucleotide, ddRNAi, or a dominant negative inhibitor of GSK3 (GSK3 DN). Preferably, the GSK3 β inhibitor is a small molecule GSK3 β inhibitor, such as SB216763, 1-Azakenpaullone, 6-bromoisatin-3' -oxime (BIO), or TWS-119. In various embodiments, T cells may be activated and expanded, e.g., expanded upon activation or activated upon expansion, simultaneously or sequentially.

In various embodiments, the present invention provides a method of treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a plurality of activated and/or expanded autologous T cells (CAR-T cells) that express a chimeric antigen receptor protein comprising the IL13 variant il13.e13k.r109k, wherein said activating comprises contacting the CAR-T cells with a tumor antigen, and said expanding comprises contacting the activated CAR-T cells with a small molecule GSK3 β inhibitor, e.g., SB216763, 1-azakellauone, 6-bromoisatin-3' -oxime (BIO), or TWS-119, wherein said activated CAR-T cells express a chimeric antigen receptor protein, and wherein said chimeric antigen receptor protein binds to the tumor antigen. In various embodiments, T cells may be activated and expanded, e.g., expanded upon activation or activated upon expansion, simultaneously or sequentially.

In various embodiments, the invention provides a method of treating a glioma in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a plurality of activated and/or expanded T cells (CAR-T cells) expressing a chimeric antigen receptor protein, said chimeric antigen receptor protein comprising a molecule that binds to a tumor antigen, wherein said activating comprises contacting the CAR-T cells with the tumor antigen and said expanding comprises contacting the activated CAR-T cells with a GSK3 β inhibitor. In this embodiment, the activated CAR-T cells preferably express a chimeric antigen receptor protein, and the chimeric antigen receptor protein binds to a tumor antigen expressed in glioma, such as IL13R or a variant thereof. In various embodiments, the glioma is glioblastoma multiforme (GBM), anaplastic astrocytoma, or pediatric glioma. In some embodiments, activating comprises contacting the CAR-T cell with a glioma tumor antigen and expanding comprises contacting the activated CAR-T cell with a small molecule GSK3 β inhibitor, wherein the activated CAR-T cell expresses a chimeric antigen receptor protein that binds to a glioma tumor antigen. The GSK3 β inhibitor may be a small molecule inhibitor or a genetic inhibitor. In some embodiments, the GSK3 β inhibitor is a small molecule, such as SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO). Alternatively or additionally, the GSK3 β inhibitor is a genetic agent comprising an siRNA, miRNA, antisense oligonucleotide, ddRNAi or a dominant negative inhibitor of GSK3 (GSK3 DN).

In various embodiments, the invention relates to a method for generating tumor-specific memory T cells comprising: transducing T cells isolated from a biological sample of a subject with a nucleic acid encoding a chimeric antigen receptor (CAR-T) comprising a molecule that binds to a tumor antigen; contacting a CAR-T cell with the tumor antigen and a GSK3 β inhibitor; detecting a first marker specific for memory cells and a second marker specific for a tumor antigen, thereby generating tumor-specific memory T cells. Preferably, the CAR-T cell is transduced with a nucleic acid encoding IL13 or a fragment or variant thereof (e.g., il13.e13k.r109k), wherein the CAR protein binds to a tumor antigen comprising an IL13 receptor or ligand binding domain. In various embodiments, activating comprises contacting the CAR-T cell with a tumor antigen, and expanding comprises contacting the activated CAR-T cell with a small molecule GSK3 β inhibitor, such as SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO). In some embodiments, the memory cell specific marker is selected from the group consisting of CD45RO + and CD45RA +, and the tumor antigen specific marker comprises expression (e.g., cell surface expression) of a protein that binds to the tumor antigen. In various embodiments, the tumor-specific CAR-T cells have specificity for IL 13R-positive tumor cells, as determined by a functional assay, comprising binding to IL 13R-positive cells and optionally disrupting IL 13R-positive cells. In various embodiments, the tumor-specific memory cells are CD8+ T cells. In some embodiments, the memory T cells in the CAR-T cells are further selected by activating the CAR-T cells with a tumor antigen and expanding the CAR-T cells in the presence of a GSK3 β inhibitor, which exhibit increased specificity and memory for tumor cells expressing the tumor antigen.

In various embodiments, the present invention provides a method for generating tumor-specific memory T cells, the method comprising: transducing a T cell (CAR-T) isolated from a biological sample of a subject with a nucleic acid encoding a chimeric antigen receptor comprising a molecule that binds to a tumor antigen; contacting a CAR-T cell with a tumor antigen and a GSK3 β inhibitor; detecting a first marker specific for memory cells, a second marker specific for a tumor antigen, and a third marker of memory CAR-T cell homeostasis; thereby generating tumor-specific memory T cells. In one embodiment, the third marker is IL13R expression, T-beta expression, and/or PD-1 expression in a CAR T cell, wherein increased T-beta expression and/or decreased PD-1 expression indicates that CAR-T cell homeostasis is improved. In particular, the methods provide improved T cell homeostasis, which includes reduced T cell failure, sustained cytokine expression, T cell clonal maintenance, and/or promotion of CAR-T memory development.

In various embodiments, the invention relates to a method of modulating T cells using the aforementioned transduction, activation, expansion, and optional selection steps, wherein the CAR-T cells are activated by a tumor antigen and expanded in the presence of a GSK3 β inhibitor, further selecting memory T cells from the CAR-T cells that exhibit increased specificity for tumor cells expressing the tumor antigen and improved memory, and further exhibit improved CAR-T cell homeostasis. In particular, the methods provide expanded populations of activated CAR-T cells with improved T cell homeostasis, including reduced T cell failure, sustained cytokine expression, T cell clonal maintenance, and/or promotion of CAR-T memory development.

In various embodiments, the present invention provides a composition comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor. Preferably, the T cell expresses a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T). In particular, T cells express a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k.

In various embodiments, the present invention provides a composition comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor, wherein the chimeric antigen receptor protein binds to a tumor antigen. Preferably, the T cell expresses a chimeric antigen receptor protein comprising interleukin 13 or a variant or fragment thereof (IL13 CAR-T). In particular, T cells express a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k.r109k.

In various embodiments, the present invention provides a composition comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor. In some embodiments, the compositions comprise a CAR-T cell and a small molecule GSK3 β inhibitor, such as SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO). Alternatively or additionally, the composition comprises a CAR-T cell and a genetic agent comprising an siRNA, miRNA, antisense oligonucleotide, ddRNAi or a dominant negative inhibitor of GSK3 (GSK3 DN).

In various embodiments, the present invention provides a separately administered formulation comprising a T cell expressing a chimeric antigen receptor protein (CAR-T cell) and a GSK3 β inhibitor. Preferably, the GSK3 β inhibitor is a small molecule GSK3 β inhibitor, e.g., SB216763, 1-Azakenpaullone, TWS-119, or 6-bromoisatin-3' -oxime (BIO). Alternatively or additionally, the agent comprises a genetic agent comprising an siRNA, miRNA, antisense oligonucleotide, ddRNAi or a dominant negative inhibitor of GSK3 (GSK3 DN).

In various embodiments, the present invention relates to a kit comprising, in one or more than one package: a nucleic acid construct encoding a Chimeric Antigen Receptor (CAR) encoding interleukin 13(IL13CAR-T) or a variant or fragment thereof; GSK3 β inhibitors; optionally comprising a first agent for transducing a T cell by the CAR nucleic acid construct; and further optionally a second agent for activating T cells. Preferably, the kit comprises a nucleic acid construct encoding a Chimeric Antigen Receptor (CAR); GSK3 β inhibitors; a first agent that transduces a T cell with the CAR nucleic acid construct; a second agent for activating T cells. In this embodiment, the first agent is a retroviral vector. Still further, in this embodiment, the second agent is IL13R α 2-Fc. In particular, the nucleic acid construct comprised in the kit encodes a chimeric antigen receptor protein comprising the interleukin 13 variant il13.e13k. r109k, and the GSK3 β inhibitor comprised in the kit is SB216763, 1-Azakenpaullone, TWS-119 or 6-bromoisatin-3' -oxime (BIO). Alternatively or additionally, the kit comprises a genetic inhibitor of GSK3 β comprising an siRNA, miRNA, antisense oligonucleotide, ddRNAi or a dominant negative inhibitor of GSK3 (GSK3 DN).

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